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The Action of the Clam Principle on Thiamine

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r

I HI AMI NE

Hi Su

S S E R 1 A I t 0 N

b m itted

as

Pa

r t ia l

lfilm e n t

THE

of

KEqUIREMENTS

Fu

FOR

THE

DEGREE

OF

DOCT OR

OF

PHILOSOPHY

IN

CHEMISTRY

AT

hORDHAM

Un

iv e r s it y

BY AUGUST 0

L% T E N M A T A Y

D e p t ".

l

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d

ProQuest Number: 10992995

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 10992995 Published by ProQuest LLC(2018). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

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n

ACKNOWLEDGMENT

T

h is

work

GRANT

FROM

C

Re

l

i t y

,

was

THE

pu b lic

made

p o s s ib le

UNIVERSITY of

the

Ph

OF

through

THE

i l i p p i n e s

a

fellow ship

PHILIPPINES, *

QU E Z O N

r

“i F O K B V OR O

tHE

NATURE

SERTATION THE

TO T H E THE

,

IT

HIGHLY

OF

in

THE

the

I

GIVEN,

OF

THE

IS

the

PRESENTED

METHODS

WHICH

THE

OF

F OR

THE

DEDUCTION

MECHANISM

FOR

THE

C L A M -T H I AMINE

Section

V III

of

tions

Part

d at a

ing

IT

in

this

this

part

part

Only

is

the

and

deduced

established instrument

DIS­ TO THE

a

continuity

data

is

f e lt

and

THE

EXPERIMENTAL

I WO .

I t

was

considered

I NTO

THE

THEORETICAL

IN

TO

I NTO

C L A M - T H I AMI NE

the

F OC U S

SOME

REACTION,

WO R K I N G

I

hypothetical IX

ihis

is

and

by

the

to

v a ri­

be­ consid­

DISCUSSED go

into AND

EXISTING

DISTINCTIVE IN

of

introduc­

CONSIDERATIONS

AL T H OU GH

X

mechanism

th e o re tic al

THE

to

re­

preserved

judicious

BY

D a TA

observa­

INVESTIGATIONS

SUGGESTED

ARE

Sections

that

OF

PRESENTED

correlating

the of

THE

IS

in

facts.

point, set

OF

(Sections

FOR

approach

APPRAISAL

pertinent

it

INTERPRETATIONS

THE

and

sections,

POSSIBLE

L OF

IN

EMPLOYED

REACTION

and

BRI NG

WORK

A SATISFACTORY

AND

DETAIL

ORDER

OF

b rief

then

AN

ANALYSIS

uNE,

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THIS

CLOSELY

p r e l im in a r y

AFTER

Introduction

these

A useful

AT

erations

SOME

from

as

ous

in

in

mechanism

serves

the

uNE.

made

ONE)

tween

of

Part

are

action

IV

IN

ADHERE

ACCUMULATION

REQUISITE

in

TO

EXPERIMENTAL

,

n t r o d u c t io n

T HE

AFTER

DESIRABLE

REPORTED

RESULTS.

INVESTIGATION

LITERATURE.

THEN

INVESTIGATION

DEVELOPMENT

PRESENTATION

u s

THE

MADE

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Th

OF

THE DATA

FEATURES

ISOLATED

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BY

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PRESENTED

in v e s t ig a t io n s

PREDICTED

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E N C U MB E R E D

LITERATURE*

ANALOGUES

PRINCIPLE

DISCOVERED

THAT

U SE D

IN

THE

SOULD

BE

ASSAYED

OF

IN

of

HYPOTHESES

APPRAISAL

THIAMINE

CLAM

OF

,

IN

in c id e n

-

HYPOTHETICAL

MECHANISM*

THEIR

A SEPARATE

L

WITH

the

WAS

ALOGUES ACTION

ON

BEING

IT

CRITICAL

COMPARATIVE

STUDIES

TALLY,

MULTIPLICATION

STUDY

SEVERAL OF

AT

THE

THIAMINE

SPECIFICITY

OF

PHOTOFLUORIMETRICALLY.

FLUORESCENCE

SUPPLEMENT

THE

OF

THE

BEHAVIOUR END

OF

IS

THIS

AN­

CL AM THE

PRESENTED

IN

DISSERTATION*

j

r

i

TABLE

CONTENTS

of

I NTRODUC TI ON P AGE

. 111. I i

Re

view

Pr

e l im in a r y

Ex

perim enta l

of

T he

L

Ap r r o a c h

Pr P R P P

Ex

e l im in a r y

to

T he

III.

El

of

L

the

Ch

struction

18

ONE

of

r t it io n

by

D

Po

V.

T he

Nat u re

VI .

T he

L

ssible

im it a t io n

n d ic a t io n

Reactio

of

Re

of

the

De

action

27

struction

31

ia l y s is

the

OF

De s t r u VARIOUS

ction

Th IN

of

FACTORS

ia m in e

THEIR 35

Mo d e

of

Wo r k i n g I SM

a

c h a n

a r a c t e r is t ic s

T he

VI 11 .

De

of

Me t h o d

periments

T h e Pr o d u c t s of AND T H E E F F E O T FORMATI ON

I

i m i t

eneration

u c id a t io n

IV.

VI I .

8

of Ac t i v i t y a c t iv i t y

Me

Pa

n v e s t ig a t io n

r e p a r a t io n of Crude Tissue Suspensions esults of P relim inary Experiments r e p a r a t i o n o f A D e s i c c a t e d Powder r e p a r a t i o n o f Aqueous E x t r a c t s

T he

.

I

the

no

PART

1 1

1

Me t h o d s

De t e r m i n a t i o n Ex p r e s s i o n of De t e r m i n a t i o n T he Ye a s t Re g IV.

iterature

of

Formation

Re a c t a n t in

An

the

I

of

I

c t h ia m in e

X De

40 43

struction

nterm ediate

St e p

of

in

Th

ia m in e

47

the

n

52

T h e E f f e c t o f Ac c e l e r a t o r s - t h e A c t i v a t i o n P r o c e s s a n d t h e P o s s i b l e Wo d e o f A c c e l e r a t i o n OF T H E C L A M - T H I AM I N E R E A C T I O N BY T H I O G L Y C O L l i c

Ac i d

56

r PAGE IX*

X•

Summation

of

the

A Hypothetical

Reaction

PART I.

It*

Studies

o retic al

the

T

with

Be h a v Clam

Thiamine Co

iour

71

Mechanism

vm

P r e l i m i n a r y R e m a r k s on t h e t i o n s on t h e C l a m System

The The

111*

67

Results

Further

Investiga­

77

Analogues

81 81

n s id er a tio n s of

T

h ia m in e

An a l o g u e s

with

Sy s t e m

Concentra tion Principle in Clam

he

89 of

the Th Tissue

i a m in e

-D

estroying

102

The u n i t o f A c t i v i t y Acid P u r i f i c a t i o n , Procedure for Preparation o f a D e s s i c a t e d Powder from th e Acid E x t r a c t , Procedure for Ammoni um S u l f a t e P r e c i p i t a t i o n , P r o c e ­ dure fo r P u r i f i c a t i o n w i t h A l u m i n a C ~ . kl l J4, P r o * C E D U R E FOR ®AMMA

104 113 114 116 118

CONCLUDI NG REMARKS

Su p p l e m e n t the Th i a m

on in e

BIBLIOGRAPHY

L

the

223

Fluorescence

An a l o g u e s

Be h

aviour

of i

-

ix

1

r

i

L I S T OF FIGURES

F l GURE 1*

Page

T he

pH De p e n d e n c e Re a c t i o n

2,

the

Ef

fect

of

3*

The

Effect Pr i nc i

of ple

urve

Clam- T

ncentration

of

5.

The

Temperature De s t r u c t i o n Pr i n c i p e e

De p e n d e n c e C urve for of Th i a m i n e by the C

Temperature

St

6.

T

7*

T he

he

The

the

Re a c tio

a b i l i t y

n

cal

Curve FOR

e

30

f

30

g

30

h

Curve

for

and

Hy

a

and

81A

ction c l

30

the

o r etic a l

Nu

pH Depe n d enc e Be t w e e n T h i a m i n e e o p h il ic Re a g e n t ,

d

lam

C urve

Co m p a r is o n of t h e pH De p e n d e n c e T H E AC I D I M E T R I C T I T R A T I O N C U R V E T h I AMI NE The Re a

30

Clam

the

T

of

h ia m in e

Co nc en tr a tion

The

ime

Course

the

30c

Su b s t r a t e Co

for

4*

8,

L

C

the

p o t h e t i

­

^ 0

J

LIST

OF

TABLES

Table I*

Page T he

De

t e r m in a t io n

in

T II.

T he

Re

east

Ac

Clam- T

the

De

E

ffect

str uctio n

Ex t r a c t s

of

t h

De

T he

Results

IV*

T he

Re a c t i o n Re a c t i o n

Pr o d u c t s R un a t D

Re a c t io Th ia m in

n

Pr o d u c t s

e

by

Ef f e c L im it

of

V I.

V II*

V III*

T he

The

The

The

Re

t

Ex

E

of

fit*

V*

of

of

are

Th

Re

ia m in e

of

in e

Products by

AND

CHART

l i t .

LIKE

THIA­

AND

GROWTH

OF

SULFITING DECREASE

WHILE

A S S UME D IS

SHOWN

SULFITING

THE

ICTHIAMINE

DESTROY

BARNHURST

USING

THIAMINE-DEFICIENT RESULTS

ON

powder,

AS

ABOUT

SHOWN

WHICH

TO

PH 7 . 2

AND

RESYNTHES I ZES

SULFITE-SENSITIVE

MORE

the

P O WD E R ,

RESPECT I V E L Y ,

ICTHIAM INE*.

FRACTION,

of

YEAST

THE

NOT

SULFITE-SENSITIVE. EITHER

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REGENERATION,

A MEASURE D OES

.-

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THIAMINE TO

s t r u c t io n

COMPLETELY

THIOCHROME

21 B

HENNESSY

GM OF

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aqueous

THIAMINE

THIOCHROME

T he MINE

of

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TO

7 2 . 5 fo OF T H E

AND

O N E

TO

IN

IS

THE COR­

COMPLETELY

A POSSIBILITY

THAT

SULFITE-INSENSITIVE, DERIVATIVE).

* T h is assum ption is r e a s o n a b le . The s u l f i t e - s e n s i t i v e F R A C T I O N I S A B O U T 5 0 - 6 0 j £ OF T H E T O T A L R E G E N E R A B L E P Y R I M I D I N E FRACTION. I C T H I A M I N E HAS B EE N R E C E N T L Y I S O L A T E D I N 3 5 - 4 0 f o Y I E L D , AND T H E L O S S E S A T EACH S T E P I N T HE I S O L A T I O N ARE G R A D U A L AS SHOWN BY Y E A S T R E G E N E R A T I O N S T U D I E S . (BARNHURST AND H E N N E S S Y 2 1 B ) . L

-s

N — C - N H 2 •HC1

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( I S - 2 7 %)

36

r

I

t

has

already

t h ia z o l e

m o ie ty

MlNE

THE

AND

TO

THIAMINE,

IS

NEVER

is

THE

COMPLETE.

THIAMINE

SINCE

TO

THAT

THE

TAINED

)

He

c)

d

I

n

)

INCOMPLETE

ACTING

SHOW T H I S

EFFECT

"PYRIMIDINE

THE

ACTION

THIS

THIAMINE (AT

BO,TH

o teo lytic

3

OF

DAYS

ating

on

CARP

LATTER

MIXTURE p

EXTRACT

3.6

H

AND

(

d ig e s t io n

p a p a in

,

BE

TO

the

water

bath

for

one

-

half

hour

added

fresh

clam

tissue

OF

ICTHIAMINE

ALONE

OR W I T H

CLAM

TURE

NOT

IS

THUS

a

week

ANY

TOTAL THE

YIELD

WHEN

TISSUE

at

room

SIGNIFICANT

THREE

OF

PYRIMIDYL INCUBATED

TO

THAT

THE

THE

temperature

)

suspension. over

FOR

CARP

ONE

PYRIMIDYL

UNRECOVERED

to lu e n e

DAY

BE

OR

IN

OBSERVED.

VISCERA AT

.

THANl5f£)

PYRIMIDINES

FRACTIONS WITH

.

suspension.

C H A N GE ( G R E A T E R

REGENERABLE

SUSPENSION

CONVERTED

APPEARS

FOLLOWING

p a n c r e a t in

with

RATIO

THE

,

Incubation

THE

CLAM-

t r y p s in

viscera

IN

OB ­

R E G E N E R A T I ON J -

carp

C OU L D

SIMILAR

BEFORE

fresh

CASE

IS

|T

ROOM T E M P E R A T U R E .

AT

for

AS-

ABSENT»

THE

added

nding

BE

THIAMINE.

SUBJECTED

7.2)

-

ACTION

INTERMEDIATE"

ON

c t h ia

REGENERATED

FRACTION

PO SSIBILITY, WAS

C L AM

CANNOT

THE

TO

1

the

I

both

AFTER

ON

of

QUANTITATIVELY

RECOVERY

AND W O O L L E Y

St a

IT

CONVERTED

THIAMINE

ACTIVITY,

CONTROLS

although

w ith

EITHER

L

THE

recovery

Incubation

NO

THE

ARE

,

KRAMPITZ

FOR b

OF

ever

PYRIMIDINE

BY

Pr

Ho w

the

UNRECOVER&D

OPERATIONS

)

THIS

that

THE

INACTIVATED

a

.

ALCOHOL

C L AM

INVESTIGATE

TO

c o m plete

RECOVERY

RESIDUAL

BE

m entioned

PYRIMIDYL

C RI BE D TO

MAY

been

ROOM

SUSPENSION TEMPERA­

ALCOHOL.

PYRIMIDINE

FRACTION,.!

37

AS

WELL

TO

THE

AS

KRAMP I T Z

IATE".

Th OF

c a tio n

THE

OF

ICTHIAM INE,

is

unrecovered

AN

AS

YET

DEGRADATION

THE

CLAM-INACTIVATED

B.

The

t h ia m in e

A SERIES

THE

U N DE R Th BE

is

ct

was

an

an

of

Va

to

THE

THE

Thus BOTH

pH

BELOW. CUBATED

TWO

THREE

AND

(UNLESS WITH

AN

BY

out

OUT TO

if

AT BE

the

A CHANGE

OF

THE

THEREFORE OPTIMA

PYRIMIDINE

was

BOTH

OTHERWISE AQU EOU S

THE

OF

­

AS

out

of

the

DESCRIBED

THE

WELL

process

expected

to

BY

THE

7.2

could

CONDITIONS. appear

REGENERA­ SULFITE-

FRACTION.

INSIGHT AS

AND

PRESENTLY.

REACTION

PYRIMIDINE AN

THIAMINE

PH 3 . 6

reaction

IN

OF

GIVE

INTO

MODE

ANY

THE

OF

SIG N I­

F OR MA ­

FRACTIONS.

incubated

U NDE R

d ia ly ze d

SULFITE-SENSITIVE,

UNRECOVERED

PH

7.2

DESTRUCTION

THIAMINE

MIGHT

t h ia m in e

3.6

i n d i

AMONG

Fo r m a tio n

the

RECOVERY

THE

OF

an

.-

THE

CARRIED

in

TOTAL

AND

TION

be

MIXTURE.

be

INSENSITIVE

THE

SIMILAR

"INTERMED­

to

be

co nceivably

RATIO

OF

s t r u c t io n

f in d

can

m ig h t

THE

FICANCE

EV EN

FRACTION

INCUBATION

CONDITIONS

SUFFICIENTLY

CHANGE

rather

a c t iv it y

Factors

rio us

WAS

REACTION

IN

MARKED

seems

OR

INACTIVE

PYRIMIDINE

THIAMINE

De

the

a lter a tio n

AND

fra c tio n

EXPERIMENTS,

of

IN

BIOLOGICALLY

regenerable

of

attempt

A CHANGE

TION

-

PRINCIPLE

VARIOUS

ALTERED

Such IN

CLAM

WITH

PRODUCTS.

the

|N

IDENTICAL

UNIDENTIFIED

Al l

Effe

NOT

AMD W O O L L E Y

Products

BY

ARE

w ith

the

REACTION

STATE, EXTRACT

200

UG

clam

p r in c ip l e

CONDITIONS OF

EQUIVALENT

SHOWN

THIAMINE TO

at

0.125

WERE

IN ­

GM OF

THEj

38

dE s s i c a t e d

' A) WERE Al l

T

clam

ime

the

T tures

Re

of

USED ,

powder

a c t io n

I.E .,

samples

10

u s e d

THE

SAMPLES

FOR

FOUR

,

DAYS,

RUN

AT

HEATED

FOR

F OUR

THIAMINE

WAS

EXTRACTS

SHOW

56°.

400

ug

STAND

,

|N

re n t

u g

STAND

ONE

d if f e r e n t

THE IN

WEEK.

WHICH

WITH

DESTRUCTION

Al l

OF

THE

SAMPLE

WAS

TIME

ALL

THE

H E A T -IN A C T IVATED OF

THEN

THIAMINE

TESTED

­

TEMPERATURE

CASE

THE

tempera

56°.

and

GIVEN

THE

CASE,

AFTER

WAS

AND

28-30°,

AT

LATTER

(BLANKS

D A YS

Four

EXCEPT

,

C qncentration.-

’ OWDER WAS LEVELS 1000

and

D A YS

STRUCTION

WAS

COMPLETE

FOR

UNDER

CL AM THE

REGENERA­

AT

U SE D

FOR

EMPLOYED ug

.

T

he

IN

EACH

WERE:

THE

aqueous SAMPLE.

100

samples

ROOM T E M P E R A T U R E EXCEPT

An

UG,

were

U NDE R

SAMPLE

200

extract THE UG,

allowed

TOLUENE.

to

DE­

CONTAINING

UG.

Dif

f e r e n t

WERE

USED,

C lam ALL

Pr

e p a r a t io n s

SAMPLES

Crude t is s u e s u s p e n Ce n t r if u g e d aqueous 1 0 $ SODIUM CHLORIDE L

.-

4 -8 °,

56°C,

Substrate

TWO

t io n s

TO

4

per io ds

28-30°.

at

a c t io n

SAMPLE

FOR

1000

HOURS,

reactio n

IMMEDIATELY.

THIAMINE

800

Re

THE AT

THE

GM OF C L AM

DIFFERENT

48

TOLUENE

NEGLIGIBLE

BEHAVIOUR

d if f e r e n t

12-15°,

DESTROYED.

TION

F ROM 0 . 3

the

H OURS

CONDITIONS.)

Pif f e

of

AL LOWED

SAME

Four

incubated

UNDER

SAMPLE

.-

I.E .,

WE RE

n

HOURS,

were

emperature

were

).

.-

BEING

T

he

follow ing

prepara

­

EQUIVALENTS

sio n extract

from

EXTRACT

OF

t is s u e

CL AM

suspension

TISSUE -J

39

Aqueous susp en sio n or a c e t o n e - d r ie d powder Aqueous e x t r a c t o f th e d e s s ic a te d powder 1 0 fo S O D I U M C H L O R I D E E X T R A C T OF T H E D E S S I C A T E POWDER A C I D B U F F E R E X T R A C T ( P H 3 M c l L V A l N E B U F F E R ) OF T H E P O WD E R .

|N ER

NO

THAN

DINE

CASE 30^)

OF

FRACTIONS,

NOR

IN

I T WOULD

s tr in g en t

THE

I

CASES,

NEARLY

ONLY

C A SE

RECOVERY

MIDINE

ARE

PYRIM I­

PYRIMIDINE

THAT

THE

FORMATIONS

RELATED

ONE

TO

could

not

be

THE

used

OTHER. because

OF

ASSAY

AS

BY

THE

NATURE

OF

THE

TWO

REACTANTS.

OF

THE

PORTION

WAS

AL WA Y S

THIAZOLE

OF

OF

MET HOD

WH E RE

AN

WAS

IN

THE

in

Section

VII,

S U MMA RY :-

So f a r ,

the

RESULTS

OF

PRESENTED

THE

EXHAUSTIVE ABOVE

APPEAR

MORE,

THE

DA T A

OR

A PROTEIN

BE

ARE

IN

ONLY

RESULTS Part

THE

RE­

WAS

OBSERVED

OF

THE

THREE

PYRI­

WHERE

THE

PH

THE

RATIO

OF

THIS

ONE o f

THAT OF

PART

STUDIES, THE

DEGREE

IN

EITHER THE

OF

IN

ARE

d is se rta tio n .

of

the

reaction,

AND

THE

OBSERVATIONS

VARIATIONS AND

OF

EXPERIMENT

th is

ch aracteristics

SUGGESTIVE

TAKING

THE

C H AN GE

EXPERIMENT

DIALYSIS

INDICATE

TO

APPRECIABLE AND

VARIED.

OF

THEREFORE

THREE

THE

THE

WAS

discussed

L

YIELD

THE

FROM

BY

OF T H I A M I N E

F R A C T I 0 NS

REACTION

7 .2

OF

CHANGE(GREAT­

QUANTITATIVE.

The

PH

RATIO

IMPOSED

A MARKED

OF T H I A M I N E

c o n d it io n s

AS W E L L

THE

DETECTED

FRACTIONS

reaction

LIMITATIONS

ALL

THE

APPEAR

PYRIMIDINE

PRODUCTS

THE

YIELD

TOTAL

ACTION n

BE

THE

THREE

More

THERE

IN

FRACTIONS* THE

COUL D

NOT

OF

AT

PH

KIND.

ENZYMATIC

REACTION.

3.6

the

AND

FURTHER-

BEHAVIOR

40

P A R T

,V *

POSSIBLE

It

was

MUST

HAVE

TISSUE* clam

REACTION MENT

ITS WE

Kr

OF

as

IS

DEGRADED

THAT

TO

TO

A S S U ME

OF

THE

AS

CLEAVAGE THE

BY

AMMONIA^A L

IN

ARE

THIAZOLE

THIS

BY

AND

MOIETY

OF

SOME

in

MOIETY

IN

THE

BUT

CLAM

CONSTITUENT

OF

IN

THIAMINE IV.

THE

KNOWN

REACTIONS

NUCLEOPHILIC

THIAMINE AN

ARE

ATTACK

REAGENTS

THIOGLYCOLLIC

IT

IS IS

BY

THE

A

8 UC H

ACID*?,

BY

DEMONSTRAT­

THAT MOS T

A

THE

THE AS

PROBABLY

REACTANT

THIAMINE.

THEREFORE,

IN

C L AM

REACTION KNOWN

DISPLACEMENT

WATER^, THE

X

THE

METHYLENE

GIVING

TWO

DISPLACEMENT

ESSENTIALLY

ON

THEN

LIVERMORE^

DISPLACEMENT

OF

PRESENT

WHICH

LOGICAL,

T H RO UG H

SUCH

DISPLACE­

C A R P - T H I AMI NE

REPORTED

ICTHIAMINE

CHART

BY

THE

t h ia m in e

AND

MECHANISM IN

-

MATERIAL

SEALOCK

THIS

F ORMED

carp

INTERMEDIATE”

CARBON.

IS

the

UNKNOWN

HENNESSY5 ^ IN

for

PRELIMINARY

ALCOHOL*

IN

OF

"1^

THE

LIBERATED

C g H ^ N S *

MOIETY

AND

INVOLVE

SUPPORT

IONS

REACTIONS

THIAZOLE

CARBON

BY

C2H603NS*

PRESENT

TO

A "PYRIMIDINE

ICTHIAMINE

SHOWN

SUPPORTED

TO

5-METHYLENE

THSI AZOLE

TISSUE, IS

THE

THAT

FROM T H E

REFER

PYRIMIDYL

BARNHURST

AT

fragm ent,

mechanism

MOIETY

GIVE

THE

CARBON-FRAGMENT,

TO

Wo o l l e y

and

H Y D R OGE N

ATTACHED

the

REACTANT X *

EVIDENCE

REACTION.

tCTHtAMINE,

OF

A CONSTITUENT

HENCEFORTH

THIAZOLE

TISSUE

th at

ARISEN

IN

POSTULATED

THE

CARP

ING

SHALL

IS

IN

HAVE

S OUR C E

a m pitz

PRESENTED

e a rlie r

CANNOT

tissue

T he

MODE OF FORMATION

stated

ICTHIAMINE

O N E

BRIDGE BISULF IT E 27

PRODUCTS

OF

40

C HA R T

IV

N u c l e o p h i l ic D i s p l a c e m e n t i n t h e R ea ctio n s of Thiam ine

ch

N= C-NH2 *HCl

t

I

Hq C - C tt

(

St e

ps

3

c = c - c

2

h4 o h

ch2

II

I .

N — CH

Reaotant

C leavage

c— s

a)

H2 Q

)

NH3

X

b

??)

c)

Na HS03

d)

h s - c h 2 - co o h

a)

P y r - C H 2 0 H •+■ T h 1AZOLE

b)

P y r ~ C H 2 NH2

c)

Py r -C H 2 S03 H

d)

Py r - C H 2 - S C H 2 C 0 0 H + "

I c t h iamine ( p y r -C H 2 —

"t

*

( c 2 h 6 o 3 n s )J +

»

-tTHIAZOLE

N .B .

(

PYR-

)

REFERS

To:

2 - M E T H Y L - 4 - A M IN O -P Y R IM ID Y L

PORTION

L

OF T H I A M I N E .

J

a

41 r shown

To

OBTAIN

P R OO F

CLAM-THJAMINE PRINCIPLE

FOUR

DAYS

M INIMIZE

RESULTS OF

AT

THE

WAS

THIAZOLE

METHOD.

REACTION

AT

OF

THIAMINE

THE

KNOWN

STABLE

IN

THIAMINE

at

OF

BY

T H AN

pH

THE •

8 . 6

DURING THE ZOLE

THE

CLAM

recovery

PRINCIPLE

MAY

BE

The

LOW

IN

THE

(45^

MOIETY THAN

OF

THE

ON of

THE

RATE

THE

TH I S

ACTS

th at

ON

THIAZOLE FACT

RECOVERY

SATISFACTORILY

OF

the

OF

THE

IS

IS

THE OF

FASTER THE

BLANK)

of

the PH9.1

LIBERATED

HYPOTHESIS

IN

THE



incubation

DISPLACING

BY

MORE

AROUND

WAS

IT

UNDER

8 # 6

THE

THIAZOLE

EXPLAINED

HEAT-

FAR

optimum

pH

MOIETY

BY

RE­

CLAM-

A MUCH

pH

AS

THE

RECOVERY)#

SIDE

the

SO

YEAST

DESTRUCTION

AT

SUPPORTS

THIAMINE

IN

THlAMINE(lN

AL KA L I ME in

THE

WHILE

ITSELF.

PROCEEDS

th iazo le

FOR

THIAMINE

OF

BY

THAT

THIAZOLE

FOR

RECOVERY

CORRESPONDING

THIAMINE

recalled

THAT

LOW

COMPLETE

DESTRUCTION

REACTION

MEAN

IS

FACT

THE

THE

THE

SIGNIFICANT

PRINCIPLE

be

CASES,

IS THE

BY

8 . 6

THIAMINE.

FINDING OF

THE

EMPLOYED

MEASURED

EXPERIMENT,

may

AND

AS

RECOVERY

MEDIUM

ALL

7.6

BEING

OF

IN

THIAMINE

COMPLETE

THIAZOLE

C L AM

|N

VIEW

VERY

REACTION.

PORTION.

BLANKS L

WOULD

IV.

MOIETY

ALKALINE It

com plete

MIXTURE

THE

THE

C L A M -T H I AMINE The

THE

IS

ALKALINE

CONDITIONS

RATE

BLANK

THAT

IN

THE

PH 7 . 2 ,

DESTRUCTION

THIS

THIAZOLE

3 .6 ,

OF

LOW T E M P E R A T U R E

IS

8 . 6

REACTION,

INACTIVATED IS

PH

MECHANISM

DESTRUCTION

pH

TABLE

MOIETY

REGENERATION

COVERY

AT

THE

ON

DISPLACEMENT

THE

ALKALINE

SHOWN

RUN

THE

RUN

8 -1 2 °, THE

ARE

FOR

REACTION,

CLAM

TO

1

IV.

Chart

in

THE FACT

THE

THAT

THIA­

HEATED THAT

THE Jf

42 r

T H I A Z O L IUM FORM

IS

RING

VERY

OPENS

IN

REACTIVE

ALKALINE

MEDIUM;

EASILY

U N DE RGOE S

AND

THE

OPENED

FURTHRR

i

THIOL

DEGRADA-

pq

TIO N. RING

ACCORDING

TO

WILLIAMS

THIAMINE

IS

OPENED

IN

STANDING IT

IS

DOES

KNOWN

THIAMINE

IN

POSSIBILITY RING

N OT

THAT

GIVE

THE

AND

BE

B E T WE E N

THE

OF

TO

A COMPLEX

PORTED ASSAYED

RECENTLY BY

THE

GI VE BY

YEAST

AT

OR

AND

IF

OF

THIAZOLIUM

FROST3 1 • THAT

pH 8 . 6 )

AGREN1 ^ ) , THE

THERE AND

BONVICINO)*

WHILE

STABILIZE

WHEN

PROTEINS

AFTER

THIAMINE.

COMPLEXEs(lN

REGENERATION

THE

ACIDIFICATION

COMPOU N D S

H ER E

DENATURED

HENNESSY

,

RECOVERY

AND

BLANK

INTERACTION THIAMINE

ALKALI,

MENTIONED

HEATED

RUEHLE

OTHER

(M CIN T I R E

SOLUTION

OPENS(IN

WITH

COMPLETE

PROTEINS

S H OU L D

AND

THIAZOLIUM

MAY THE

THE

WHICH

THE

OCCUR THIOL

MANNER IS

AN FORM RE­

NOT

METHOD.

U . J . H E N N E S S Y AND G . B O N V I C I N O , A B S T R A C T S , A . C . S . M e e t i n g a t A t l a n t i c C i t y , N . J . , page 6 3 c . ( S e p t . 1949)

42

1

r Table

The

Re Re

Products

actio n

R un

a c tio n

at

Re g

Sample

ACTIVE Extract

He a t e d Extracts

p

H

D

IV

in

the

if f e r e n t

Clam p

T o ta l Regen. P y r 1M 1 DINES

ia m in e a

H Values

Va l u e s

enera tio n

Th

as

ug

T o t a l Re g . Th ia z o l e

Th

ia m in e

®

SULFITE1 N S E N S I T 1 VE

3.6 7.2

156(78#) 148(74#)

2 0 0

( (

1 0 0

2 0 0

1 0 0

#) #)

24(1 2$) 56(28$)

7.6

106(53#)

2 0 2

(

1 0 0

#)

56(28$)

8 . 6

92(46#)

2 0 0

(

1 0 0

#)

6 0 (3 0 $ )

3.6

2 0 0

(

1 0 0

7.2 7.6

2 0 0 ( 186( 124(

1 0 0

8 . 6

#)

#) 90#) 62#)

198(100#)

2

(1 0 0 #) 195(100#) 90( 45#)°

0

2 0 0

(

1

$)

8 (4$) 54(27$)

A) 5 ML OF A QU E OU S E X T R A C T E Q U I V A L E N T T O 0 . 1 2 5 GRAM OF C L A M POWDER W I T H 5 ML OF T H E G I V E N B U F F E R AND A D J U S T E D TO T H E G I V E N PH WAS I N C U B A T E D W I T H 2 0 0 UG OF T H I A M I N E FOR FOUR D A Y S AT 8 - 1 2 ° , P R E S E R V E D W I T H A FEW DROPS OF T O L U E N E . BEFORE RE­ G E N E R A T I O N , T H E S A M P L E S WERE F I L T E R E D .

THE

l

The d e s t r u c t io n ACTIVE EXTRACT. Heated b la n k a t Heated b la n k a t Heated b la n k a t Heated b lank a t

was

3 .6 : 7 .2 : 7*6: pH 8 .6 : pH pH pH

b)

The

c)

Unsupplem ented(no

fig u r e s

complete

in

in

all

samples

c o n t a in in g

No d e s t r u c t i o n o f t h i a m i n e . No w n B 2 5 ug o f t h i a m i n e d e s t r o y e d . 1 5 0 ug o f t h i a m i n e d e s t r o y e d .

parentheses

th iazo le

show

or

the

per

pyrim idyl

cent

recovery

.

alcohol j

a

42 b

ADDED

B E F O R E R E G E N E R A T I O N ) S A M P L E FOR T H I S H E A T E D B L A N K A T C A V E A R E G E N E R A T I O N V A L U E OF 8 3 UG# I n A L L OTHER CASES 9 T H E U N S U P P L E M E N T E D SAMPL ES CAVE R E G E N E R A T I O N V A L U E S A L M O S T I D E N T I C A L TO T H O S E G I V E N I N T H E T H I R D COLUMN FOR " T O T A L REGENERABLE P Y R I M I D I N E S #

PH 8 * 6 ,

L

J!

43

r

“i

PART ONE

V.

THE NATURE OF REACTANT X »>

A•

The

SHOWN

THAT

results

THE

OF

THE

|T

OF

INTEREST

DUCTS

FORMED

DESTROY REMOVE

MOLECULE* A NCE

OF

WHICH

WHEN

SUCH

BE

TO

SCRIBED

in

INCUBATED

COMPLETELY WOULD

SHOWN

of

250

the

UG

T he OF

clam

IN

non

EQUIVALENT

TO

BE

IN

TO

90^

ASSAY

OF

TWO

SHOWED

SENSITIVE PRESENT

IN

DAYS THAT

DESTROY AT

-

OF

THE

OF

IS

IS

REACTION

AL LOWED

IS

IN

A

THE

TO TO

SMALL DISAPPEAR­

FRACTION)

REGENERATION.

powder

was

MANNER

TEN

THE

(PRESUMED THE

therefore

PREVIOUSLY portion

ML

GM OF

AMOUNT

PRODUCTS.

PRO­

EXPECTED

LATTER

OF

THE

THE

YEAST

OF

THE

TO DATA

BE

sub

­

DE­

was

then

N O N —D I A -

POWDER

THIAMINE

ROOM T E M P E R A T U R E * .

FRACTION

EXTRACT.

REACTION

PORTION

d ia ly za b le

0 .5

A SIGNIFICANT

PYRIMIDINE THE

CL AM

THE

THE

THIAMINE.

EXTRACT,

PERIOD

ORIGINAL

REFLECTED

YEAST

DIALYSIS

II.

c tio n

BY

had

A SIGNIFICANT

FORMATION(SULFITE-SENSITIVE

extract

ORDER

IF

exper im en ts

RETAINED

DIALYSIS

BE

LYZABLE U SE D

THE

NATURE

EXHAUSTIVE

A REMOVAL

EASILY

WITH

OF

NON-DIALYZABLE

X

d ia l y s is

FRACTION

ACTIVITY

THE

EXHAUSTIVE Se

exh a u s tive

KNOW T H E

THE

REACTANT

aqueous

jected

TO

ICTHIAMINE

MAY

T he

ORIGINAL

THIAMINE. THE

the

NON-DIALYZABLE

AMOUNT WAS

of

HAD

A DDE D

TO

IN

A

REGENERATION

SULFITEICTHIAMINE)

SHOWN

ON

TABLE

WAS V.

* T H E A C C E L E R A T I O N OF T H E R E A C T I O N BY A D D I T I O N OF A C C E L E R A ­ T I N G R E A G E N T S C OUL D N OT BE E M P L O Y E D S I N C E T H E R E WAS A GOOD I N D I C A T I O N T H A T T H E S E R E A G E N T S A C C E L E R A T E T H E R E A C T I O N BY P A R T I C I P A T I N G I N T H E N U C L E O P H I L I C D I S P L A C E M E N T OF T H E T H I A Z O L E 4 0 I E T Y UNDER T H E C A T A L Y T I C I N F L U E N C E OF T H E CL AM P R I N C I P L E , j

43

r

T able

T he

Re

actio n

Th

of

ia m in e

Po

rtion

Extract

5.5

p

&Q.

Ex

e q u iv a l e n t

to

ml

0.28

GM

Pr o d u c t s

t

.

POWDER

V

in

Non-

by

the

of

Clam

H

the

De

str uctio n

d ia l y z a b l e

Extract

T o t a l Regen$ P y r 1MlDINES

3.6

218

7.2

178

3 .6 s

178

7.2°

113

3.6

206

7.2

164

6 .5

2 4 5 |ug

Re g e n . a f t e r S U L F I T 1 NG

36

ug

UG

81

'

5 . 5 ml Non- d i a l • P o r t i o n ( 0 . 2 8 GM P O W D E R ) , PLUS D 1ALYSATE

He a t e d

59

ug

UG

to

Bl a n k



CO c—

10 ml No n - d i a l . Po r t i o n e q u i v . 0 . 5 GM POWDER

UG

40

ug

75

UG

6

No t e s

a

)

Re

b

)

AND

genera tio n

C)

30

UG

from

250

THIAMINE

ug

t h ia m in e

REMAINED

o r ig in a l l y

UNDESTROYED

pr e se n t

AT

W H I L E 2 4 UG R E M A I N E D U N D E S T R O Y E D A T p H 7 . 2 A F T E R TWO R E A C T I O N AT 2 8 - 3 0 ° * | N T HE OT H E R S A M P L E S , E X C E P T I N H E A T E D B L A N K , T H E D E S T R U C T I O N WAS C O M P L E T E *

L

PH

3.6

D A YS THE

*

a

44

r This

i appears

to

be

a

clear

indication

th at

R eactant

X

is

NON-DIALYZABLE*

B*

Su p p o r t

DIALYSATE. 100°

FOR

ABSENCE

for

THE

FIVE

t h is

was

DIALYSATE

MINUTES

AT

o btained

A L ON E PH

3.6

OF

REACTIVE

NUCLEOPHILIC

BISULFITE,

CYSTEINE

AND

THIAMINE DITIONS IN

AT AS

EXCESS

THE

DUCTS

TO

A QU E OU S

C.

THE

WITH

RATE OR

LOW

DISCUSSED

INCUBATION IN

VERY

OR

THE

REACTION

THIAMINE

HAD

NO

STRUCTION,

T he

a ter

,

he

d e r

was

t u r e s

der

L

(

p

AND

THIAMINE

AND

7.2

INDICATING OF

ACID,

WHICH

THE

DIALYSATE

WHEN

ACTIVE

AQUEOUS

THERE OF

THE

WAS

CL AM

NO

C OMP A R E D

TO

REACTION

OR

OF

DESTROY CON­

WAS

OBSERVABLE

THE

TYPE

EXTRACT

DESTRUCTION

AT

AN

THE

EARLIER.

ON

THE

MIXTURE

,

m o is t

then

TH I A M I N E .

p

(

one

-

OF

THE

THE

A DDE D

PRIOR

TO

DIFFERENCE

IN

THE

P R O­

UNSUPPLEMENTED

in

,

G .1N

a

vacuum

H 7.2) THERE

water

and

NO

100°)

at

C L AM

THE

when

EXTRACT

LIM IT

OF

various

and

0 .1 N

DE­

0.5

gm

of

solvents

NaO H).

dessica tor

over

the

in c u b a tio n

0.1

gm

of

EITHER

the

ON

T he

P 2 O5 .

clam

to

EFFECT

AND

FORMED.

added

c o n t a in in g

WAS

ON

w ith

HOT

to

hour

ACTIVE

PRODUCTS

extracted

e t h e r

in

half

RATE,

corresponding

redispersed

and

was

d ried

,

ON

REACTION

powder

m a t e r ia l

H 3.6

e x t r a c t s

INFLUENCE

clam

were

d r ied

,

OR

alc oh ol

extracts

T

DESTROY

SUBSTANCES

the

EXTRACT.

TO

(w

N OT

MILD

AS

aqueous

u.

DID

AND

LIM IT

He a t e d

A D DE D

w ith

THIOGLYCOLLIC

THIAMINE,

DESTRUCTION

s t u d ie s

CONCENTRATIONS

FRESH

THE

in

M IX-

clam

THE

pow­

pow­

RATE

OR J

45 r ON

~i THE

LIM IT

These LEAD

TO

OCCURS

OF

DESTRUCTION,

o bservatio ns

THE IN

together

TENTATIVE

THE

C L AM

SUPPOSITIONS

SYSTEM,

the

a c c u m u l a t e d da t a

THAT REACTANT

X

AS

IT

Is:

A)

NON-DIALYZABLE

B)

HEAT-LABILE

C)

I N S O L U B L E I N A L C O H O L AND M O I S T E T H E R , OR AT L E A S T I N A C T I V A T E D BY O R G A N I C S O L V E N T S A N D / O R D R Y I N G ,

D)

SH OU L D H A V E A S U L F U R ATOM ( A N D VE R Y P R O B A B L Y A B A S I C N I T R O G E N G R O U P ) A T OR NEAR I T S R E A C T I V E C E N T E R , THE B A S I C N I T R O G E N GROUP I N T H E S I D E C H A I N OF I C T H I A M I N E I S SHOWN BY E L E C T R O M E T R I C T I T R A T I O N ( S E E S E C T I O N 1 1 ,

TWO).

P a rt

E) F)

PRECIPITATED

BY

ACETONE

PRECIPITATED

BY

AMMONIUM

A D S O R B E D ON A L U M I N A H ? , 4 o r p H 8 Me I

G)

p

The

BEHAVIOUR

TISSUE

WHICH

MAY

BE

EXPLAINED

NOW

ARISES

NISM.

I

17

MOIETY

t

may

AN

SE COND

THIS

SUPPLIES

be

INVOLVES BY

OF

BY

REGARDING

"PYRIM IDINE A

CftAMMA l v a in e

AT

5,2

PH

b u f fer

P R E C I P I T A T E D AND I N A C T I V A T E D AND M E T A P H O S P H O R I C A C I D .

H)

(SEE

SECTION1 1 1 ,

(

AND Se c

see

E L U T E D BY 111,

tio n

TWO)

P a rt

NISM

SULFATE

TWO).

PART

BY

w ith

THE

COMPONENT

IT

BE

A CONSIDERATION that

A PRELIMINARY

the

TO

WHICH

GI VE

Kr

THE

IN

A OF

IS

OF

OF

THE

CLAM

THE

QUESTION MECHA­

Wo o l l e y

and

TISSUE

THE

mecha

THIAZOI^E

TO

DEGRADED

PYRIMIDYL

THE

REACTION

OF

ACID

1C T HI AMI NE

PROTEID.

a m pitz

C ARP

THEN

X)

FRAGMENT

DISPLACEMENT

"REACTANT*

INTERMEDIATE" ENZYME)

TO

TRICHLORACETIC

(REACTANT

TWO- CARBON

ASSUMING

recalled

UNKNOWN

BY

GI VE

A

(PRESUMABLY

ALCOHOL.

IN

THE

-

46

r

CASE

OF

THE

UNKNOWN

CLAM

REACTANT,

Wh a t e v e r S E E MS

TO

the

BE

PLACES

T HE

CARBON

TO

SE C ON D

STEP

ENSUING

THE

REST

OF

MORE

true

IF MO I E T Y

LEAST

SPLIT

REACTANT

CLEARLY,

WE

GIVES

IT

MAY

THAT

THE

T H IA M IN E

REACTION

MAY

WRITE

( C 2 Ha 0 3 N 3 ) P ------------- 1

BE

THE

,

one

THE

X DIS­

METHYLENE

POSTULATE

A

COMBINATION,

I.E ., FROM

TO

ILLUSTRATE

X AS X

Reactant

WRITTEN

"i

p o in t

FRAGMENT,

REACTANT =

b e

REACTANT

I C T H I AMINE#

GIVE

NOW

may

TO

THE P R E L I M IN A R Y

THAT

tCTHIAMINE*

WITH

NECESSARY

^(C e% 03N S )pJ SO

TO

STATED,

COMBI NES

IS

A S S UME D

reaction

TWO-CARBON

X TO

HAVE RISE

WE H A V E

AND

AFTER

OF T H E

WE

of t h e

AS

PYR-Cf^-X,

AT

X,

nature

EVIDENT,

FORM

HOWEVER,

REACTANT

THIAZOLE

THE

THIS

SYSTEM,

AS

FOLLOWS:

|p Y R -C H 2-(C 2H 6 03N S )pi

+

T h ia zo le

Mo I E T Y ( p Y R I M l D I NE Com plex) Second (Kr

a m pitz

second

step

and

enzyme

Wo o l l ???)

ey

'

s

V

[ p y r -C H 2 -(C 2H 603N S ))

+

P

I C T H I AMINE WH E RE

The

above

Wo o l l e y IS

L

PYR-

THE

suggestion

m ight

HIGHLY

IS

be

PYRIMIDYL

that

involved

SPECULATIVE

AND

the

in

PORTION

second

BE

THIAMINE,

enzyme

thissecond

CANNOT

OF

step

TAKEN

UP

of

Kr

a m pitz

is

in v it in g

IN

THIS

and

but

SECTION,

47

P A R T

V I.

THE L I M I T A T I O N

The CLAM

l im it a t io n

the

In tro d uctio n

ET

AL.

OBSERVED

Wo o l l e y ^ TIME

They

reported

THE

IN

THE

REACTION

ALCOHOL THE

AND

LIM IT

Ho

wever

THE

NOR

we

EARLY

which

a

IN

ACTION

,

6 0 ft

is

be

AFTER

due

the

EXCESS

A MOUNT S

HAD

NO

DESTRUCTION,

never

observed

the

amount

g iven

clam

THE

of

of

A FEW H O U R S .

THE

N OT

EFFECT

REACTION.

WHI CH

can

BEEN

AND

m ec h a n ism s

in

is

d estro y

ON

THIS

KRAMPITZ

present

by

PYRIMIDYL

INHIBITING

t h ia m in e

HAS

regular

e q u il ib r iu m

preparation

DESTRUCTION

OF

*1 5

KRAMPITZ

in h ib it io n

h yd r o lytic

t h is

Sealock

is

CEASES to

g iven

CARPf

destruction

BUT

in d ic a t iv e

OF

a

PREVIOUSLY

L ite ra tu re ).

THE

MOIETY

which

MENTIONED

T H E ' E Q U I L I B R I U M ' 0 F THE

have

amount

BEEN

carp

cannot

SINCE

t h ia m in e

the

IN

STAGES

t h is

VALUE',

provided

of

the

THIAZOLE

ON

a c tio n

LIM ITATION

th a t

of

HAS

LIM ITATION

that

r epo r ted

,

DESTROY

PRODUCTS

'EQ U ILIB R IU M Wo o l l e y

A

amount

(Review

found

WITH

the

CAN

in

and

I N THE DESTRUCT1OMr

in

PREPARATION

O N E

.

clam

below

.

the

The

SATISFACTORILY

EXPLAI NED.

I THE OF

n

the

case

PROBABLE

Re a c t a n t

OF X

THE to

CONSIDERATION L

IN

CLAM

the

CAUSE

IMPORTANCE

BECAUSE

of

BOR

THE

THE

OF

THE

RELATION

c essa tio n

SHOULD

MAKE

at

of

THIS

IN

NATURE OF

the

,

in v e s t ig a t io n

THE

DESTRUCTION

l e a s t

LIMITATION

STUDY

POSSIBLE the

actio n

THE

OF

.

THE

IS

REACTION

EXHAUSTION

r e a c t io n

EVIDENT:-

THE

of

The

OF follow ing

MAN N E R

OF

*•

48

r ACTIVATION PHILIC

OF

THIAMINE

REAGENTS

MUST

BRIDGE-CARBON( I . E . , ELECTRON-RICH) PHlLlC CLAM

GROUP*

PREPARATION

THE

AN

THIAMINE

(OR

A PRECURSOR)

VELY,

WOULD

BEEN

S H OU L D WITH

THE

EXHAUSTED,

INCREASING IN

THE

OBSERVED* TOTAL

OF

DESTRUCTION

THE

7.2,

DOESN'T

OF

TEMPERATURE,

AND

VARY

8*6

THE OF

THE TO

IS

FOR

IS

EXPECT

T HE

OF

BY AND

SIGNIFICANTLY.

OF

LESS

A NUCLEOIN

THE

OF

SH OU L D

THE

BY

SHOW

A GIVEN

ONLY THE

(STUDIES

HAS

AT

MORE

FORMED

ALTHOUGH

THE

TO APPAR­

ALCOHOL

AMOUNT

OF

NEVER

CONSTANT^

ACTIVITY

OF

THE THE

MEYHOD)

AT

THE

RATE CLAM VARIES

LIM IT

PH 3 * 6 ,

DESTRUCTION

ALKALINE

PH WERE

* Th i s r a t io as w e l l as th e r a t io of the p y r i m i d y l A L C O H O L FORMED TO T H E T O T A L R E G B N E R A 8 LE P Y R I M I D I N E F R A C T I O N O B T A I N E D A T PH 3 * 6 AND 7 . 2 S T A Y S R E A S O N A B L Y C O N S T A N T E V E N I N D I F F E R E N T C O N C E N T R A T E S OF T H E C L A M P R I N C I P L E ( S E C T I O N 111, P a r t TWO o f t h i s d i s s e r t a t i o n ) *

L

X

PROGRESSIVE

CONCENTRATION,

LIM IT

REACTANT

EXTRACT

THIS

SLIGHTLY*

IC-

ALCOHOL

ALCOHOL

THUS,

THUS

RESPECTI­

IF

PYRIMIDYL

FOR

GIVE

A CLAM

REASONABLY

SUBSTRATE

EXAMPLE,

MADE

PYRIMIDYL

PYRIMIDYL

ASSAY

AFFECTED

OF

T H A T . EVEN

THE

E XH A U S T C D •

THE

IS

ALCOHOL

HOWEVER,

STAYS

THIAMINE

METHYLENE—

SYSTEM

PYRIMIDYL

INCUBATION

FORMATION

X

APPROACH

ENZYME

THIAMINE

DESTROYED

MEASURED

DESTRUCTION

7*6

AND

OF

REACTANT

THE

X AND WAT ER TO

REACTANT

RATIO

THE

OF

i NUCLEQ-

WITH

METHYLENE-BRIDGE-CARBON

THE

WHEN

OF

THE

ALCOHOL.

CEASES

PH,

OR

PYRIMIDYL

THEREFORE,

WITH

THE

FORMATION

AMOUN T S

THIAMINE

PR|NCIPLE(AS

THE

HENCE,

ENTLY,

THE

BY

REASONABLE

CONTINUE*

INCREASE SEEN

ATTACK

REACTIONS

ACTIVATION

ENZYME

"ACTIVATES*

AN

HAS

AN

FAC I L I T A T I N G

NOW,

BE

CLEAVAGE

METHYLENE-BRIDGE-CARBON

FACILITATING

IT

ITS

INVOLVE

THUS IF

IN

49

r

"i

NOT

FEASIBLE

DESTRUCTION

BECAUSE OF

THE

THIAMINE

HIGH

BLANKS

RENDER

THE

DUE

TO

THE

ALKALINE

EXPERIMENTAL

RESULTS

VERY

UNCERTAIN).

AN THE TO

INVESTIGATION

CESSATION FIND

OU T

OF

IF

AN

AN

STRUCTION

OTHER

OFFERED.

THE

OF

THE

MORE

ENZYMATIC

REACTION

EXPLANATION

THAN

THE

FOLLOWI NG

GENERALLY

FOR

THE

EXHAUSTION

WAS

NOW

CAUSES MADE

LIMITATION

OF

EXPERIMENTS

KNOWN

REACTANT

WERE

IN

IN X

FOR ORDER

THE

C OU L D

DE­ BE

R UN ! -

A) 1. (

Excess

p y r im id y l

WE R E

TO

2.

I

I

WHEN

TO

a

n

WITH

EXHAUSTIVELY

PRODUCTS

AND

DIALYSATE

REGENERATION

WAS

KEPT

U N DE R

A D DE D

EXHAUSTIVE ADDITION Table

V I.

EXCESS

OF

THE

THE SAME

INSTEAD DIALYSIS,

OF

I

FRESH

r

OF

,

THE

FROM

A

OF

m o ie t y

CLAM

IN H IB ITIO N WERE

IN

extract

ONE

AT

TO

DAY

REMOVE

FRESH

20°

THE

UNREACTED.

BE

(4x)

EXTRACT

OF

INCUBATION

SOLUTION,

A PART

OF

AFTER

ITS

RESULTS

WAS

ADDITION

DID

CONDITIONS

after

REACTION

THE

IDENTICAL

THE

C OU L D

aqueous

AN

REGAINED

)

POWDER

PRESENT

A CTIVITY.

THIAMINE

products

PRESENT.

FOR

THIAMINE

th ia z o l e

NO

an

COL D

reaction

EXTRACT

THIAMINE

DIALYSATE.

f/*)

the

PRODUCTS

THIAMINE IN

known

THIAMINE.

OF

PREPARED

ANY

and

A QU E OU S

THREE

EXCESS

THE

three

e x p e r im e n t

DIALYZEO

WA T E R

OF

ALL

related

the

AN

CONCENTRATION

INCUBATION

FRESH

of

c t h ia m in e

ADDITION

EVEN

MOL AR

,

IN ITIA LLY

THE

OBSERVED THE

alc oh ol

ADDED

PRIOR

amounts

N OT

WHICH

WITH

SIMILAR

ACTIVITY

ARE

CAUSE

EXTRACT BUT

OF

SHOWN

UPON IN

THE

50 r

v b

FOR

)

Three

LIM ITS

DITIONS THREE

IN

SAMPLES

the

TO

I

n

STRUCTION

20

)

I

MGS

n

REACH

same

THE

OF

DAY

THE

s im pl y

SAME

THE

tested

TO

T HE

IS

USUALLY

0.1%

DESTROYED IN

denaturation

CON­

TEMPERATURE).

w ith

LIM ITATION

a

DAY

ROOM

preserved

THIAMINE

to

ADDED

(ONE

AT

were

EXPERIMENTAL

WAS

EACH

VALUE

28-30°

at

HENCE,

due

THE

extract

SOLUTION

ONE

CASES,

SAME. be

UNDER

LIM ITATION

kept

THREE

aqueous

THIAMINE

THE

were

another

WHILE

the

of

try p s in ,

papain,

pepsin,

48

hours THE

DECREASE

IN

THE

THE This

N OT

8-12°

INCUBATED IN

EACH

e xtr a c ts

incubated

at

pH

6

CASE,

in d icates

DUE

TO

THE

THERE

that

any

PROTEOLYSIS,

IN

THE

OF

AN

.

ENZYMES WITH

,

WAS

THE due

DE­ to

stand

e q u iv a le n t

separately

p ancreatin,

THIAMINE-DESTROYING

LIM ITATION

MERELY

at

PROTEOLYTIC

SAMPLES HOWEI TER

PROBABLY

clam

were

TURBID.

PARATION.

,

powder,

WITH

THE

e x p e r im e n t

clam

FOR

CUBATED

L

THE

INTERVALS

cannot

GM o f

SALIVA

ED

the

­

.

c

0.1

AT

all

APPROX I M A T E L Y

of

DESTRUCTION

THAT

samples

TOLUENE.

in g

THE

EXCEPT

SUFFICIENT Al l

samples

6

.

A ll

AND

the

w ith

1

ML

A L MOS T

SALIV8

REMAINED

WAS

APPRECIABLE

POWER loss

OF T H E

in

the

OF

SAMPLES

BECAME

NO

to

INr

WATER-CLEAR VERY

C L AM

PRE­

a c tiv ity

is

ETC.

DESTRUCTION

THEN

CANNOT

BE

EXPLAIN­

BY:

1.

ATTAINMENT

EQUILIBRIUM.

2.

In h ib itio n of the reaction P R O D U C T S OF T H E R E A C T I O N .

3.

A D E N A T U R A T I O N OBSERVED I N ENZYME IN G , P R O T E O L YSIS, O X I D A T I O N , ETC.

by

an

accumulation

ACTION

DUE

of

TO

the

STAND­

J

51 r

“ i

The

most

lo g ic a l

e xp la n a tio n

DESTRUCTION

APPEARS

TO

SUPPLIED

THE

ISM

THEREFORE

ALCOHOL

X

L

IS

BY

IN

CLAM

SHOULD

SUCH

BE

THE

TISSUE

THAT

the

EXHAUSTION

(l.E .»

EXPLA IN

A M AN N E R

for

THE ITS

l im it a t io n

OF

REACTANT

FORMATION FORMATION

in

the

A LIM ITING

X). OF

THE

STOPS

ANY

FACTOR

MECHAN­

PYRIMIDYL WHEN

REACTANT

EXHAUSTED.

•J

51 a r

i

The

St u d y

or on

Clam

Ex

:

t r a c t

Effect

the

L

the

im it

of

of

Pr o d u c t s

R eaction

the

d e s t r u c t io n

.

1 ml c o n t a in e d the s o lu b le p o r t io n of 0 . 1 2 5 GM OF a c e t o n e - d r i e d c l a m p o w d e r . ( 1 ML OF T H I S E X T R A C T W I L L D E S T R O Y A B O U T 2 5 0 UG T H I A M I N E I N 2 4 HOURS A T ROOM T E M P E R A ­ TURE, p H 7 . 2 )

SAMPLES: I.

6

ML OF

I I .

6

ML C L A M

EXTRAC

H I .

6

ML CL AM

EXTRACT.

6

ML C L A M

EXTRACT.

IV .

Pr

ocedure

0 . 1 fo

:

C L AM

Al l

toluene

for

24

DENATURATION

Then,

Samples AGAINST

8-10°

AND

I, 10

IN

CARE

TAKEN

WAS

CLOSE TO

TO

FIND

OUT

SINCE and

CHANGES

SAMPLE

THE

TO

THE

B uffer

250

(IN

DIALYSIS.

SAMPLE

IV

T he

total

of

d ia l y s is

6

l

ML

OF

EACH

COL D

VOLUMES.

PRINCIPLE

,

WAS

all

EACH

VOLUMES

DILUTION

d i a l y s is

ML

added

SAMPLE(EQUI VALENT

N OT

order TOO

DISTILLED

OUT.

OF

in

THE

w ith

WA T E R

WA T E R

IN

ALL

CASES,

DlALYZABLE

PORTION

WAS

RUN

IN

S A MP L E

II)

HAD

ANY

EFFECT

T HE

CONDITIONS

UNDI A LYZED

was

ORIGINAL

ORDER

USED

ON FOR

EXTRACT.

.

two

days

samples

were

TO

GM OF

0.25

AT

UNTIL

Itl

UNDER

to

STABLE.

SAMPLE

THE

the

OF

28-30°

cellophane

DISTILLED

DIALYZED

THE

the

not

in

THE

SOLUTI O N ( 2 0 0 0 U G ) .

at

dialyzed

were

ALL

the

was

stand

III

STABILITY

t im e

to

ARE

I

THE

THIAMINE

EXTRACTS

THE

Af t e r

OF

allowed

RUNNING

KEEP

ML

BUFFERED

OF

WAS

4

4 ML W A T E R .

WITH

ORIGINAL IF

were

hours.

II,

FINALLY

THIAMINE

PLUS

samples

AVOID

bags

EXTRACTPLUS

d ilu te d

C L AM

to

18

m l

J

POWD E R ) -i

51

WERE OF

TESTED

THIAMINE

FOR AS

ACTIVITY SUBSTRATE.

FROM T H E

ORIGINAL

shown

T

on

DESTROYED TOTAL

VOL.

A QU E OU S

AT

ptf IN

3.6 ALL

EXTRACT

able

Y1,

where

the

FROM

200

UG

THIAMINE

OF

20

ML.

OF

AND

PH 7 * 2

CASES, WAS

IN

DIALYSATE

ADDED.

destructio n

24

USING

THE

200

PREPARED RESULTS

represents

HOURS

AT

UG

25°

ARE

t h ia m in e

IN

A

b

TABLE

Sa m ple

Extr a c t

p

*

1

n

Ex t r a c t

Extr a c t

Extract

L ...........

-

-

11

! II

-IV

H

VI

Th ia m ir e

3*6

0

7 ,2

0

3*6

104

7 ,2

60

3 .6

114

7 .2

55

3 .6

187

7 .2

152

UG

ue

ue

ug

destroyed

52 r

P A R T

VII.

aspect

regarded

of

the

LACK

for

A CONSIDERATION

TO

DO W I T H

THE

ICTHIAMINE

17

THE

CARP-THIAMINE OF

THE

TO

ACT

MIDYL

alcohol

IN TIONS

AN

OUR OF

LY

BECAUSE I.

SHOWS

THE

AN

THE

B E C OM E S

PYRIMIDINE

THE

NATURE

NOW

MORE

OF

ALKALINE,

a

UP

?s e c o n d

NO

PRIOR

THIS

V) .

HA S

enzyme*

in

DETERMINA­

GIVE

OT H E R

OF

KRAMPITZ

ENZYME* TO

­

FORMATION

h y po th esis

OBTAINED

STEP

SEEM

BUT

TO

REACTANT OBTAINED

THE

DECREASES

THE

CONTAINS

HAVE

RE-EXAMINED,

ON

SECTION

"SECOND

t h is

SATISFACTORY

PRODUCTS

FRACTION

ACCELERATED

WE

OBSERVATIONS

REACTION IS

A

THE

d i s

IS THE

have

PREPYRI­ not

REFERENCE

REACTION.

INTERMEDIATE OF

IN

INTERMEDIATE* on

TAKEN

MECHANISM.

STEP

of

BE

been

MICROBIOLOGICAL

THIS

s t u d ie s

LITERATURE

INTERMEDIATE

E .,

REACTION)

L

Further

ELUCIDATION

STEP,

IS

.

THE

THE

.t

A "PYRIM IDINE

SUCH

THE

F ROM

PRODUCTS.

INVESTIGATION,

IN

in d icatio n

REACTION

NOW

REACTION

(p A G E ^ \

ON

REPORTED.

SUCH

an

heretofore

MAY

INTERMEDIATE

EARLIER

REACTION

has

PROOF

A WORKING

obtained

1 1

SUMED

BEEN

OF

DISCUSSED

W o o lley

w hich

DEFINITE

POSSIBLE

and

TION

r eactio n

OF

TO

PR

1

INDICATION OF AN INTERMEDIATE STEP IN THE REACTION.-

An

TO

O N E

ALKALINE

IT

THESE

SUCH

X.

TABLE

AT

IF

AN

YIELD

OF

THE

NOTED

GREATLY G .,

MAIN­

INTERMEDIATE (WHICH

PH

THAT

TOTAL AND

CONSIDERED

I S/

DIFFERENT BE

E.

BE

INDICA­

MECHANISM

DEMAND

TO

SIDE,

MEAGRE

MAY

REACTION

IS

VERY

ON L Y

VALUES AS

THE

REGENERABLE

THE

DECREASE

OF

53 r p

H

of

Rx

the

3 .6 7 .3 7 .6

44

the

94 108

values

thiam ine

IS

GREATLY

CARBON

PLANATION

WE AT

THAT

HAVE

EITHER

SUBSEQUENT CARP

OF

THROUGH

pH 3 . 6

OR

PH 7 . 2

ADDITION

THE

PYRIMIDYL

THE

UNRECOVERED

L

THAT

AND

THE

DECREASES

HAS

CLAM

NO

V / 1 TH

AN

YIELD

THE

X, IN

OF

THE

APPEARS

TO

BE

ORIGINALLY

ON

I I I ,

IS

EXTRACT

PART

EFFECT FORMED,

NOR

THE

THE

EX­

IS

OF

IN

OBVIOUS.

O NE).

pH OF

RUN

THIAMINE,

FRESH THE

TOTAL

INDICATING DC

N OT

ICTHIAMINE

SULFITE-SENSITIVE IN

METHY­

N OT

OR

AND

THE

REACTION

APPARENTLY

FRACTION

A ST R ON G

ALCOHOL

DESTRUCTION

CLAM

AL­

SULFITE-

RESPECTIVELY. I C T H I AM I N E

OF

DECREASES

ATTACK

SIGNIFICANT

INCREASE

YIELD

PYRIMIDYL

PYRIMIDYL

PRINCIPLES

THE

THE

ICTHIAMINE)

FRESH

(SECTION OF

OF

COMPLETE

PYRIMIDINE

YIELD

THE

C L A M - T H I AM I N E

ALCOHOL

ALCOHOL, THE

TO

EITHER

SUSPENSION

VERT

PYRIMIDYL

OF

THE

AND

INDEPENDENT

DECREASE

ug

DESTROYED

THIS

OF

200

from

thiam ine

BE

REACTANT

WHEN

CARP

GREATLY

THE

THAT

THE

HOWEVER,

AND

LARGE

THAT

THE

FORMATION

(46$) (25% ) ( 1 6 fo )

the

THE

pH.

u g (68$)

cases,

YIELD

TO

INCREASING

SEEN

VISCERA

YIELD

THE

a ll

YET

(PRESUMED

BY WA T E R

FOR

THE

136 92 50 32

recovered

COMPLETELY

CONSTANT

THE

ARE

In

SULFITE-SENSIT. FR AC T I 0 N •

ug

thiam ine

COMPLETE.

WITH

ICTHIAMINE

LENE

WAS

FRACTION

INDICATION OF

WAS

us

present.

REASONABLY

SENSITIVE VERY

in

PRESENT

THIAZOLE

COHOL

are

o rig in a lly

ORIGINALLY T HE

24 56 56 60

ug

52

8.6 A ll

SULFITE-INSENS I T I V E FRACTN

Un r e c o v Py r i m . Fr

THE

CON­ TO

FACT,

FRACTION THE

54

r REACTION

MIXTURE

Thus, p lete OF

FRESH

C L AM

reaction

EXTRACT

WAS

USUAL

INCUBATION

DITIONS

BUT

TRACT.

A

A D DE D

TO

TO

STAND

FOR

AND

NO

RUN ON

TO THE

THE

REACTION

M INE,

THE

SULFITE-SENSITIVE

THE

ALSO,

AS

FROM

THE

MAY

BE

D A YS

ADDITION

OF

THE

FRACTI i ON

TOLUENE THEN

WERE

FROM C ON ­

FRESH

C L AM

EX­

V.

TO

AT

MEASURED

SAME

SEEN

FROM T A B L E

THEN

THE

PORTION

PH

INCUBATION

OBTAINED

REACTION

IS

AND

ACIDIFICATION

N0 N - D I ALYZABLE

com­ THE

OBTAINED

UNDER

PYRIMIDINE

SEEN

WERE

VALUES

8

THAT

THE UNDER

VALUES

PH

to

8

PH 3 . 6

TO

PRODUCTS

THE

AT

SHOW

|N

DECREASED

OF

THREE

pH

8 -1 2°C ).

MIXTURE.

REGENERATION

SUBSEQUENT

EFFECT

THE

at

AT

ADJUSTED

THE

RUN

run

HOURS

REACTION

MIXTURE

WAS

was

THE

DIFFERENT

WITHOUT

BLANK

HAD

WAS

”i

EXPERIMENT.

(24

THEN

MANNER.

SIGNIFICANTLY

3.6

THIAMINE, WAS

AL L OWE D

SIMILAR

pH

OF

MIXTURE

TEMPERATURE,

THE

NOT

thiam ine

clam

REACTION

MIXTURE

IN

ANOTHER

DESTRUCTION

THE

ROOM

the

SUGGESTED

A

TO

PRODUCTS.

WITH BE

THUS

THIA­

GREATLY WE

N OT E

FOLLOWING:

EXT RACT

p

O Rig • E x t r a c t N . D . AND D I A L Y ­ SATE

N .D .

A L ON E

ORIG.

E xtract AND D I A L Y ­ SATE N« D . AL ONE

H

S U L F I T E - S E N S 1 T 1 VE Py r i m i d i n e Fractn

3 .6

182

3 .6 3 .6

166 119

7 .2

97

7 .2 7 .2

89 40

Per c e n t Re c o v e r y

Y3$

ug

66$ 48$

40$

ug

N .D .

The

L

values

represent

recovery

from

3 6 fo 1 6 fo 250

ug

t h ia m in e

J

55

r

ORIGINALLY

“i

PRESENT.

N.D. REFERS 0 1 ALYSIS.

TO

THE

NON-DIALYZABfcE

PORTION

AFTER

EXHAUSTIVE

SUMMARY The

above

oata

do

not

allow

any

in te r p r e ta tio n s

NATURE

OF T H E

UNRECOVERED

PYRIMIDINE

NATURE

OF T H E

SE COND

INVOLVED

ASPECT ED THE

OF

SINCE

OVER-ALL AS

YET

BEFORE 1* VALUES

LEAVING T he

THE

The

THE

H

values

SHOWN

3.

OUR

In

OF

THE

of

BEEN ANY

WE

EACH

LARGELY ME T H OD

EXCEPT NOTE

MOIETY

NOR

BY

A

FEW

AT

to

the

THE THIS

UN I N V E S T I G A T ­ OF

MEASURING

DIFFERENCE. OBSERVATIONS:

ALL

INDICATING

DID

ON

REACTION.

LEFT

CASE,

MOIETY

THE

DIRECT

MAY

THIAZOLE IN

IN

FRACTION

thiam ine

IS

THE

GIVEN

THAT

PH

DIS­

OCCUR.

destroyed

APPROXIMATELY

THE

by

a

SAME

given AT

amount

THE

of

DIFFERENT

ABOVE.

STUDY

OF

THE

REACTION,

DIALYSIS

OF

THE

C L AM

PYRIMIDINE

EFFECT

ONLY

WS U L F I T E - S E N S I T I VE

wUNRECOVERED

L

THE

THIAZOLE

PRINCIPLE

C L A M - T H I AMINE

HAVE

PROBLEM,

OF

amount

CLAM

PRIOR

N OT

IS QUANTITATIVE

THE p

DO

HAS

PYRIMIDINE

YIELD

P L A C E M E N T OF 2.

PROBLEM

WE

UNRECOVERED

STEP

FRACTION

as

THE

OF

VARIOUS

PH OF

EXTRACT

THE

APPEAR

PYRIMIDINE

FACTORS

REACTION TO

AFFECT

FRACTION”

AND

ON

THE

AND

A

THE

YIELDS

OF

THE

F R A C T I O N 1* .

J

56 r

P A R T

VIII.

THE E F F E C T OF A C CE LERA TO RS ;

A*

P relim inary

BISULFITE

MINE

TEMPERATURE

AT

TIONS,

ROOM AS

SHOWN

CYSTEINE

BY

AND

AND

n

THE A C T I V A T I O N

C o nsid eratio n s*-

GLUTATHIONE,

THAT

O N E

C ysteine,

TH I 0 G L Y C 0 L L I C

AND

U NDE R

TH I OC H ROME GLUTATHIONE

VERY

PROCESS. -

p h enylhydrazine,

ACID MILD

DESTROY

THIA­

REACTION

32

ASSAY.

AGREN

DESTROY

THIAMINE

CONDI-

DEMONSTRATED USING

THE

\

Mz

l n ic k

F

and

ield

57 ACIDJ ,

THIOGLYCOLLlC PHILLIC

DISPLACEMENT

PRODUCTS

|T

IS

THIAMINE

HAVE

BEEN

POSSIBLE IN

A

HAVIOUR, cule. ly

since

The

or

M E N D OU S

FORTY Section

2-3 L

III,

P O WD E R , MGS

BECAUSE

OF

OF

case

of

IS

THIAZOLE

b is u l f it e

UNDOUBTEDLY

MOIETY,

AND

AND

WOULD

X appears complex"

IN

WITH

2^*38

and

A NUCLEO-

THE

REACTION

to

ma y

be

INVOLVE

THIS

a

a

WITH

A

SPEED

TO

PROTEIN

p ro te in -lik e be

s p lit

mechanism THE

does

X.

BE­ m ole­

enzym atical­

ENDOWMENT

REACTANT

FORM

not OF

TRE­

A BRIEF

CON­

EVIDENTI-

pH 3

this

0.1344 100

SUCH

FINITE

then

IT

ON

REACT

SHOW T Y P I C A L

such

TWO o f

CONTAINS

MANNER.

However, WOULD

X MAY

REACTANT

PR OC E E D

REACTIVITY

MAKE

THIAMINE

THAT

CAN

A MclLVAINE

Part

the

NON-CATALYZED

Reactant

S H OU L D

ML

THE

COMPLEX"

NUCLEOPHILIC

SIDERATION

n

MECHANISM

THEREFORE

"pyrim idine

LIKELY

I

ISOLATED.

spontaneously.

APPEAR

CLAM

OF

REACTION

"PYRIM IDINE

.

THE

SIMILAR

STOICHIOMETRIC THE

method

ML

BUFFER

thesis) GM OF

OF

E X T R A C T ( D E S C R I BED to

1 gm o f

WILL

DESTROY

equ ivalen t

PROTEIN

REACTION

AND

IN

MIXTURE.

EVEN J

57 r ASSUMING WILL

-i THAT

GIVE

X.

WITH

MOL E

ATTACKED GIVE

THIS

A MOLECULAR

Reactant ONE

ALL

Now

BY

WEIGHT

0 .7 5

if

OF

PROTEIN

TO

THE

5

OF

of

(0 .2 5

GIVE

A CONCENTRATION

2 0 ,0 0 0

OF

mole

THIAMINE

WA T ER

REPRESENTS

X

Reactant MOLE

-5

3 0 ,0 0 0

TO

OF

M FOR

X,

reacts

com pletely

BEING

ALCOHOL),

X

REACTANT

THIS

FOR

THIAMINE

PYRIMIDYL

X TO

REACTANT

THIS IN

WILL

THE

SOLUTl O N . * ON THE

THIS

BASIS,

REACTION

OF

IT

X occurs.

REACTION

RATE

MAY

OF BE

THAT CLOSE

THE

THE

OF

A

WA T E R

FRAGMENT

IN

THE

ACTIVATION

OF

ELECTRON

IS

effect

SEEMS

THE

BRIDGE

THE

ICTHIAMINE

OVER

TH IS WILL

favoring

the

attack

OF

SINCE THE

IS

X

BE

MADE of

THE

OF

SENSE INTO

THIAMINE THIAMINE.

APART

and

BRIDGE

CARBON

ELECTRON-POOR THE IN

CLEAR

Re a c t a n t FORMATION

PYRIMIDYL

THE

AND

be

MADE

COURSE,

B R OU GH T

METHYLENE IS

OF

IN

CARBON

DISTINCTION

AND

INESCAPABLE

X

FACILITATING

IMPORTANT

THAT

OF

would

CARBON

DISPLACEMENT,

REACTANT).

OVER

fast

REACTANT

FROM

OF

re la tiv e ly

BRIDGE

B E T WE E N

IN

the

REACTANT

METHYLENE

THAT

ACTIVATION

ACTIVATION,

process

^PREDOMINATES

L

THE

AN

S T E R I C ...................... . S T E R I C

FORMATION

( I.E .,

POSTULATE

YIELD

a ctivatio n

ACTIVATION

OF

HIGHER

the

NUCLEOPHILIC

an

explain

ALCOHOL.

THE

TO

THIAMINE,

of

THIAMINE

Such

would

THE

AND/OR

TO

COMPLEX

phase

THROUGH

AL SO

TWO-CARBON

PROXIMITY

D IS T IN C T

This

PYRIMIDYL

ENERGETIC

THROUGH

Th is

AND

LOGICAL

X WITH

REACTANT

Reactant

THAT

APPEARS

ALCOHOL

APPROACH THE IN X

MODES

OF

A MOMENT. over

OF

OF

that

ICTHIAMINE

E YEN

THOUGH

T h e SAME R E S U L T MAY BE A R R I V E D AT O B V I O U S L Y BY A C A L ­ C U L A T I O N BASED ON T H E S I D E C H A I N OF I C T H I A M I N E , (C2H603NS

j

58 r WA T E R Th

is

THAT OF

“i IS

IN

INFINITELY is

in

fact

the

THE

WA T E R

MUS T

BE

MINED

ATTACK

BY

WELL

RATE

OF

AND TO

THE

DOWN

THE

PYRIMIDYL

THE

"PYRIM IDINE

IN

THIS

IS

REQUIRED

AS

10

DESTRUCTION EXPERIMENTAL

The

OF

proof

CASE, THE

OF

of

such

be

up

Su c h

CLEAVE

and

OF

THIAMINE

APPROACH

TO

THE

RATHER

ARE

DETER­

BEEN

CON­

SEPARATE THE

OF

BOTH

CONDITIONS

HAVE

LATTER

SECONDARY

THE

FROM

PYRIMIDYL

THEN

BREAK­

COURSE

THAT

FORMED

FROM

REACTIONS.

EVEN

"ACTIVATION"

OF

CYSTEINE

PHENYLHYDRA-

TEMPERATURE

THE

RATE

IS

THE

IS

IS

POSSIBLE

ROOM

an is

I I ) .

VERY

C L AM

OF SUCH

activatio n best

in

What

should

UNDER SUCH

THE an

le f t

Part

THIAMINE

a c t iv a t io n

DEMONSTATFI ON

L

taken

ACTIVATION an

IS

ICTHIAMINE

BY

THE

RESPECTIVELY,

|T

THROUGH

OF

WE

WATER

postulating

AND IN

SLOW

REACTANT

X

CONCENTRATIONS

C OMPARED

PRINCIPLE

UNDER

TO

T HE

THE

SAME

CONDITIONS.

and

THE

X.

X.

REACTANT

for

BUT

AT

THIAMINE

X

I

OF

COMPLEX*,

HOWEVER,

BUT

U 9

b a s is

REACTANT

GIVE

TO

INDEPENDENT

OBSERVATIONS.

Reactant

(Sections

AND

THIAMINE -3

TO

COMPLEX"

best

ATTACK

ICTHI AMINE•

ALCOHOL

BY

OF

THE

X

COMPARED

CONCENTRATION,

ATTACK THAT

fqvoring w ill

OF

lTP Y R I M I D I N E

GIVE

LATTER

DESTROY

LOW

AS

REACTANT

ING

AS

OF

HERETOFORE

ATTACK

ALCOHOL

AS

THE

SIDERING

ZINE

EXCESS

c o n s id e r a tio n

REACTION

THE

LARGE

TWO

and/or

for of

concerns ITSELF be

a

IN

p r e s e n t

,

A DEMONSTRATION

IS WAS

steric

for

d issertatio n

SENSE water

THEREFORE

here

DEFINED alone

CONDITIONS

MADE

e ffe c t

con sideration

im m ediately

THE

EXPERIMENTAL

INFLUENCE

la te r

th is us

a

is ABOVE.

cannot

USED.

DESIRABLE.

THROUGH

THE

A AN

59

r INVESTIGATION

T he

nature

POSSIBLE

OF

THE

EFFECT

of

the

accelerators

MODES

BY W H I C H

OF

THEY

ACCELERATORS*

MAY

im m e d ia te ly

ACCELERATE

suggest

THE

three

REACTION

RATE ,

I.E ., a

)

A R E D U C T I O N P R O C E S S WH E RE B Y " A C T I V E C ' E N T E R S " CL A M S Y S T E M ARE R E N D E R E D MORE R E A C T I V E * REMOVAL

OF

THE

C)

A PARTICIPATION IN THE D I S P L A C E M E N T R EACT ION IN C O M P E T I T I O N W I T H WA T E R AND R E A C T A N T X TO FORM T H E C O R R E S P O N D I N G P Y R I M I D I N E D E R I V A T I V E , W H I C H WE MAY DESIGNATE A S . * . .

A CONSIDERATION SINCE CLAM

WE

HAVE

NO

SYSTEM.

POISONS*

PROPOSED

THE

CLAM

SYSTEM

T

FACT

AI^SO

REDUCING

SOLUTION REACTANTS

BUT

THE

STUDY SOON

WAS

THAT

OR

AND

OF

FIRST

K N OWL ED GE

THE

WAS

AS

THE

B)

(P y r -C H 2 — Ro t

he

IN

OF

THE

THE

COMPLEX THE

NATURE

AND

OF

WHENIT

POTENTIAL SUCH

HAVE

THE

ON

AT

THlE CENTERS" THAT

CAN

THE

p

ACT

H OF

REACTANT

THIS

EASY

REACTIONS.

FOUND

OTHER

A STUDY

IN

EVIDENT

SEVERAL

DEPENDING OF

NOT

"ACTIVE

BECAME

WE

IS

CENTERS" THE

INVOLVED

REAGENTS

AGAINST

PO SSIBILITIES

"ACTIVE

ACCELERATORS

REDOX

MITIGATED

TWO

ABANDONED

OXIDIZING

THE

OF

)

S T A GE

THE

OR OF

THE

INVESTIGAT!ON•

B.

A

Po

ss ib le

REACTION

The was

BY

Mo d e

a t t r a c t iv e

(SULFYDRYL p o s it iv e

AND

.

of

the

T he

AMINE-TYPE

methylene

Ac

celeratio n

THIOGLYCOLLIC

in v e s t ig a t io n

more

of

group

of

the

Clam - T

h ia m in e

ACtP. -

th ir d

p o s s ib il it y

a c t iv it y

of

CARBONYL

REAGENTS)

increases

as

most

the

of

p

H

shown

above

these

reagents

T OWARDS of

the

THE SOLUTION^

60 r

n

GOE S

T OWARDS

THE

acceleration acid

pH

ture

observed

(Table

(above)

a

ALKALINE

I I ,

lik e ly

CONTAINING

GIVEN

REAGENT,

WHILE

IN

is

p a g e

one. THE

IT

THEN

THE

TISSUE

ACTIVATED

The

d e f in it e

REAGENT MIDINE

WOULD

if

ISOLATION A BETTER

BE

THE

FROM

YEAST

FOR

TION

VALUE

WHE N

THIS

ONLY

KNOWN

IS

THIAMINE

BE

THE

FOR

IF

DERIVATIVE

DERIVATIVE

IS

than

HAVE

SUCH

OF

AND

WAS

IT

ENOUGH BEING

N OT

TO

PLANNED,

COULD

BE

F ORMED

UTILIZED

THIAMINE.

PYRI­

THE THE

OFFERED IN

SO,

THE

ANY BY

THE

REGENERA­

P Y R - C H 2 - S O 3 H WAS

AND BY

IM­

INTERPRETATION.*

USEFUL

REACTION

CL AM

WAS

BY

ME T H OD

AFFECT

ALLOW

IN

DURING

INCOMPLETELY

MIGHT

THE

g iven

THIS

AVOID

REGENERATION

ONLY

a

ATTACK

(PYR-Ch^-RGT)

OR

THE

TO

OF

IN

EXPECTED

MIXTURE.

NON-SPECIFIC

THE

FORMED,

REACTION

by

THE

m ix­

A

A FACTOR

p a r t ic ip a t io n

IMPOSSIBLE

at

PARTICIPATED

OF

OF

incubation

THIAMINE

INFLUENCE

YEAST

WHICH

ACCELERATED

MUST

RATE

p o s s ib ility

an

NO

ISOLATION

SYNTHESIS,

WAS

BLANK,

a

THE

UTILIZED ,

EXPERIMENT

pH

PYR-CH2 -RGT

INCUBATION

ALMOST

SIGNIFICANTLY

BISULFITE

PYRIMIDINE

NOT

THE

SINCE

from

THE

METHYLENE-BRIDGE-CARBON

CLEARLY

WOULD

THAT

THE

such

APPROACH,

CASE

THE

th ird

EXTRACT,

HEATED

U NDE R

PROCEDURE.

GIVEN

SHOWN

of

HOWEVER,

ACCELERATOR

*

then,

proof

DERIVATIVE

PRACTICAL,

IS ,

Now,

CLAM

THAT

a lk a lin e

the

ACCELERATOR

REACTION

at

FACT

makes

BE

DISPLACEMENT WHICH

greater

ACTIVE

CAN

THE

SOA)

A CORRESPONDING

OCCURRED,

SIDE.

TO THE

THE

THE

U S .............. T H A T CORRESPONDING

YEAST

FOR

I C T H I A M I N E AND T H E P Y R I M I D Y L A L C O H O L ARE C O M P L E T E L Y C O N V E R T E D TO T H I A M I N E BY Y E A S T I N THfE P R E S E N C E OF T H E THIAZOLE MOIETY.

61 r THIAMINE

SYNTHESIS#

NON-CATALYZED EVEN

AT

VERY

INCUBATED

WITH

VIt.

Table

REACTION LOW

ACCELERATING

CLAM

RUN

TION

EFFICIENCY

THAT

THE

FICANT TION shown

Cy

SAMPLES

s t e in e

T he

OUT OF

THI

as

T

0

IF

THE

IN

IN

WITH

THE AND

are

in

and

case

DID

THE

,

WERE

c o nfined

NOT

th at

THE

VI!

CAUSE

SHOW ANY WITH

WER

SIG N I­

PYRIMIDINE (THE ?

were

REGENERA­

FRAC­

RESULTS ob-

results

ESSENTIALLY ,

the Blanks

VALUES.

extr act

OF

ON

occur.

THERE

th e r e fo r e

to

SHOWN

show

REAGENT.

aqueous

ADDITION

TABLE

REGENERABLE

PORTION

HENNESSY**(57)

ON

THEREFORE

AS

not

FOR T H E

APPRECIABLE

T HE

AFFECTED

DATA

in v e s t ig a t io n s

was

did

HOWEVER,

WITHOUT

a c id

to

REGENERATION

TOTAL

the

WAS

T#2

AND

USED,

WAS

WITHOUT

REAGENTS

ACID,

for

the

each

THE

THE

t h io g l y c o l l ic

tools

AND

thiam ine

YEAST.

BE

THIAMINE

REAGENTS

CARBONYL

in v e s t ig a t io n

BONVICINO

of

NOT

BLANKS

PH 3 . 6

in

THE

NON-DIALYZABLE and

HEATED

BOTH

GLYC0 L L I C

Vtt

able

COUL D

WITH

run

DIFFERENCES

AND

THE

selves

FIND

AMINE-TYPE

in

USING

were

DIFFERENCES

IN

AT

destru ctio n

ALSO

CYSTEINE

THE

EXTRACT

REAGENTS

non-catalyzed

SIGNIFICANT

IN

IT

CONCENTRATIONS#

Blanks

TO

HOWEVER,

THE

offered

SAME).

them

­

*

t h io g l y c o l l ic

SYNTHESIZED

a c id

.

(PYR -C H2-SCH2 -CO O H)* * r

AND

THIS

COMPOUND

WAS

FOUND

TO

BE

LARGELY

N ==C-NHo

H3 C 6 UNAVAILABLE

TO

THE

YEAST

FOR

T HE

SYNTHESIS

II

C-CH

-SCH

(I

N -— CH OF

THI AMINE(TABLE

VIVES

^

L

SULFITING.

a n a l y t ic a l

V III) Such

d a t e

:

AND a

APPARENTLY

regeneration

Calculated

for

SUR­ BEHAVIOUR

t s MORE OR

COOH

r LESS

TO

CHAIN,

EXPECTED

BE

THE

I . E . ,

NE-CARB0NE

MQRE

NUCLEO-PHIL L I C

The

GREAT

THE

FROM

NEGATIVE

NATURE

CARBOXYL

ELECTRON-RICH ATTACK

BY

THIAZOLE

DECREASE

IN

THE

PYRIMIDINE

FRACTION

ED

INCUBATION

MIXTURE

A

THE

CLEAR

BLANKS ALONE NOR

INDICATION SHOW

HAD

ON

NO

NO

THE

TO

REACTION IN

THE

BE

THAT

CLAM

THI

THE

0

AND

DAVIS'^

EFFECT

PRINCIPLE

"THE

ACCELERATION

ON

M-NITROANILINE

THE

GLYC0 L L I C OF

FOR

TOTAL

ACID

TABLE

IS

V III)

RE ADD

IS

HEATED

PYRIMIDYL

T HE

ACID

AN

VALUE

THilOGLYCOLL I C ACID

OR T H E

THE

FOR

THIAMINE.

AND

THIAMINE.

SIDE

METHYLE

REACTIVE

REACTION,

ON L Y

OF

SUGGESTED

YEAST

LOGICAL

ACTIVATING

ALCOHOL

UNDER

THE

CONCLUSION

PARTICIPATED

THIAMINE. IS

DU E

WITH

C OU R S E

OF

THE

E N Z Y M E —S U B S T R A T E

THE

TO

THE

IN

REACTION

SIMILAR

THE

A

IN

FACTOR

THE

PRESENT

WHILE

THE

TO

THESE OF

LATTER

PYRIMIDINE

OF

THE

THE

AUTHORS,

THE

IS

FOR

OF

AN I MO THE

BEING

GROUP

PYRI­ SPLIT

M -N IT R 0 ANILINE

FOLLOWING THEY

ACTION

CARBON

REACTION;

STAGE

MECHANISM

THE

COMBINATION

5-METHYLENE

COMPLEX"*

CORRESPONDI NG

ON

ACCORDING

ENZYMATIC AT

A

M - N I T R O AN I L I N E

THIAMINE

ISOLATED

VII

USED.

OF

THE

(TABLE

OF

THE

THIOGLYC0 LLIC

OF

MOIETY

THE

LESS

EFFICIENCY

MIDINE

ENTERS

WHEN

ENTERED

AGE N C Y

RENDERS

GIVE

TO

5-METHYLENE

EXTRACT.

CARP

THE

THUS

IC T H I AMIN E

CONDITIONS

ACCELERATING

OF

ON

REGENERATION

THROUGH

SEALOCK

IT

DESTRUCTION

EFFECT

EXPERIMENTAL APPEARS

THAT

GROUP

REGENERATION

GENERABLE TO

AND

THE

OF

THE

REPORTED

FORMATION THAT

COMPOUND,

THEY

OF

63

r IN

74.5J&

YIELD

F ROM

AN

N ~

C -NH2

t

in c u b a tio n

m ixture

THE

C ARP

AND

M-NITROAN I L IN E *

The

EXTRACT*

reported

PRINCIPLE FROM

DRIED

HAVE

BATCHES

BATCHES

WQS

- c h 2 - nh

b in

the

a c t iv it y

SOURCES

MAY

BE

T h IS

EMPLOYED

OR

DUE

IS

FOR

THE

WIDE

VARIANCES

OF

CLAMS

IN

THE

E ffec t

of

Th io g ly c o llic

COURSE

FROM

TO

THE

OBSERVED

T he

C.

varianc es

ACCELERATORS.

POWDER

c

THIAMINE

DIFFERENT

DIFFERENT

SIMILAR

WE

FROM

I

h3c - c

c o n t a in in g

of

a

THE

THE

g ive n

SAME

WHY

AN

OF

THE OUR

A CTIVITY

BUT

OF

ACETONE-

INVESTIGATION,

IN

f is h

SOURCE

OC C U R R E N C E

REASON

(1 0 ^

BECAUSE OF

DIFFERENT

WO R K .

Acid

on

the

Lim it

of

D estructio n .

I

t

was

APPEARS

stated

TO

FORMATION BUT

not

tack

THE BY

WA T E R

ACTIVATE THAT

ATTACK

( i . e . ,

t

X

has

OF

the

CEASES

THE

IN

EXHAUSTION

THE

WA T E R

IS

been

the

DISTINCT in d ic a t e d

X IS

a

HAS AND by

OF

and

X.

THE

AFFECTED

THE

pyrim idyl

IN D I­ ATTACK

alcohol

th a t

the

a t­

EXHAUSTED.

separate

BEEN

factor

SHOWN

SEPARATE the

destruction

CONDITIONS,

interm ediate)

MOLECULE

IS

INDEPENDENT

and

the

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APPARENTLY

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that

in

OF

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REACTANT

in d ic a t io n

FACTOR

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same

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not

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THE

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ON

VARIATIONS

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that

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tures

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35 64

uG

3.6 7.2

160 124

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35 65

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3.6 7.2

162 123

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38 60

ugb

H Y D R A Z 1 NE R Y D R A Z 1NE

3.6 7.2

152

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35 87

ug

HISTAMINE HISTAMINE

3.6 7.2

161 123

UG

35 90

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C Y S T E 1NE C Y S T E 1NE

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50

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52 65

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p ro c e d u re used THE N E X T PAGE .

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D U R I N G T H E T H I O C H R O M E P R O D U C T I O N AND I N T H I S C A S E , A D E E P ORANGE RED H U E . T H E E F F E C T OF H Y D R A Z I N E ON P R O D U C T I O N WAS NOT I N V E S T I G A T E D . Ge n e r a l P r o c e d u r e U s e d f o r E f f e c t o f R e a g e n t s on t h e R e g I ncorporated into the Clam- T h

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regenera­ : - t ncubaT I O N M I X T U R E S S I M I L A R I N a l l R E S P E C T S TO H E A T E D B L A N K S BUT W I T H NO T H I A M I N E ADDED WERE P R E P A R E D AND I N C U B A T E D UNDER T H E SAME E X P E R I M E N T A L C O N D I T I O N S . T H E N , AN A P P R O P R I A T E A L I ­ Q U O T WAS ADDED TO A Y E A S T S U S P E N S I O N C O N T A I N I N G T H E P Y R I M I D Y L A L C O H O L AND T H E T H I A Z O L E M O I E T Y , AND T HE T H I A M I N E S Y N T H E ­ S I Z E D A N A L Y Z E D FOR I N T H E U S U A L MANNER G I V E N U N DE R E X P E R I ­ MENTAL M e t h o d s . The r e s u l t s a re g iv e n below in a s u p p l e ­ m e n t tfo T a b l e V I I . t io n

Bl a n k s

for

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the

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of

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ct

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None Hy d r o x y l a m i n e S E M I C A R B A Z I DE Hy d r a z i n e HISTAMINE Cy s t e I ne THIOGLYCOLLIC *

of

a

were

of

as

Table on

the

Th

on

the

follow s

VI I

the Re g e n e r a Yeast

ia m in e

tion

Re g e n e r a t e d

175

ug

173 161 1 54* 172 171 175

ACID

C OL OR

WITH

HYDRAZINE

PRESENT(SEE

NOTE

C,

63c

V II I

Table The

E ffe c t

Thiam ine

of

Th i o g l y c o l l i c

R eaction

as

e ra tio n Sa m p l e

p

H

Shown

Acid by

! Total

He a t e d C l a m E x t . 2 5 0 UG T H I A M I N E TH IO G L Y C O LLIC ACID

I 3.6

250

j 7.2

245

C l a m E x t ( ^ 0 . 3 gm D E S S . P O WD E R ) 2 5 0 UG T H I A M I N E

| 3.6

202

! 7.2

141

3.6

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7 .2

130

3 .6

48

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a

)

T he t u r e

48

Ext.

c o n c e n t r a t io n was

C lam -

Regen­

Re g .

SULFITEINSENSIT PYRIM.

T o ta l Re g . Py r im id in e (C alc u la ted )

250

ug

0

ug

250 250

ug

UG

4 13

ug

UG

|

C l a m E x t ( «~>0. 3 g m D E S S . POWD E R ) 2 5 0 UG T H I A M I N E TH I O G L Y C O L L I C A C I D

Clam

the

S tu d ies.

|PYRIM lDINE ! (F ound)

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on

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1

x

10

•*3

of

&t .

th e

I n

52

250

250

ug

30

ug

250 237

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53 40

ug

237 237

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cases

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ug

t h io g l y c o l l ic a l l

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the

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r e a c t io n

T I M E WAS 2 4 HOURS AND T H E T E M P E R A T U R E WAS 2 8 - 3 0 ° . ALL T H E T H I A M I N E P R E S E N T WAS D E S T R O Y E D I N T H E 2 4 H R . P E R I O D . t N A L L C A S E S , T H E T H I A Z O L E M O I E T Y C OU L D BE A C C O U N T E D FOR A L M O S T Q U A N T I T A T I V E L Y . T H E B L A N K FOR N O N - S P E C I F I C D E S T R U C T I O N OF T H I A M I N E BY T H I 0 G L Y C 0 L L I C A C I D AS W E L L AS T H E B L A N K FOR P O S S I B L E E F F E C T OF T H I O G L Y C 0 L L I C A C I D ON T H E R E G E N E R A T I O N E F F I C I E N C Y OF T H E Y E A S T SHOWED T H A T T H E S E E F F E C T S ARE A B S E N T . WHEN T H E N 0 N - D I A L Y Z A B L E P O R T I O N ( A F T E R E X H A U S T I V E D I A L Y S I S ) WAS USED I N S T E A D OF T H E O R I G I N A L CL AM E X T R A C T , T H E R E S U L T S WERE E S S E N T I A L L Y T H E SAME AS T H E D A T A SHOWN A B O V E . b

)

c)

L

Ca lcu lated

For

form ula

as

of

t h i a m i n e

.

P y r-C H 2-TG A ,

see

p a g e ^ /•

J

64 r THE

so

i ACTIVATION long

MAY

as

SHOW

TING

FACTOR

AND

ful

X

IS and

THE

ACTIVATING

CAN

ACID

STAND WAS

FOR

N OT

ONLY

IN

Bl a n k s

n

30

X

10

DAYS

UG

TO

THE

OF

-5

AT

3Y

BE

a

related

the

0

X 10 THE

28-30°.

there

H had

no

was

extract

of

powder

vo id

t h ia m in e

-

was

REACTANT

WOULD

IN

LARGE

THEN

LIM IT

OF

0.1

-

in h ib ito ry

INCREASE GM OF

TO

OF

CLAM

ACID

THI 0 GLYC0 L L IC

AS

ACID.

s p e c if ic

destructio n

WAS

SHOWN

ALSO

e ffe c t

resid ue

is

a c t iv it y

C0 N C E N -

AL LOWED

TH I O G L Y C O L L I C

A DDE D

Th

TO

THEDESTRUCTI 0 N

PER

.

EXCESS

WAS

DESTROYED

u sed

THE

M I XTURE(TH I OGLYCOLLIC

AN

the

THAT

X.

MIXTURE

|T

TH I 0 -

BECAUSE

MEAN

THIAMINE

ACID.

success

INCREASE

THE

THE

which

ACTIVA­

THAT

M WHILE

non

destro ying

E .,

FROM

no

EXPERIMENT,

I.

WAS

WITHOUT

not

INTEREST

THERE

CONTAINING

THE

FACT

IT

THE

AFFECTED.

evidence

were

OF

ADDED

ON L Y

"POISON*

THEN

THERFORE

M )•

IS

EXIST

FROM

THE

DESTRUCTION,

INCUBATION 2

TO

alcohol

REACTION

DISTINCT

WAS

TO

SEPARATE

ATTEMPTS

DESTROYED,

THIOGLYCOLLIC

“ S-CH2 C0

NOT

ABANDONED.

OF

THIAMINE

SAMPLE

that

2

of

WAS

T H E SAMBLE

showed

THIAMINE Pyr-CH

7*4

F OU R

TO

COMPARED

L

ACID

SIGNIFICANTLY

20

POWDER

I

MAY

CONCENTRATION

THE

LIM IT

THIAMINE

FACTOR

WAS

IS

SOON

ENTER

E X T R A C T -T H I AMINE

TRATION

OR

only

WAS

THE

THIOGLYCOLLIC A C L AM

IS

SEEMS

Experim ental

p y r im id y l

APPROACH

OF

present.

obtain

AFFECT

A MOUN T

is

EARLY

IF THE

X

MOLECULE

DESIRABLE.

ACID

CAN

THIAMINE

REACTANT X

GLYCOLLIC IT

THE

Reactant

THAT

Re a c t a n t

OF

.

on

from

the

T HE

r esid u e

Ho w

ever

is

,

of

THAT reaction.

A QU E OU S almost

if

de

­

for

-1

­

65

r exam ple,

“1

ACTIVATING RESIDUE WHEN THE

MIGHT

AS

WE L L

FAR

AS

ACID

SHOW

IS

RESULT

ADDED WAS

SINCE

NEGATIVE

THE

ACTIVATING

C A R B ON OF

in

activatio n THIAMINE

BY

THIAMINE as

soon

some

way

as

by

the

thiam ine

is

lo st

BY WA T E R

ATTACK

(AND

OF

OTHER

EXTRACTED,

THE WITH

separate

THEN

LATTER

THE

ACTIVITY MAY

ENTER

TH I 0 G LYC 0 L L I C

INFLUENCE

SEEMS

and

THE

a

REAGENTS*

X,

of

w hile

THIAMINE-DESTROYING

THEREFORE,

the

OF

INCOMPLETELY

OTHER

Reactant

PENDENT

ONLY

extracted

THE

altered

ATTACK

com pletely

WITH

(or

OF

IS

THE

METHYLENE w ith

is

CONCEIVABLY

REACTIION.

SO

up

FACTOR

THIOGLYCOLLIC

ACID

THE

X

Reactant

as

TO

BE

and

WA T E R

of

is

X

a

2

CEASES.

I

n

TIED

exhausted

-carbon

furth er

ACCELERATORS)

LABI L I Z E S

INTIMATELY

Reactant loss

WHICH

fragm ent)

destruction ADDITION,

APPEARS

TO

BE

THE

INDE­

CONCENTRATION.

NOT E A.)

I

THE

n

ASSUMED

THAT

ARE

FORMED

and

Re a c t a n t

THIAMINE, BEING

DISCUSSION THE

BY

INDPENDENT X

on

the

POSSIBLE BY

APPEARS

TIED

TO

BE

EMPLOYED

EXPERIMENTAL L

f

PRECEDING

ALCOHOL,

ATTACKS

l a b il iz e d

AN

T H ROUGH

AN

ACTIVATING UP W I T H

HERE

TO

OBSERVATIONS

*

PYR-CHg-TGA

INFLUENCE

X.

FACILITATE ITS

-

USE

OF

IS

HAS

-

BEEN

I C T H I AM I N E

carbon

ACID of

REACTIONS

THE

WHOSE THIS

THE

AND

b r id g e

DISPLACEMENT

ACTIVATION

IT

THIOGLYCOLLIC

m ethylene

REACTANT

AND

SECTION,

OF W A T E R ,

R E P E C T I V E L Y ....................... T H E

MADE

BEEN

THE

PYRIMIDYL

BRIDGE-CARBON

HAS

IN

METHYLENE-

ACTIVITY

ASSUMPTION

PRESENTATION

OF

N OT

IMPLY

MEANT

TO

THE

66 r

n

THAT

THE

ABOVE

EVIDENCE UNTIL IS

ON

ITS

MECHANISM

HAND.

BY

NEW

DISCUSSIONS

MORE

DETAILED

W ILL

BE

IN

ONLY

A

TOOL

FIN DING S, THE

IT

THE

SUGGESTED

EXPLAIN

F OR

BE

SENSE

POSSIBLE

SUMMATION

ALL

OF

BY

THE

THEFACTS

CORRELATING

SHALL

QUALIFIED

OF

A F T E R T HE

ONE

HOWEVER,

AS

DISCUSSION

PRESENTEO

THE

DOES,

SERVICEABILITY

DISQ UALIFIED

ENSUING

IT

IS

USED

THE IN

A ND

EVIDENCE THE

B E S C R I BEDA B O V E .

WO R K I N G THE

A

MECHANISMS

RESULTS

(NEXT

SECT I O N ) •

IT

B. TORS

MA Y

POSSIBLE

IS

EXERCISE

Table

on

t y p e

CARBONYL

YEAST FECT

L

VIt

REGENERATION OF

THE

do

EFFECTS IN

n o t

REAGENTS

Hy d r a z i n e

N E E D L E S S TO

THEIR

PARTICIPATION

data

ACTION.

PERHAPS

and

,

DO

in

THROUGH

OUT MEANS

DISPLACEMENT f a c t

ENTER

,

r e v e a l

INTO

s e m ic a r b a z id e

KffiTHOD.

AMINE-TYPE

THE

POINT

FURTHER

CARBONYL

THE also

THAT

OTHER

ACCELERA­

THAN

REACTIONS. w hether

th e

DISPLACEMENT in t e r f e r e

INVESTIGATIONS

REAGENTS

THE

WERE

NOT

THE a m in e

-

RE­

w it h

ON

THE

THE

the

EF­

MADE.

Jf

67 r

P A R T

IX .

IN

ONE

SUMMATION OF THE R E S U L TS * -

T he

evid en c e

T HE

DESTRUCTION

VOLVES

A

X,

(

Reactant

X

ITS

AS

TWO

appears

to

PRIMARY GIVE

PROBABLY ASS

a

be

A

WI TH*

"PYRIM IDINE STEP

TO

THE

GIVE

ENZYMATIC.

WE

reaction

PYRIMIDYL

| C T H I AMIN E • PICTURE

AND

ALCOHOL

m olecule,

METHYLENE MUS T

IN­

PRODUCTS*

END

complex

COMPLEX"

MAY

all

BY WAT ER

DISTINCT

THE

-

PRINCIPLE

MOIETY

re la tiv e ly

COMBINATION

over

CL AM

THIA20LE

RESPECTIVELY,

A S E C ON D

REACTION

THE

the

THE

GIVE

AT

IS

OF

BY

TO

TO)

STEP

THIAMINE

that

^,

THIAMINE LEAST

OF

in d ic a t e s

p[c2H603NSj

ICTHIAMINE,

HENCE

far

DISPLACEMENT

Re a c t a n t AND

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CARBON

BE

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THIS

S E C ON D

THE

OF BY

OVER-ALL

-

Pyr - CH2- T h I A Z O L I UM (T H I A M I N E )

Re a c t a n t

Py r i m i d y Al c o h o l

Py r i m i d Complex

l

4

X

in e

(PYR-CH2 - [ c 2 H6 0 3N sj

TH I A Z O L E MO I E T Y

P)

*

THIAZOLE MO Mo I E T Y

SE C ON D STEP

I C T H I AM I N E

4

P

(P y r-C R 2 - { C 2 H6 03NS ) T

he

PYRIMIDYL

IS

MOST

LIKELY

FORMED

BY

AN

INDEPENDENT

68 r ATTACK

1 OF

WA T E R ,

LAB I L I Z A T I ON AN

OF

ACTIVATING

INVOLVED ACTION LITY

THE

(OR

THAT

THE THE

PYRIMIDYL

IN

SOON

PYRIMIDLY

WH I C H

AS

PH

ALCOHOL

TO

BE

DEPENDENT

OF

OF

THE

UNDER

X

IS

REACTION SAME

IS

APPARENTLY

THE

FROM

THE

RENDERED

FORMATION

MIXTURE,

THE

CONDITIONS

THE

THIAMINE

STOPS*

IS

ON

IN

EXHAUSTED,

BORMED

I C T H I AM I N E

THE

IS

THIAMINE)

DEGRADATION

WHILE

X

REACTANT

REACTANT

ALCOHOL

FURTHER

THAT

THE

SE E MS

M E f f B Y L E N E - B R I D G E - C ARSON

AS

T H RO UG H

BY

ATTACK

DESTRUCTION

OBSERVATION

AFFECTED

ITS

INFLUENCE

BECAUSE

COMPLEX" THE

AND

BY

THE

RE­

POSSIBI­ "PYRIMIDINE

UNLIKELY

IS

GREATLY

YIELD

STAYS

BY

OF

THE

REASONABLY

CONSTANT.

-r OF

e

T he

pr o per tis

THE

C L A M - T H I AM I N E

ACTION. THAT

Ye

t

,

THE

TO

THE

LABILIZES MORE

X

THE

OESTROYED

BY

D EFINITIO N, IN

IT

ITS

OF

THE

Reactant

or

to

THE CAN

INDICATIVE

THE

REACTION

OF

AN

ENZYME

SYSTEM

REQUIRES

PROPERTIES

WITH

CAUTION.

SUCH

BEHAVIOUR

MAY

THE

BREAKDOWN

OF

o

TO

R MAY

If

ACT BE

IN DUE

PROCESS HARDLY IN

BE

SIMPLY

X

Reactant

NUCLEOPHILIC

OBSERVED

IS

IN

separate

is

REAGENTS,

factor

the

THE

AND

T HE

AS

ACTION).

present

in

BE "THE

NATURE

MAKING FACT

which IT

THAT SINCE

A BIOCATALYST WHETHER

IS

REACTION

e n tity

T H O U G H T F P R O V OK I N G ,

CLASSIFIED

VITRO

TO

THIAMINE

METHYLENE-BRIDGE-CARBON a

c h a r a c t e r is t ic s

OBSERVED

thiam ine.

IN

ACTIVATION

l

OF

the

THE

ANd/

T OWARDS

IS

X

MADE

ARE

and

METHYLEN-BRIDGE-CARBON

REACTIVE

LEAST

FROM

POSTULATED

and

IT

(AT

3E

system

REACTION

COMPLEX",

R eactant

clam

COMPLEXITY

ENZYME

PYRIMIDINE of

the

INTERPRETATION

CHARACTERISTICS DUE

of

DU E the

THE

TO clam -1

69

r

“i

SYSTEM

REMAINS

SATISFACTORY THE

Ap a r t

from

THIAMUNE ,

IN

SOME

WAY of

THIAZOLE

IN

VERY

A

OR

FOR

OR

OF

STERIC

in

SUCH FOR

ALTERED

THE

ELUCIDATION

C ARBON

SUGGESTS

OF

of

X)

D EMANDED

MU S T

STOPS

OF

m ethylene

WO R K I N G

OF

EXPLAIN

ANY

WHY

THIAMINE

INFLUENCE

MAY

DE­

BE

FACT

MUST

THE

BE

FAST

WHEN

be

BUT

account

IT

ELSE

X

EXISTS

SUCH

MAY

AN

AL SO

L A B I L I Z A T I 0 N OF REACTANT

IS

THE

BE THE

EXHAUST­ THE

TWO

ACTIVA­

c a re fu lly

REACTION the

X

B E T WE E N

AND

considered

for

the

REACTION

REACTANT

CONNECTION

HYPOTHETICAL

OF

RE­

of

OR

MIXTURE.

BRIDGE-CARBON

should

ACCOUNT

THE

PRESENT

THAT

THE

carbon

MUST

FAVORS

RATE

ENERGETIC THAT

bridge

displacem ent

REACTION

INTIMATE

also

WHICH

CONSIDERING THE

-

MECHAN I S H M

nuclcophilic

IN

AN

and

must

the

DISAPPEARS

A WORKI NG

mechanism

APPARENTLY

RELATIVELY

THE

Reactant

of

IS

I S ................ I T

INFLUENCE

EFFECT.

TION

in

the

MECHANISM.

fo llo w in g

:

a

)

an i n t e r m e d i a t e R eactant X w ith

b

.

Fo r m a t io n of t h e p y r i m i d y l a lc o h o l in such a manner T H A T I T S F O R M A T I O N C E A S E S WHEN R E A C T A N T X t S E X H A U S T E D OR A L T E R E D . ONCE F O R M E D , T H E P Y R I M I D Y L A L C O H O L AND I C T H l A M I N E ARE NOT C O N V E R T I B L E ONE T O T H E O T H E R BY CLAM

c)

step a f t e r the prim ary com bination the m ethylenb-bridge-carbon.

of

ACTION.

The OF

L

AN

X

(LABI L IZ A T IO N

a

the

REACTANT

EFFECTS

Such

OR

CONDITIONS

BRIDGE

X

THING

THIOLS.

EFFECT

X

ONE

THAT

SATISFACTORY AN

MILD

AND

LOW C O N C E N T R A T I O N

METHYLENE ED

ANY

BUT

a c t iv a t io n

ACCOUNT

ACTIVATION ONLY

the

MOIETY.

VERY

SEEN.

REACTANT

R eactant

CANNOT

UNDER

OF

BY WA T E R

IN

ACTION

BE

MECHANISM

EXHAUSTION

STRUCTION

WE

TO

THE

C H A R A C T E R I S T I C S OF T H E R E A C T I O N AND C L AM S Y S T E M ; S P E C I A L A T T E N T I O N M U S T

T H E P R O P E R T I ES B E P A I D TO J

70 r T H E S I G N I F I C A N C E OF R E L A T I O N TO T H E TWO DUCTS* d

)

e

) An a c t i v a Reactant X

of

f

The

)

l im it a t io n

The OTHER

CLAM

ESSENTIAL OF

of

the

for

IN IT

the

d e s t r u c t io n

m ethylene

manner

an

THE IS

SINCE FROM

the

d e fin e d

adequate

-

POSSIBLE REACTION

PR O­

.

b r id g e

-

carbon

p r e v io u s l y

explan ation

for

as

w ell

as

. the

unrecov

­

FRACTION.

APPEARS TWO

N OT

DIFFERENCES

KIND

PRINCIPLES

L

in

in

PRINCIPLE

SOURCES

ICTHIAMINE.

»©T

t io n

Al l o w a n c e PYRIMIDINE

ered

"i T H E TWO p H O P T I M A AND I T S KNOWN D I S T I N C T P Y R I M I D I N E

PH

YET OR

DIFFER

OPTIMA

AND

POSSIBLE

ARE

COMPARATIVE

OT H E R

TO

SOURCES

MERELY DATA ARE

TO

FROM IN SAY

THOSE

THE IF

VARIATIONS FOR T H E

NOT

FROM

FORMATION THESE OF

OF

REPRESENT

DEGREE

AND

THI A M IN E -S P L IT T IN G

AVAILABLE.

71

P A R T

X.

HY PO T HE T IC A L

T he

f ir s t

B E F OR E

cussed

Me

AND

INDICATED

OF

AND

VOLVING

WA T E R

THE

PH

BE

THIAMINE

MAY

BE

3 .-

AND

OPTIMA

SIMILAR BY

u n r ela ted

X,

REACTANT AND

THE

TWO

EXPLAIN

SATISFACTORILY

PYRIMIDYL

ALCOHOL

CEASES

ADDITION,

IT

IATION OF

pH

REACTION OF

THE

ON T H E

SENTED

EVIDENCE

SPLITTING ERED

IN

FOR

D I S S E R T A T I ON.

IN

AND

CYSTEINE

WAS

AN

­

of

IS

WHY

THE

THIS

IN

NOT

NOT

ONL Y

IN­

SUGGESTED

BY

IS

C A SE

THE

THE WHERE

WAS

MUS T

IN BE

REC E N T L Y , R E D D I

SEPARATION

SECTION

OF

MUSSELS.

III,

SINCE

PART

OF

THE

EXHAUSTED. IN

IN

CLEARLY

WA T ER

A8 D

FORMATION

X

CHANGES

concurrent

REACTIONS

LIKELY

VARIATIONS

MECHANISM

BECAUSE ACTUAL

ARE

but

PRODUCTS,

WHY T H E

OBSERVED

FRESH

DETAIL

APPARENTLY

REACTANT

SIGNIFICANT

HOWEVER,

ENZYMES

GREATER

WHEN

EXPLAIN

REACTION.

CONSIDERATION,

d is

N0 N-CATALYZED

overlapping

ENZYMES

PRODUCTS.

RATIO

THE

PYRIMIDINE

C A N N OT

TWO

,

AS

IT

THE

DIFFERENT

NO

less

reaction

DISPLACEMENT

BY

CAUSE

TO

or

DISREGARDED.

Two

CANNOT

more

ONLY.

BISULFITE

CATALYZED

CONDITIONS

TWO

been

s t o ic h io m e t r ic

SIMULTANEOUS)

AND

have

SUMMARIZED

sim ple

THIAMINE

chan ism

(INDEPENDENT

A

i

MECHANI SM. -

mechanisms

WILL

A .-

X WITH

DE S T R U C T I ON

TWO

three

chan ism

REACTANT

Me

REACTI ON

ONE

TWO THIS

REACTION

RATIO SUCH THE

OF A

VAR­

EFFECT

KEPT ET

IN

UNDER

AL

20

PRE­

THIAMINEIS

CONSID­

TWO OF T H I S

72

C. -

Mechanism BY

A SINGLE

WA T E R

AND

GIVEN

U NDE R

TO

THE

ENZYME

THAT

THE

LATE

ALSO

THAT

AN

of

the

EFFECT

ALSO

CANNOT

IT

IN

above

TOO

CAN

UNWIELDY

WOULD IS

THE

BY

AN

ENZYME

BY

REACTANTX

BREAKS

DOWN

X;

Reactant OF

UNDER IN

THE

QUIRED

A SE COND

|F

THAT

FOR

ATTACK

THE

SPECIAL

THE

BY

REASONS

REFERENCE

FORMATION

FOR

also

OF

THE

REACTION

THE

an

THE

POSTU­

PYRIMIDINE

COMPLEX.

explan ation

X WITH

for

THE

IS

MECHANISMS

IN­ B ECOMES

METHYLENE-BRIDGE-CARBON

ATTACK

be

ON

THE

COMPLEX",

made

ALCOHOL F OR

THE

s a tis fa c to rily REMAINS

MIXTURE.

RUN

THE

EXPLANATION

THE

CONDITIONS

REACTION

TO

S U G­

EX P E R IM E N TA T I ON.

OF

cannot

WHICH

INVOLVED(ENZYMATIC

PYRIMIDYL

ALCOHOL

MECHANISM

AN

"PYRIMIDINE

must

NECESSARY

REACTANT

OF

FUTURE

ROOM

it

BE

OF

OF

SUBSEQUENT THE

VARIATIONS

STEP

EACH

AGAIN,

AND

WHY

THE

SUCH

THE

THE

H

inclu des

B OT H

THE

COMPETITIVE

T

COURSE

GIVE

DIFFERENT

BY

THE

PYRIMIDYL

PH OF

OF

ACTIVATION

GIVE

and

THE

SERVATIONS L ONE*

TO

He r e

AMINE.

MELD

TO

I

AND

REACTION

SE EN

AN

AND

W

DEGRADATION

THE

GUIDE

MECHAN I SM P . -

m ethylene-bridge-caR bqn

PLAUSIBLE,

EXPLAIN

mechanisms

BE

AS

E

MECHANISMS

FAVORING

IT

NOT

B AB0 V

METHYLENE-BRIDGE-CARBON. CLUDED,

”1

the

A SIMULTANEOUS

IS

THERE

SPONTANEOUS)

of

CEASES.

ABOVE

THEMSELVES,

None

X

IT

ALCOHOL

GEST

OR

AND

MECHANISM

FACT

ALL

a ctivatio n

REACTANT

PYRIMIDYL

tN

An

AHEAD

WHICH AND

ESPECIALLY

THE

MECHANISM,THEREFORE,

THEN

IC TH I-

ACTIVATION explain

REASONABLY

THE OF

BRIDGE-CARBON

WITH

AN

the

CHANGE RE­

EXPERIMENTAL NOT

why

CONSTANT

ASSUMPTIONS

IS

OF

OB ­

ATTRACTIVE j

73

r

“i w

Me c h a n i s m

WHICH IS

WE

MADE

E- | . -

HAVE THAT

THE

THETICAL (

IS

THE

MENT,

THIAMINE

IS

P

0

CgH^OgNS,

PYRIMIDINE

THIAZQLIUM

IS

) ,

THE

WHE RE

ATTACHES

PORTION

INTO

ACTIVATES

THE

C O M P L E X ’*

THUS

CLOSE

IN

PENDULUM TO

B R l D G E —C A R B O N ;

THE

STERICALLY

R E A C T I 0 N • • . • . . . . RATHER

GETIC

OR

SUMED

THAT

AND

THE

( t .g .,

FOR

ELECTRONIC THE

INCREASING

THROUGH T H E

SAME

THEN

F OL L OWE D

DOWN

OF

THE

ACTION),

TO

EFFECTS.

LABILlZATION

ACTIVATION

BY

OF ITS

THE

IN OF

MECHANISM. THE

FOR THE

SECOND

THE IS

IS

SO

THE HYPO­

REACTANT

X

THE

GROUP

NITROGEN THE

IN FRAG­

METHYLENE-

THUS

FAVORED

THROUGH

THEN

WE

ENER­

HAVE

AS­

METHYLENE-BRIDGE-CARBON F R A G ME N T

REACTION)

STEP

THE

SAME

LABI LI Z E D ,

IN

ARE

PRELIMINARY

C 0 M P L E X " , ( PROBABLY AT

A

4-AMfNO

THAN

POSTULATED

I C THI AMIN E •

METHYLENE-BRIDGE-CARBON

THE

V.

BRINGING

EFFECT,

TWO-CARBON

CHANCES

"PYRIM IDINE GIVE

FRAGMENT

METHYLENE-

REPRESENTS

THE

TO

assum ption

ITSELF.

QUATERNARY

PROXIMITY

the

WHEREIN

EFFECTIVELY

TWO- CARBON

X

CHART

PICTURED

THE

m e c h a n is m

THE

REACTANT

ITSELF

AND

r e a c t io n

ME CHANI SM,

SCHEMATICALLY

M OIETY*,

CgH^O^NS,

OUR WORKING WHICH

PRESENTED

BY

FRAGMENT, THE

IN

AS

h y p o t h e t i c a l

PRINCIPLE

“ ACTIVATED

DENOT ED

IN

t h i s

CHOSEN

BRIDGE-CARBON MECHANISM

I n

IN AN

X

EXERCISED

C OMBI NAT I ON T HE

IS

BREAK­

ENZYMaTIC

TIM E, A

REACTANT

WHILE

THE

SIMULTANEOUS

Such a c o n c e p t is not w i t h o u t f o u n d a t io n s i n c e the 4-am inoH C l GROUP AND T H E Q U A T E R N A R Y N I T R O G E N E X E R T A ST RONG I N D U C ­ T I V E ON T H E M E T H Y L E N E - B R I D G E - C A R B O N , I . E . , M A K I N G I T L E S S ELECTRON-RICH. T H I S CONCEPT I S D I S C U S S E D I N GREATER D E T A I L *

IN

L

THE

NEXT

SECTION.

74

r

~] ATTACK SOON THE

BY

ACTIVATING OF

THE

ENZYME,

MENT

OF

WHICH,

POWER LOSS

AMI NO

IN

Reactant

X,

YIELD

ICTHIAMINE

IS

OF

DISPLACEMENT OF

THE

A

CARBON HENCE

IS THE

(

IS

IN

Me OF

under

THE

TO

AND BY

ITS

THE

CONSTANT

THIAMINE

He

TO

WHI CH

r e

.

we

E .

The

OF

ON

OF

DISTINGUISH NOW

may

OF

THAT

high

THE

INDEPENDENT OF

IS

m e d iu m

UNDER­ )

is

METHYLENE-BRIDGEOF

REACTANT

PYRIMIDYL THIS

IT

IN­

X,

ALCOHOL

CONSIDERATION

CLEARLY

FROM

A N OT H E R

PRESENTED.

pic tu r e

AND

IS

REGION

A T T a CK

AC­

ALCOHOL.

INCREASE

THE

OF

THUS

re la tiv e ly

a lk a lin e

ATTACK

RATIO

METHYLENE-BRIDGE-CARBON Mechanism

in

DESTROYED.

IS

ANYMORE.

Wa T E R

ALKALINE

IN

OF

ALTERATION

SLIGHT

LOST,

ARRANGE­

L OSS

MECHANISM

BY

AS

APPARENTLY

the

ABOVE

ion

THE

IS

BE

ALTERATION

PYRIMIDYL

THE

RATE

IN

OR

as

MOIETY

THE

hydroxyl

ORDER

Eg* -

IN THE

GOING

or

MECHANISM

chan ism

THE

TO

WATER.

THE

WHICH

(JUST

AS OFT,

TO

PECULIAR

REACTION

COMPARED

REASONABLY TOTAL

INTO

EXHAUSTION

THIAZOLIUM

SPLIT

ASS UME D

SOME

RESULTS

w ell

CONDITIONED

F ORMED TO T H E

CORftLLARY

ENTER

T HE

BE

MOLECULE,

as

A BYSTANDER

NECESSARY

THE

ON

DESTRUCTION,

OF

ALCOHOL.

CONFIGURATION

for

IN

Wa t e r

SENSE

IN

WITH

THE

MAY

DEPENDS

INHERENT

ALCOHOL

STANDABLE. IN

OF

PYRIMIDYL

X

CHEMICALLY,

CONCENTRATION

PYRIMIDYL

SPECIFIC

accounted

COURSE

THE

A PRECURSOR.. . . . . . I S

POWER

THE

CONNECTED

is

GIVE

REACTANT

CANNOT

TIMATELY

|T

OF

OR

WA T E R

LIM ITATION

OF

OF

ACIDS

PHYSICALLY AND

TO

•••••O R

CATALYTIC

THE

TIVITY) THE

OC C U RS

ASfCgH^OgNS)

BECAUSE AN

WA T E R

OF

wp y r i m i d i n e

the

same

REACTANT com plex*

a c t iv a t io n

X ma y

DISCUSSED then

1

L

~J

75

r

BREAK

DOWN

VATER

THEREFORE

step

TO

rather

PLACEMENT SULTS VI I I (

GIVE

.

ACID

P Y R - C H 2 “TGA

THE THE

the

may

ON

THIS

MECHANISM

SCHEME, ED

IS

merely

Mechanism dous

EFFECT

IDYL

ALCOHOL

LY

A WEAK Both

NOT

"PYRIMIDINE

ON

ALL

THE

IT

FOR

WEL L

AS

COMPLEX"

S T A GE

THE

TO

TO

TO

FORMATION,

REASONABLY

THE

t h io

-

THE FOLLOWING

IS

IS

SE E N

T H aT

FIRST

F ORM­ THE

AGAINST

pH

has

YIELD

CONSTANT.

RE­

SECTION

ALCOHOL.

w hile

-

AND W O O L L E Y

MITIGATES

that

of

WITH

KRAMPITZ

PYRIMIDYL

WHICH

d i s

GIVE

|T

INTERMEDIATE" T HE

IN

IMMEDIATELY

BE

observation

ON

THUS

E-j

TWO

HAND. FACTS SERVES

CORRELATING A GUIDE

CONTRARY

and

E2

are

DEPARTURES

THE

OBSERVED

ME N T

attack

COMPLEX".

ICTHlAMINE

RADICAL

EVIDENCE

THE

the

the

(

EXPERIMENTAL

PRESENTED

"ACTIVATED AKIN

r e a c t io n

A COMBINATION

THE

INDICATION

REMAINS

B E T WE E N

TIONS.

AT

SECONDARY

OF

THIS

a

trem en­

THE

IS

PYRIM­

ADMITTED­

ARGUMENT.

THE

TO

is

THAN

DEGRADED

Mechanisms

MAKE

CHOICE

1

E2

due

"PYRIMIDINE

EXPERIMENTAL

THE

to

THE

THEN

M OIETY).

. I IC T H I A M I N E •

AND

T HE

destruction

be

THE

A

DURING

ACID

WOULD

WHEREIN

WHI CH

ONLY

OF

ALCOHOL

THIOGLYC0 LLIC

. . . . . . .RATHER

FORMATION

REACTION

primary

METHYLENE-BRIDGE-CARBON THE

PYRIMIDYL

THIAZOLIUM

WITH

54-57)

GLYCOLLIC

in

THE

OBTAINED p p

ENTERS

than

OF

BOTH

FOR IS

highly

FROM

h ypothetical

ESTABLISHED

MECHANISMS

IS

HARDLY

HOWEVER,

MECHANISM

FROM

SMALLEST

AS

THE

THE

A SIMPLE DATA.

FUTURE

AND

MORE

E-j

OBTAINED,

POSSIBLE DOES OF

CONVENIENT

IMPORTANT,

MECHANISM

1

IT

UNTIL

WILL

8

do

A

THEORY.

NUMBER

EXPERIMENTATION.

but

FROM

EXPLAIN ASSUMP­ INSTRU­ SERVES EVIDENCE

E U SE D

AS

i

76

r

~i A

L

TOOL

F OR

THE

DISCUSSION

OF

THE

EXPERIMENTAL

OBSERVATIONS,

oleophilic Pe-\£ent

( D f /♦///
2 ^

SHIFTED

OF

correspondence

INFLECTION

THIAMINE

CASE

WITH

SHOUL D

ALL

WHICH

REACTION

THIAMINE

WHICH

THEPH

OF

THE

THE

AT

OPTIMUM,

ONE

C L A M —T H I A M l N E

The

BE

IS

VARY

B E T WE E N

THE

pH

VARY

CONCENTRATION

THE

RELATION

BECAUSE

C OMP OU N D S

THE

RATE

FACTORS,

STRUCTURE

OPTIMUM

REACTIONS,

SUBSTRATE.

THE

OF

GREATEST

REACTION

|N

A pH

RATE

REACTION

BET WE EN

THE

RAPIO

VERY

ABOVE

9.05

^

2

3

4

S '

6

7

6

3

10

11

pH Upper

Curve:

Titration Curve

lor

Thiamine(Williams

Lower

Curve:

pH dependence Curve for destruction by the C l a m Principle

of

and

Ruehle)

thiamine

82

*1

r

COINCIDES ATED. OF

THE

THE

THE

OF

THE

RATE,

QUATERNARY AND

HENCE

REACTION

THIAZOLE

CARBON;

DE GR E E

OF

THE

IN

DEGREE

NITROGEN

RELAYED

TWO

T HE

OF

THE

VINYLENE

MAKE

THE

METHYLENE-CARBON

LESS

DECREASING

THE

REPULSION

FOR

NUCLEOPHILIC

REACTANT

METHYLENE-CARBON THE

CLEAVAGE

OF

WITH

ENERGY

AND

INCREASING

THE

ATTACKING

THIAMINE

BY

A

Any

MAKE

THE

METHYLENE-BRIDGE-CARBON

IN

SENSE,

A

NUCLEOPHILIC WHICH

INFLUENCE

lmade

in

ON

THE

or

in flu e n c e ,

THE

STRUCTURAL

AMINO

Me c h a n i s m

Et

GROUPS. t h a t

th e

BOND

two

ON

N ET

THEREBY

OF

A OF

OTHER

which

THIAMINE

WOULD THE

a m ino

OR

WOULD MOS T

BY

E

RESULT

ENERGY |N

TH

SIMILAR

A

IS

THE WORDS, FACIL­

tends

ELECTRON-RICH,

FACTOR*

THUS

S E E .....TH E

REACTANT

POINT

WILL

EFFECT

THE

THE

THAT

EXERTS

th e re fo re ,

THE

METHYLENE-CARBON

TO

REAGENT.

REAGENT.

TWO

EASY

APPROACH

THE

OF

OF

OF

ELECTRON-RICH,

CLEAVAGE

THE

IS

LESS

IN

CLAM-

BRIDGE-CARBON

CHAIN.

THE

"ACTIVATING

RATE.

INDUCTIVE

"ACTIVATE*

EXTRANEOUS

AN

THROUGH

e ffe c t

THE

CHARACTER

NUCLEOPHILIC

ITATED.

MOIETY

DISPLACEMENT

4-AMtNO-HYDROCHLORIDE

THROUGH

HALF

METHYLENE-BRIDGE-

THIS

GROUPS

LIBER­

IMPORTANCE

PROCESS.

REACTION

ST RONG

A

THE

IS

PYRIMIDINE

POSITIVE

THE

SALT

THIAZOLIUM

THE

ON

OF

AMI NO

EXERTS

THE

THE

IN

A NUCLEOPHILIC ATTACK

AN

SUGGEST

ACTIVATION

INFLUENCE

THE

BY

UETHYLEN-CARBON;

TO

THE

QUATERNARY

GROUP

ELECTRON-RICHNESS

INFLUENCED

EFFECT

BY

WILL

THE

STRONGLY

INVOLVES

THEREFORE,

QUATERNARY

IS

WHd CH

NITROGEN

IN

MOIETY

METHYLEN-CARBON

BE

AT

CORRESPONDENCE

THE

T H I AMINE

THE

PH

4-AMINO-HYDROOHLORIDE

AND

THE

WITH

to

WOULD,

A

POINTS EXERCISE

LOGICALLY

T HROUGH ITS BE

EARLIER A S S U M P T I O N groups

are

j

83 r | NVOLVED ON

IN THE

ACTIVATION

A CREDITABLE

THEORETICAL 1

SEALOCK DICATE ING

THE

THE

TION THE

AND

INHIBITORY THE

OF

CARP

DISPOSITION

THlAZOLtUM AMINO T R OGE N

IN

IS

THE

THE

OF

TWO

RESULTS AMINO

PRINCIPLE

ON

THIAMINE,

THE

4-AMINO

S E E ME D 3

TO

BE

CARBONS

THIAZOLIUM

THIS

THIAMINE

ANALOGUE

Thus

Compound

R and

GR OU P

X

IN

INHABITS

Compound

C— /

THEY

IN

FROM

S

THE

ON T H E

REPORTED

I . E . ,

THE

THE

"IF

THE

QUATERNARY

ANALOGUE,

caused

5 6 fo

CIPLE,

CH

REACTION".

and

^

,

TO

THAT

THESE

OF

C-NHo

'

U

n

3c^

ch

/

j

i

TWO

MECHANISM TION

AND

HIBITOR C U L A T t ON Bu r k

DESTRUCTION WHEN

ASSUMED

PRESUMABLY, WAS OF

N OT

SPLIT

MlCHAELIS

t r e a t m e n t

for

TO THE

THIAMINE

PRESENT

THEY WE RE

OF

DID

NOT

SPLIT; H OL D

IN

FOR

SINCE

c o m p e t e t i v e

USING

MADE

S WAS THE

i n h i b i t i o n

WH E T H E R

AND W O O L L E Y

TH aT

USED

REAC­

THE

IN

enzyme

IN­

THE

LlNEWEAVER in

PRIN­

EQUAL

C A R P - T H I AM I N E

WAS

C OMPOUND

CONSTANTS

CARP

HOWEVER,

KRaMPJTZ

THE

ASSUMPTION

THE

CONCENTRATIONS

REPORT,

THE

BY

3

S)

(Cpd .

C OMP OU N D S WAS

H

R)

THIAMINE.

100^

H«C

// \

HC

RESPECTIVELY,

N I­

THEN

H

THE

AC­

H

1

OF

IN -

THAT

TO

C A R P - T H I AM I N E

below

HaN-CHp-CHo-N7

IN H IB ITIO N

1

INVESTIGAT­

RELATION

A GIVEN

CHo

( cpd.

RESTS

STRONGLY

ANALOGUES

IMPORTANT, AW a Y

RING

WHICH

GROUPS.

THIAMINE

TO

2

REPORTED

OF

OF

REACTANT

BASIS*

EFFECTS

MOIETY

GR OU P

BY

g

GOODL AND

IMPORTANCE

THIAMINE

OF

CAL-

AND r e a c t i o n s

.

84 r

4 NO THE OF

REFERENCE VARIATION

THIAMINE

CASE AGE

OF OF

IN

WITH

THAT

ITE

WAS

HR.

PERIOD.

EFFECT

ON

THE

IN

PH

S C H U L T Z 2 *^ E T

THIAMINE

BEST

THE

AT

OF

PH

PH 5 . 2 OF

.

BRIDGE

THAT

THE

THIAMINE AT

AND

HAS

GURIN

BY

SULFITING,

MOIETY.

This

ON

THE

APPEARS

made BY

PYRIMIDINE

CLEAVAGE AGENT

THAT

BEEN THE

REPORTED

DERIVATIVES

REAGENTS. BENZYL

SNYDER

BISULFITE,

SODIUM

THIOSULFATE

NARIES

ARE

ALSO

ARE AND

THE

IS

IN

38

SPECK BY

AND

OF

BISULF­

IN

EFFECT

FEW OF

AND

FOUND

SALTS,

IN

pH

ON

WITH

EASE.

the

e a rlie r METHYL­

THE

|T

SUCH

SPLIT

SODIUM

AS

BY

OB­

RATE

MAY

OF

THE

RE­

BE

N OT E D

BENZYLPYRI—

NUCLEOPHILIC

FACILE SALTS

CLEAVAGE SUCH

AS

HYDROSULFIDE,

THIOCYANATE. NO

LABlLlZlNG

A NUCLEOPHILIC

SULFUR-CONTAINING

POTASSIUM

NOT

PYRIMIDINE

QUALITATIVE

OBSERVED T H E

SULFITE,

IS

BISULFITE

THE

on

LITERATURE.

READILY 51

e ffe c t

A 24-36

LITTLE

34

THE

from

OB­

4-AMINO-HYDROCHLORIDE

THESE

THE

THE

IMPORTANT

THIAMINE

ALSO

SODIUM

SPLIT

the

QUATERNARY

QUATERNARIES

SODIUM

A FUNCTION

i.e .,

THAN

ON

THAT

UNDERSTANDABLE

MOIETY

B E T WE E N

BENZYL

BE

CONJUGATED

OTHER

STUDIES

REACTION

HAVE

DINIUM

NO

is

THE

SUGGESTED

TO

above,

BRIDGE-CARBON.

SERVATIONS,

THEY

suggestion

E N E - B R I d g e - c ARBON GR OU P

AND

THE

CLEAV-

BY

pH 4 - 5

WITH1

CARBON |N

WILLIAMS

BISULFITE

CLARKE

DEALING

SOLUTION.

.

DESTROYED

fe.1 P M A N N ^ ? 7.8

THE

STATED

DESTRUCTION

TO

LITERATURE

METHYLENE

AL.

AT

OF T HAT

ACCORDING

con sid eratio n s

L

CHAN GE

ONE-HALF

REACTION

OF

THE

T HE

PROCEEDS

CLEAVAGE

HERE

IN

4 —M E T H Y L —5 - ^ J —HYDRO X Y E T H Y L - T H I A Z O L I UM E T H I O D I D E

SPLIT

THE

FOUND OF

PH 1 ,

AT

ON L Y

BE

REACTIVITY

B ISULFITE, THIAMINE

SERVED

THAT

C OU L D

STUDIES

ALLYL WERE

QUATER­

MADE

WITH

n RESPECT

TO

NOTED

THE

U NDE R

VERY

POSE,

THE

THE

ABNORMAL MILD

AMI NO

OF

PH

OF

REACTION

NITROGEN GROUP

THE

INSTABILITY

CONDITIONS,

IN ITIA L

QUATERNARY THE

EFFECT

IN

AMIN0BEN2YL

THESE BEING

BOND. THE

OF

THE

BENZYL

cp RAISONJ t

SOLUTION.

COMP OU N B S T HE

INSTABILITY MOIETY

WAS

QUATERNARY

RAPIDLY

CLEAVAGE IS

IN

"i

OF

D E C OM­

THE

BENZYL-

GREATEST

THE

SALTS.

PARA

WHEN

POSI­

TION.

I

n

a d d it io n

VARIATION THIAMINE THAT

WE

IN

HAVE

ENTERS WE

CANNOT

ACTIVITY

PH, NO

X,

INTO

PRESENTED

and

Me

I.

e

OF

Et

chan ism

m e c h a n ism

;

IT

-

be

ANY

ATTEMPT

AT

by

TWO a

OF

THE

OF

THE

VERY

ster ic

WHICH

a

up

hich

AND RE­

POSSIBLE WITH

VARIATION

PR W I L L WHY

NOW

CHAIN

BE

R E A C T A N T S ( T H I AM I N E as

shown

p o s s ib le

an

BE

in

reaction

in t e r e s t in g

S HOUL D

OF

OF

A STOICHIO­

BORNE

aspect

IN

INVESTIGATION.

SIDE

HENCE

H.

effect

as

opens

REACTION

NEATLY

COMPLEX

GROUP

, ................ w

NUCLEOPHILIC p

OF

DIFFICULTY

FUNCTIONAL

fragm ent

WITH

co nsidered

REACTION

THE

THE

X AND T H I A M I N E

KINETIC

THAT

OF

FURTHER

DISPLACEMENT

CENTER

c o n s id e r a tio n

IN

THE

carbon

UNDERLINES

favored

C LAM -THI AMINE

KNOWN

NATURE

two

B E T WE E N

THE

IS

WITH

VARIATION

REACTANT

OF

IT

the

THE

METHYLENE-BRIDGE-CARBON

A CONSIDERATION

should

the

THE

THE

REGARDING

info rm a tio n

FACED

REACTION

COMBINATION X)

OF

THE

BECAUSE

Re a c t a n t

OF

ARE

.,

of

NUCLEOPHILIC

THIS

REACTION

METRIC

WE

PICTURE OF

lack

IDEA

THE

NEVERTHELESS, THE

the

REACTIVITY

WITH

Reactant

in

to

I C T H I AM I N E

HAS

A

MIND

SULFUR GROUP F OR

ATOM IS

AND

A

REVEALED

BY

ILLUSTRATION

CONTAINS

THE

ASSUME

THE

STBUCTURE .

AS

OF we

WE

IS

WRITE

GO

w r i t e

FROM

SIDE Ac i d

THIS

CENTER

THE

THIAMINE may

GROUP(THE

ELECTROMETRIC MA Y

(Z R )

THE

Thus

THIAMINE

WE

NITROGEN

NUCLEOPHILIC

AND

known

BASIC

IN

TITRATION SIDE

the

THE

THE

f o llo w in g

TO

WHICH

CENTER.

THE

(NZH)

CHANGES ARE

(NZH)

for

ALKALINE

CLEARLY AS

ALKALINITY

CHA IN

pH

J C T H I AM I N E * ) .

X,

forms

THE

NITROGEN

REACTANT

INCREASING

ACID

OF

CHAIN,

NUCLEOPHILIC WITH

BASIC

and

REGION!

THI AMINE w//*T

• * £ f n il

id . a/* pH 4 . 5 PH 9

n



/

z r t

Cffi

to

4-

H ' c fh *

M 9/ f * S 9

.1



_______ ’A b o v e

p

H

9

*/t ■ c /L

3 re f

/✓



Z

v

M S fl

ch-*

ffo sfiff

I /J /L

S ' *

elk-

P / C S

e>

C ft*

*

Ba r n h u r s t and He n n e s s y , u n p u b l i s h e d w o r k . T he e l e c t r o ­ T I T R A T I O N C U R V E S HOWS TWO I N F L E C T I O N P O I N T S

m e t r ic

CORRESPONDING

TO

TWO

EQUIVALENTS

OF

N a OH.

IN

87 Curve

a

N S°

N

(N °2 “ )

♦• ♦ I • 1

Curve

C

• •

*



M°N



f

t m 0 -tu 0

r

(

N ZH )

C U R V E _ B _______I N

N °N 0 S°

ZH°

p

2 Curve

A

H

SL

4 .5

:

( — ►)

decreasing

METHYLENE TOWARD Curve

B

:

Curve

C

:

( H

I . E . ,

NUCLEOPHILIC

— ■)

p

electron

CARBON,

i n c r e a s in g

)

Re s u lt a n t

d e p e n d e n c e

BETWEEN

n u c l e o p h i l i c

Be t w e e n

p

H

for

Th ia m in e

and

A Hy

Re a g e n t ,

L

or

12 the

REACTIVITY

a c t i v i t y

and

c l e a v a g e

B:

of

(NZH) •

h y p o t h e t i c a l

r e a c t io n

(N ZH )*

AND

De p e n d e n c e

A

Curves

of

Fig. Theoretical

richness

INCREASING

U

REAGENTS.

curve

THIAMINE

10

8 C urve

for

p o t h e t i c a l

N

th e

Re a c t io n

Nu c l e o p h i l i c

ZH°

-i

88 i AT

ACID

pH,

PRESSED CARBON

the

WHILE IS

AT

WHILE

THE

FROM

ARE

F OR

.

A

A ND

8,

8

.

C.

THE

in

Th u s,

TWO

c a u t io n

CTC• ,

MIGHT

BE

AN F OR

THE

PROVIDE

POSTULATED

L

TO

WITH

TO

H

AS

IN

TAKEN IN

THE

THE

THE

METHYLENE

CURVES

REAGENT

o p t im a

f o r

. . . . .

THEY

WO U L D BE

the

THE

THE

PH

IN

THE

INCREASING

BY

q u i t e

AS

of

pH

CONFINED

THIAMINE

ANALOGUES

EXPERIMENTAL

EVIDENCE

F OR

OF

CURVE

shown

in

be

the

s t r u c t u r e s

CHANGES.

pH

OF

REACTION

THE

A

p e r t i n e n t l y

AND

THIA­

RESULTANT

can

BASIS

DEPENDENCE

BETWEEN

d ia g r a m

OF

MECHANISM

WITH

THE

INTERPRETATION

HAVE

BEST

RESPECTIVELY.

BE

SYSTEM

THE

CARBON

VARIATIONS

REACTION

AFFECTED

COMPLEX

ARE

EXHIBITED

the

C L A M - T H I AM I N E

WE

BRIDGE

r e a c t io n

i l l u s t r a t e s

IN

INCREASES

EFFECTS,

on

ARE

(NZH)

A

s i m p l i f i e d

in

ALKALINE,

CARBON

A ND

DISPLACEMENT

REASON,

BEHAVIOUR

8

SUP­

MOR E

REACTIVITIES

HYPOTHETICAL

IS

METHYLEN-SRIDGE

METHYLENE

THE

APPARENTLY THIS

CHAIN

CONCEIVABLY

c o n s i d e r a t i o n

HAS

SUBSTRATE.

p

SIDE THE

e x t r e m e l y

accounted

REACTANTS

a

MAY

the

two

ESPECIALLY

DEALING

FAR

AND

THE

BECOMES

ACTIVATION

NUCLEOPHILIC

Curve

IN

BY

THE

Such

SO

AND

OF

OF

(NZH)

OF

PH

THE THE

NUCLEOPHILIC

t h e o r e t i c a l l y

OF

A ND

DESCRIBED

STERIC

THE

WHERE

A ND

CURVES

f i g

8 ,

(NZH)

OF

UlSREGARDlNG

l i k e

OF

VARIATIONS

FlGURE

ALKALINITY

MINE

AS

REACTIVITY

REACTIVITIES

CURVE

MINIMUM.

THE

r e a c t iv it y

ELECTRON-RICHNESS

ELECTRON-RICHNESS

INCREASES* SEEN

THE A

NUCLEOPHILIC

n u c le o p h ilic

EFFECTS,

WH E R E A

WE

ARE

VERSATILE

OUR TO

why

STUDIES SEE

STERIC

IF

THEY

EFFECT

E-j •

_I

89 r

\

3•

T he Sy

T he

Be h

ste m

aviour

Th

of

An a l o g u e s

ia m in e

w ith

the

Clam

, -

following

t h ia m in e

analogues

were

used

in

the

I N Y E S T I G AT I ON : Gr o u p A) B) C)

I , N E O P Y R I T H I A M I N E - B R O M I D E - H Y D R Q B R O M I DE 2 , 4 , 6 , " C O L L I D 1N I U M AN AL OGUE 2 , 6 " L U T I D I N I U M A N A L OG U E

Gr o u p a b

) )

I I • 2 ,4 2 ,5 -

Gr o u p a b

NO T E:

) )

t h ia z o l iu m

analogue

t h ia z o l iu m

analogue

11 I . Be n z t h I a z o l i u m a n a l o g u e OXYTHI A M I N E -C H L O R I D E

F o r e a s i e r r e f e r e n c e , t h e f o r m u l a s and f u l l names o f T H E S E T H I A M I N E A N A L O G U E S H A VE BEEN P L A C E D I N A F L Y ­ L E A F A T T H E END OF T H I S D I S S E R T A T I O N , HENCEFORTH, T HE A B OVE C OMP OU N D S W I L L BE R E F E R R E D TO BY T H E NAMES GIVEN ABOVE, ( T H E A N A L Y S E S OF T H E S E COMP OU N D S ARE I N THE F L Y - L E A F ) . ALL

TO

d im e t h y l d im e th y l

GIVE

THES

ANALOGUES

FLUORESCENT

FERRICYANIDE. PORTIONAL OF the

THE

TO

THE

The

ent

CONCENTRATION

I

OF

AND

3ERGEL I

at

BEHAVIOUR

the

b

,

I

c

,

and

T O D D 4^

DEVELOP

end

OF I I

of

THE a

(SEE

is

THOSE UPON

FLUORESCENT

DEVELOPMENT

Compounds

L

C OMP OU N D S

FLUORESCENCE

Su p p l e m

GROUP

T HE

EXCEPT

IN

OXIDATION

INTENSITY IN OF

t h is

THESE

111

MEDIUM,

AFTER

IN in

view

SOME

PRO­

(THE ARE

DETAILS

GIVEN

IN

).

THE

THE

FOUND

ALKALINE

DIRECTLY

d is s e r t a t io n

unexpected

WERE

WITH

C OMPOUNDS

FLUORESCENCE

ON L Y

IS

A QUEOUS

SUPPLEMENT),

FLUORESCENCE

GROUP

CASE of

the

COMPOU N D S TIME

OF

IN

work

IN CONTACT -J

90

r WITH

i THE

OXIDANT

fluorescence M b ,

lik e

UPON

MIXING

E NT I A T E D GROUP

I

member IS

in

AT

ROOM T E M P E R A T U R E f ir s t

t h e

thiam ine, WITH

MAY

BE

Thiam ine).

ADDED,

YEAST

SE E MS

ABLE

I

M

AND

INTO

Groups

in

2,4,6-co lliD IN Iu m GIVE

FLUORESCENT

FLUOROMETER,

ARE

T

he

GREATLY

AIDED

T HE

COMPETITION

|N

THE

CONCLUSIONS WH E N E V E R MENTIONED

THE

NEXT

CLAM WITH

GROUP

DO

SPECIFICALLY.

THE

ANY

th iazo le

m oiety

ANALOGUES

IN

REGENERATION.

(w ith when

in

the

used

AS

DIFFERENCES

exception equivalent

OXIDATION

WITH

MEASURED

LESS

THAN (SEE

Gr o u p s

IN

THAT

It

and

OF

THE

IN

PLACE

AND

THE the

amounts,

THE

PHOTO-

OBTAINED

FROM

A,

,

PAGE

are

also

PROPERTIES

BEHAVIOUR OF

of

IN

ALKALINE

TABLE

I

OF

SINGLE

AFTER

SYSTEM,

OF

THIAMINE

THESE AS

WELL

THIAMINE.

NOT

It!

the

C H A R A C T E R I ST I CS

FOLLOWING

DRAWN

OF

AND

THIAMINE.

THESE

MEMBER

BY

INVESTIGATION

THE

PRESENCE

TIM E-DlFFER -

SHOWN

UPON

analogues

SENSITIVE.

COMPOUNDS W I T H

OF

THIS

AS

INTENSITIES,

AMOUNT

).

It

and

Ha a n d

im m ediately

SINGLE

$ hen

DISTINCT

Compounds

OF

ANY

CONVERT

SIGNIFICANTLY

SULFITE

IN

I

PRODUCTS

WHOSE

pplem ent

BEFORE

analogue)

F E R R I CYAN I D E

EQUIVALENT

TO

THIAMINE

compounds

L

THE

11 ( o r

READINGS

AS

IN

Group

GALVANOMETER

Su

DEVELOPMENT,

DETERMINED

NO

fluorescence

BECAUSE

of

GROU P S

AN

f u ll

OXIDANT.

FLUORESCENCE

SHOW

twenty seconds*

develop

THE

AND

EXPERIMENTS

TO

BE

PRESENTED,

III

INCLUDE

THE

GROUP

C OMP OU N D S

ARE

INVOLVED,

T HE

COMPOUNDS} THEY

WILL

BE

91 r

! a

)

Cleavage

An

aqueous

OF

THE

(200 IN

20

ML

HOURS.

OF IN

ALL

Th is

a

BY

OF

DESTRUCTION,

WHICH

BY

)

ACTEO

ARE

IT

WAS

CLAM IS

IN

It!

Th ia m in e

IN

THE

SAME

SUBSEQUENT LITTLE

PROVIDED

DESTRUCTION

Thu s, OF

an

0 .1

GM

OF

aqueous

OF

CLAM

THIAMINE-EQUIVALENT A ND

7.2

IN

10

TEMPERATURE. LOGUE L

OR

OF

GROUP

PH 3 . 6 COULD

BE

STUDY

THAT AT

THESE A

THE

are

COMPARATIVE

SIMILAR

USUAL

24

F OR

(TABLE

ANALOGUES

RATE

COMPOUNDS,

DETECTED

analog ues

OF

GM

EACH

II

7.2

AND

th ese

ASSAY

IX). d e s t r o y

WERE

TO

­

RATES DE­

THAT

METHOD.

Re a c t i o n

An a l o g u e s . -

I f

THIAMINE,

THE

GIVEN

THIAMINE

AT THE

t h e s e

THEY

SHOULD

OF OF

I ,

r eag en ts

SHOULD

AMOUNTS AMOUNT

Gr o u p s

by

EXHAUST

EACH

ANALOGUE

CLAM

EXTRACT,

IN

RELATIVELY

RESULT

THIAMINE. e x t r a c t

,

c o n t a i n i n g

the

POWDER,

WAS

INCUBATED

EACH

THE

ANALOGS

OF

REACTION

AN

AQUEOUS WA S

AND

t h a t

SUFFICIENT

ML

THIAMINE

MILLIMOLES

FLUORESCENCE

A

0 ,1

of

X.

AS

WITH OF

I

Clam-T h ia m in e

th e

MANNER

ADDITION

BOTH

I n

TABLE

and

INCUBATED

AT

USING

tl

X AND

GROUP

Pr i n c i p l e . -

p o r t io n

0.6

WITH

OF

PR I N C I P L E

SPLIT,

SHOWN

soluble

FULL

FOUND

of

BEEN

t i o n

i n d i c a t i o n

I n h ib it io n

REACTANT HAVE

THE

GAVE

C lam

the

FLUORESCENCE

PRINCIPLE.

THIAMINE

RESULTS

b

CLAM

NO

BLANKS

c l ea r

ed

STROYED

MIXTURE

by

the

INCUBATED

EQUIVALENT)

CASES,

HEATED

THE

WA S

REACTION

ALTHOUGH was

c o n t a i n i n g

P O WD E R

THIAMINE

An a l o g u e s

t h e s e

e x t r a c t

CLAM

UG

of

RUN

OF

MIXTURE

EXTRACT UNDER

F OR

24

CONTAINING THE

SAME

s o l u b l e

WITH

AT

BOTH

HOURS NO

200 PH AT

ADDED

por

­

UG

3.6 ROOM ANA­

EXPERIMENTAL -J

92

CONDITIONS. ED

IN

E ACH THE

A

SUITABLE

CASE, CASE

THIAMINE

DROPS THEN

THE

OF

TREATMENT

SAMPLES

AFTER

A DDE D

WITH

IN

A DDE D TO

LY

ADDE D

ED

OXYTHlAMINE,

OTHER

SAMPLES

WAS

REACTED

WITH

The OF

CLAM

I.

E.

INTERPRETED POSSIBLE

ER

,

PR OD U C E D

c)

EXTRACT

The

PRODUCED Clam

WAS

THAT

THE

BLANK

SAMPLE

ANALOGUES

IN

GM OF

THIS THE

th e

OF

THE

r e g e n r a t io n

FROM

EACH

A N A L OG U E

BY

NO

ADD­ THE

IN

AS

DID

REACTANT

MUS T

THE

IN ITIA L­

WOULD

ABOUT

RESULTS

X. 250

CANNOT

BE

the

ACTION

UG

BE

MADE

DURING

of

WAS

OXYTHlAMINE)

DESTROY

EXTRACT

A FEW

SAMPLE

MANNER

b e h a v io u r

THE WITH

IT

WITH

A L L OWA N CE

OF

THIAMINE

(EXCEPT SAME

AFTER

IN ITIA LLY

X I).

THE

CLAM

IN

EACH

ADDED

C OUL D

P O WD E R .

SINCE

IN

REACTED

CASE

(EXCEPT

AND

CONTAINING

THE

IN

UG

CONTAINING

(TABLE

IN

THAT

PRESERVED

COMPOU N D S

PRINCIPLE

200

MIXTURE

T HE

OF

DETECT­

FLUORESCE

THEN

DESTROYED

THE

THESE

NOT

DO

BE

S H O WI N G

DESTROYED

WHILE

APPRECIABLE

THE

r e a c t io n

OF

THE

STUDIED.

Regeneration IN

DAYS

COUL D

Be h a v i o u r

DESTRUCTION

OF

of

THE

the

Re a c t io n

THIAMINE

Products

ANALOGUES

BY

THE

Pr i n c i p l e . -

To L

He n c e

BEEN

WHI CH

DESTRUCTION

D f i N A T U R A T I ON

PRODUCTS

CLAM

THE

ACCURATELY

.

SAMPLES

INCUBATION

TWO

IN

USED

o p e r a t i o n

HAD

THIAMINE

THESE

0.1

TOO

EACH

AND

C L AM

EXTRACT

THIAMINE

FOR

THE

THE

FERRICYANIDE).

IN

N OT

THEREFORE,

ALL

EXCEPT

THE

APPEAR,

THIAMINE,

TO

THE

THIAMINE

FLUORESCENCE

COMP OU N D S

S T AND

TOLUENE.

DETERMINED.

OF

NO

A N A L OG U E

ALKALINE

A L L OWE D

OF

HOURS,

ALIQUOT

GROUP

WERE

24

6

ML

OF

CLAM

EXTRACT

(CONTAINING

THE

SOLUBLE

PORTION -J

92 ft

IX

T a b le

T he

De s t r u c t i o n by

Th ia m in e

Pr i n c i p l e

th e

UG

TH 1AMI NE ANALOGUE

of

An a l o g

in

Clams

Pr e s .

, E Q U I V . TO 2 0 0 UG T H I A M I N E

200 231 249 237 236 236

T H I AMI NE 2 ,6 -L U T ID IN IU M NEOPYRITHIAMINE 2 ,4 ,6 -C O L L ID IN . 2,4 -T H I4 Z O L IU M 2 , 5 - T H 1AZOLI UM

An a l o g u e s

Ga l v .

Rea ding

for

*

4 UG T H I A M I N E - E Q U I V . IN 1 0 ML A Q . SOLUTI ON

UG

None i» » t» » n

HEATED BLANKS T H 1 AMI NE N E O P Y R 1T H 1 A M I N E

2 ,4 ,6 -C O L L IDIN. 2 , 4 - T H 1AZOLIUM 2 , 5 - T H 1A Z O L I U M

PROCEDURE

105

200 231 249 237 236 236

2 , 6 - L U T I D I N IUM

USED:

To

6

UNITS

40 35 100 43 35

ml

clam

e x t r a c t

,

c o n t a i n i n g

the

S O L U B L E P O R T I O N FROM 0 . 1 5 GM D E S I C C A T E D C L A M P O WD E R , WERE AD D E D 3 ML OF M C l L V A l N E B U F F E R , A F T E R A P R E V I O U S A D J U S T ­ M E N T TO T H E D E S I R E D P H . S A M P L E S WERE RUN A T BOT H PH 3 . 6 and 7.2 . T he n 2 ml of an a q . s o l n . of t h e g i v e n a n a l o g , C O N T A I N I N G T H E E Q U I V A L E N T OF 2 0 0 UG OF T H I A M I N E , WERE ADDED. T H E S A M P L E S WERE T H E N D I L U T E D T O 2 0 ML AND A L ­ LOWED TO S T A N D 2 4 H R S . A T ROOM T E M P E R A T U R E . HEATED Bl a n k s

were

^An a l y s i s :

HC L - H P O 3

r u n

.

T he

r e a g e n t

d i f f e r e n t

(5

m l

)

in

samples the

usual

were

t r e a t e d

m a n n e r

.

w i t h

th e

A 1 .0

ML WATER

A L I Q U O T WAS T H E N D I L U T E D TO 2 0 ML W I T H D I S T I L L E D AND 1 0 ML A N A L Y Z E D I N T H E U S U A L M A N N E R , U S I N G 0 . 0 2 5 % POTASSIUM

L

F E R R I CYAN I D E

IN

15%

NAOH.

-I

92 IB r

i Table Co m p a r a tive

Ra t e s

Th ia m in e

and

De s t r u c t i o n

of

An a l o g u e s

Substrate

X

p

by

of

Clam

the

Th ia m in e Pr i n c i p l e a

ug

H

S ubstrate

of

O est.b

2 , 4 - T H 1 A Z O L I UM

3.6 7 .2

65 67

ug

3.6 7.2

59 60

ug

NEOPYRITHIAMINE

3.6 7.2

25 30

ug

2 ,4 ,6 -

3.6 7.2

60 58

ug

3.6 7.2

40 40

ug

analogue

2

, 5-th iazo liu m analogue

c o l l id in iu m

analogue

T H A 1 M l NE

a

) 4

b

)

ML OF A Q* E X T R A C T F OR E A C H C A S E .

De s t r u c t i o n

from

200

EQUIVALENT

ug

t h i a m i n e

F OR E Q U I V A L E N T V A L U E S ) UNDER U S I N G 1 0 ML R E A C T I O N M I X T U R E , e t c

.

Heated

blanks

run

0*1

TO

P O WD E R ( see

e q u i v a l e n t

USUAL

30°,

showed

GM

no

WA S

USED

Table

IX

ASSAY C O N D I T I O N S , 2 HRS REACTI ON T I M E ,

d e s t r u c t i o n

.

I n the a n a l y s i s , a l iq u o t s from each sample are W H I C H WO U L D b e C A L C U L A T E D t o G I V E A G A L V A N O M E T E R

ta ken

READING

1 0 0 T O 1 5 0 U N I T S ON T H E S C A L E . F OR E X A M P L E , FOR T H E 2 , 4 - T H I A Z O L I U M A N A L O G U E , AN A L I Q U O T OF 1 . 5 ML FROM A F I N A L VOLUME OF 2 0 ML C O R R E S P O N D I N G TO 1 5 UG T H I A M I N E - E Q U I V A L E N T O R I G I N A L L Y PRESENT IS USED. THE A L I Q U O T I S D I L U T E D TO 1 0 ML W I T H W A T E R , 3 ML OF 0 . 0 2 5 $ F E R R I CYAN I D E R E A G E N T I N 1 5 $ NAQH ARE ADDED AND T H E R E A D ­ OF

ING

FROM

t a k e n

.

Su p p l e m e n t .

Further As

d e t a i l s

m e n t io n e d

in

are the

p r e s e n t e d

in

the

,

th e

s u p p l e m e n t

com par

­

V A L U E S ARE N OT T OO A C C U R A T E E S P E C I A L L Y B E C A U S E T H E A MOUNT OF F E R R I CYAN I D E USED HAS D I F F E R E N T E F F E C T S ON T H E R E A D I N G S FROM EACH A N A L O G U E , BUT T H E R E S U L T S DO SHOW C L E A R L Y T H A T T H E A N A L O G U E S ARE S P L t T . . . AND AT C O M P A R A B L E RATES. a t i v e

L

J

92c

T able T he

I

n h ib it io n by

Substrate Ad d e d

I

n i t

.

Wa t e r T h 1 AMINE 2 , 4 - t h ia z o liu m 2 , 5 - t h I A Z O L I um BENTH1AZOLIUM b 0 x y t h I am I n e N E O P Y R I T H 1AMINE 2 , 4 , 6 - C O L L I D 1N I U M

the

of

the

Th

XI Clam- T h a im in e An a l o g u e s

Re a

ction

ia m in e

Am o u n t I AD DE D

2 2 0 0

236 236 298 1434 250 237

n i t

.

Th ia m in e FROM 2 0 0

ML ug

160

ug

74 76 16 190

De s tr o y e d UG T H I A M .

''

ug

1 0 0

1 1 2

80

P r o c e d u r e U s e d : - To 4 m l o f e x t r a c t , c o n t a i n i n g t h e s o lu b le p o rtio n of 0.1 GM d e s s i c a t e d powder, a d ju s ted to the desired pH (p h 3 .6 and p H 7 . 2 ) w e re added 4 ml o f M c I l v a i n e b u ffer. (T h is amount o f clam e x t r a c t w i l l d e s tro y abo u t 2 5 0 UG T H I A M I N E UNDER T H E C O N D I T I O N S USED FOR S T U D Y I N G T H E LIMITATION IN t h e d e s t r u c t i o n ) . T h e n an a m o u n t o f t h e g i v e n r e a g e n t s h o w n a b o v e is A D D E D , T H E M I X T U R E S A L L D I L U T E D TO 1 0 ML W I T H W A T E R , AND T H E N AL L OWE D TO S T A N D FOR 2 4 HCURS AT ROOM T E M P E R A T U R E j T hen s u it a b l e a l iq u o t s from each sample were a n a lyz ed for fluo rescence. I n a l l c a s e s , no f l u o r e s c e n c e c o u l d b e D E T E C T E D E X C E P T OF C OU R S E I N T H E B E N Z T H I A Z O L I U M AND OXYTHlAMINE INCUBATION M IXTURES. 2 0 0 UG OF T H I A M I N E WERE T H E N ADDED AND T H E S A M P L E S D I L U T E D TO 1 5 M L , P R E S E R V E D W I T H A FEW D R OP S OF T O L U E N E AND ALLOWED TO S T A N D TWO MORE DAYS A T ROOM T E M P E R A T U R E . T H E S A M P L E S WERE T H E N A N A L Y Z E D FOR T H IA M IN E PRESENT. T H E R E S U L T S SHOWN ABOVE ARE FOR T H E R E A C T I O N S RUN AT P H T . 2 . T H E R E S U L T S FOR T H O S E RUN A T PH 3 . 6 WERE E S S E N T I A L L Y T H E S A M E . T HE AMOUNT OF T H I A M I N E D E S T R O Y ­ ED I N EACH C A S E D I D NOT I N C R E A S E EV EN A F T E R T H E S A M P L E S WERE AL LOWED TO S T A N D F I V E MORE DAYS AT 8 - 1 2 ° . A)

B)

L

2 4 6 UG OF T H E B E N Z T H I A Z O L I U M TO 2 0 0 UG T H I A M I N E . 179

UG O X Y T H l A M I N E

IS

ANALOGUE

EQUIVALENT

TO

ISEQUIVALENT

200

UG T H I A M I N E

J

93 r

i

OF

0.15

GM

OF

THE

ADDED

3

ML

OF

MC

OF

A QU E OU S

AN

CONTAINING The

tu re. the

ILVAINE

SOLUTION

200

samples

Samples

then

were

SHOWED

THAT

ZOLIUM

ANALOGUE

he

IN

ALL

run

shown

AND

in

THE

T

was

by

OF

THE

SIGNIFICANTLY IN

THE

THOSE RUN

CASE F ORMED

TO

TEST

EFFICIENCY CONVERT ON

THE

TIVE L

THE

THEM

TO

IN

43

EACH

hours and

OF

OF

THE

CANNOT

BE

ASSAYED

WHI CH

WAS

WHEN WAS

T HE

GROUP

OF

THE

AND

THIAMINE.

OF

BLANKS

AND

WAS

IN

OF

were

sam ple

then

results

are

SIGNIFICANT AMOUNT

OF

THE

REGENERATION

SULFITING

WERE

PRODUCTS

ANALOGUES

SEE

SINCE

o x y th ia m in e

THIAMINE.

TO

BENZTHIA-

each

The

THE

AFTER

ANALOGUES

EFFICIENCY

PYRIDINE,

II

AL SO

.

for

PYRIMIDINE

DESTRUCTION

YEAST

REGENERATION

VALUES

AND

case

EQUIVALENT

ADDED

THE

EFFECT

except

THERE

AN

THUS, I

(

method

from

by

period

COMPLETE

each

ADDED.

A nalysis

C A SE

ANALOGUE)

REGENERATION

.

THE

cases

ML

tem pera­

IN

regeneration

THIAMINE

room

incubation

DESTRUCTION

all

at

WERE

the

in

n

2

a fter

produced

I

WERE

THEN

CASE

7.2

products

yeast

PH.

pH,

A N ALOGUES( AND T H I A M I N E )

pH 3 . 6

except

THE

THIAMINE,

THE

THIAZOLE,

THE

GIVEN

aliquots

GROUP

IN

OF

THE

DESIRED

s u it a b l e

L O WE R .

OF

(

THE

in

X I I .

MOIETY

PROCEDURE.

TO

for

both

assay

absent

the

able

REGENERATION

OF

OXYTHlAMINE

BENZTHIAZOLIUM

THIAZOLE

at

CASES

AND

reaction

determ ined

EACH

OF

incubated

'HOTOFLUORIMETRICALLY)

T

BUFFER

OF

thiochrom e

fluorescence

ADJUSTED

UG T H I A M I N E - E Q U I V A L E N T

were

m odified

POWDER),

ARE

PRODUCED

SIMILAR

BLANKS

WERE

ON T H E

RE G EN RAT I ON

IF

YEAST

THE

FOR

THE

POSSIBLE

THE

YEAST

BENZTHIAZOLIUM

BY

THE

TO

CAN

EFFECTS RESPEC­

MOIETIES

OF

EACH J

.

94 OF

THE

ANALOGUES(EXPECTED

EACH

ANALOGUE

WERE

ONL Y

THE

BY

THE

LOWER

THAT

INCUBATED

TION

WITH

may

be

IN

THE

SAMPLES

OF

ZOLIUM

seen

NO

WITH

THE

TION

ASSAY

MOIETY

WAS

BUT

THIAMINE,

INGS

WERE

TAKEN 24

HOURS

ANALOGUES

DO

NOT

SHOW

PYRIMIDYL THIAZOLE AND

[AND

THE

CASE

OXIDIZED

THE

DARK

(NOTE I -

FLUORESCENCE

YEAST

THE

REACTION

PRODUCTS

PYRIDINE

MOIETIES

OF

DESTRUC­

INCUBATED

YEAST

THE

REGENERA­

THIAZOLE

ANALYSIS

GAVE

WHEN

READ­

WERE

THE

AL LOWED

TO

PYRIDINIUM

CANNOT

OR

XII

Table

HEATED

IMMEDIATELY).

APPARENTLY

THE

IN

TO

EVEN

SAMPLES

THE

BENZTHIA-

SAMPLES

THIOCHROME

READINGS

in

THE

THE

OF

IN

EXTRACTS,

WHILE

THE

FROM

WITH

THESE

RECOMBINE THE

ANALOGUES

T HE

RESPECTIVE OF

GROUP

I

I I •

The SHOW

IN

CLAM

ADDITION

SUBSEQUENT

WE

INVESTIGATED*

shown

AND

OF

THE

ANALOGUES

SUBJECTED

THE

DEVELOP

THAT

PRIOR

GALVANOMETER

PORTIONS AND

THE

AFTER

FOR

THE

SINCE

FOR

OBTAINED*

WHEN

WERE

RUN

N OT

ACTIVE

EACH

OCCURRED*

EXTRACT

THE

values

WERE

CLEAVAGE

INTERPRETATIONS

OXYTHlAMINE IN

NOT

WERE

the

WITH

VALUES

COMPLETE

WITHOUT

INSIGNIFICANT

RESULTS

from

THE

REASONS

FOR

BLANKS

INCUBATED

CLAM

VERY

STAND

however

DESTRUCTION

ACTIVE

OF

HEATED

IN

WERE

POSSIBLE

OBTAINED

ANALOGUE(EXCEPT

ANALOG)

PR OD U C E D

QUALITATIVE

THE

REGENERATION

EACH

BLANKS,

THE

VALUES

It

SIGNIFICANT

IN

BE

PRINCIPLE)

VALUES*

REGENERATION

SAMPLES

CLAM

INTERESTED

REGENERATION

TO

THAT It

TO

yeast

THE

regeneration

values

YEAST

DOES

CONVERT

THIAMINE*

(THE

REASON

from

T HE FOR

the

heated

ANALOGUES INCOMPLETE

IN

blanks

GROUP

I

CONVERSION

.

94a

Table

XI I

T h e R e g e n e r a t i o n Be h a v i o u r o f Produced in the d estr u c tio n by t h e C lam Pr i n c i p l e

Su b s tr a te

Th

p

H

the of

Total Py r i m

Re a c t i o n Pr o d u c t s T h i a m i n e An a l o g s

Re g e n . a . F ractn

id

SU LFITE-IN S E N S 1 T 1 VE

3.6 7.2

178 152

3.6 7 .2

172 126

1 2

3.6 7 .2

173 138

1 2

3.6 7.2

118

16 •. •

3 .6 7.2

101

78

2 0

N E O P Y R I T H 1 AMINE

3.6 7.2

150 123

30 52

B E N Z T H 1A Z O L I U M

3.6 7.2

52m 42

11

3.6 7.2

NEGL. NEGL.

• • • •

ia m in e

2 ,4 -

t h

1A Z O L I U M

analogue

2 ,5 -

t h ia z o l i

um

analogue 2

,

6

-lu tid in iu m

analogue

2 ,4 ,6 -

c o l l

1D 1 N 1 UM

analogue

analogue

O X Y T H 1AMI NE®

HEATED Th

1

12 35

UG

34 35

. . .

6

1 2

BLANKS

AMINE°

2 , 4 - t h ia z o l iu m analogue 2 , 5 - t h I A Z O L I UM n » 2 , 6 - L U T I D 1N 1 UM 2,4,6-CO LLIDINIU M B N E O P Y R 1T H 1 A M I N E B E N Z T H I A Z O L 1 UM ANALOG OXYTHlAMINE

2 0 0

0

124 148

0

1 0 0

0

130

0

3

1 1 0

1 2

47

1 2

0

R e g e n e r a t i o n w i t h t h i a z o l e m o i e t y a d d e d , f r o m 2 0 0 ug T H I A M I N E - E Q U I V A L E N T FOR EA C H A N A L O G U E . THE T H E O R E T I C A L Y I E L D WOULD T H U S BE 2 0 0 UG T H I A M I N E # FOR T H I A M I N E E Q U I V A L E N T OF A N A L O G U E S , SE E S U P P L E M E N T # B) F o r F U R T H E R D E T A I L S ON O X Y T H l A M I N E , SEE S U B - S E C T I O N ( f ) of t h is Se c t i o n # c) U s e d a s s t a n d a r d i n t h e c a l c u l a t i o n o f t h i a m i n e R E G E N E R A T E D I N EACH S A M P L E .

a

L

)

ug

9S

r

i wot

was

13

in vestigated ).

MADE

CLEAR

1. zolium ED

TO

THE

The

BY

THE

OF

THE

EARLIER,

THESE

FLUORESCENCE

I

THE

the

VANOMETER SAMPLES

OF

BLANKS

N OT

THE

SPLIT WILL (

f

)

BY BE

).

ON L Y

IT

CLAM

OF

THIAM INE).

AMOUNT

24

SOME

OF

EVEN

IN

COMPAR­ FROM THE

MENTIONED LESS

THIAMINE.

analogues

(Group

APPRECIABLE

THIAMINE. AFTER IN

IN

REGENERATION

THE

THE

CONTACT

THE

I I ) , THE IN

GAL­

OXIDIZED

DARK.

T HE

HEAT­

DEVELOPED WITH

THE

ALKA­

REAGENT.

OF

REGENERATION IT

APPEARED

PRINCIPLE. IN

A

AFTER

THE

SEPARATE

THAT

INCUBATION OXYTHlAMINE

BEHAVIOUR

THE

BENZTHIAZOLIUM

APPARENTLY

IS

N OT

SPLIT

THE

INVESTIGATION

YEAST WAS

FOR

NOT

OF

SUB-SECTION.

OF

BY

AS

ALIQUOTS

A S

REGENERATION

TIME

th ia ­

SIGNIFICANTLY

YEAST

HOURS

YEAST

the

REGENERAT I 0 N ( IN

OF

WERE

TO

CHAN GE

TO

AFTER

GIVE

MOIETY

STAND

occurred

GREATER

YEAST

BEHAVIOUR

UTILIZED

^URTHER

TO

MUCH

TO

READINGS

NOT

EXTRACT,

DISCUSSED

|T

DID

for

EQUIVALENT

p y r id in iu m

THIAZOLE

VALUES

CL AM

THE

T he

CLEAR. IS

THE

FROM

SUBJECTED

SUBJECTED

THE

ACTIVE

the

BLANKS)

F E R R I C Y A N I DE

FROM

of

WAS

ANALOGUES

EQUIVALENT

ALLOWED

FLUORESCENCE L I NE

AN

case

READINGS

WERE

MOIETY

FLUORESCENCE

(HEATED

PRESENCE

SUBJECTED

THIAZOLIUM

thiam ine

obtained

ACTION

READINGS

THIAZOLE

THAN

IMMEDIATE

SAMPLES

ED

n

NOT

to

CONSIDERATIONS:

readings

YEAST

GALVANOMETER

PRESENCE

2.

AFTER

BLANKS

conversion

FOLLOWI NG

galvanom eter

ANALOGUES

HEATED

THE

THE

That

THE

MADE.

BY

THE

NOT

SUB-SECTION

ANALOGUE

SYNTHESIS

IS

OXYTHlAMINE

(SEE

C L AM

WITH

IS

PRINCIPLE OF

NOT NOR

THIAMINE. j

96

THAT

THE

RESULTS

THE

ANALOGUES

STRATES

FOR

FOR

INHIBITIO N

THE

THIAZOLIUM

THE

PRESENTED

CL AM

WITH

PRINCIPLE IN

THE

NOTED

THE

PROCESS.

IMPORTANCE

THE

TYPE

IN

ITSELF THE

THE

AND

TWO

SUB-SECTION ti

AND I

INHIBITION WHICH

BEING

PRESENTED AMI NO

TO

THE

EARLIER THE

TO

GR OU P S

IN

OF

THE

QUATERNARY

OF

SIMULTANEOUS

THE

IMPORTANCE

D)

The

DEMONSTRATION

THE

2 , 4 , 6 - C O L L I D III I UM A N A L O G U E # -

AND

T HE

FACT

THAT

THIOCHROME

TEST

COMPETE

THE

100

UG

OF

GETHER

pH 3 . 6

THE L

THE and

THAT

AT MENT

THE

WITH

TAINING

SHOWS

IN

4

ALLOWS

COMPOU N D S A

IN

GROUP

DEMONSTRATION

REACTION.

THUS,

TOO

IF

ML

AN

OF

7.2

for

BOTH

A QU E OU S

PORTION two

GLANCE,

SERVE

MODIFICATIONS

AS OF

A

IT

THE

at

ARE

MIGHT

GOOD

0.1

OF

hours

C OMP OU N D S

EXTRACT

BE

LlNEWE^VER

AND

THE

THE

POSSIBLE MOLECULE,

NITROGEN#

DESTRUCTION

THEY

ACTUALLY

UG

OF

ARE

OF

THIAMINE INCUBATED

CL A M THE

POWDER

POWDER

AT

f l u o r im e t r ic

(TABLE

THAT

MEANS BURK

OF

DELAYED

THAT

THOUGHT

EXPERIMENTAL

WITH

CASE)

A

the

DESTROYED

THIS

GIVE

GM OF

30°,

THE

I

2 , 4 , 6 - C O L L I D I N I UM A N A L O G U E

SOLUBLE

FIRST

COULD

THE

IN

THIAMINE

PARTICULARLY

THIAM INE

IN

LINE

AS

BENZ —

EFFECTIVELY

CENTERS

IN

SUB­

REASON

DUE

(SPLIT

ARE

THE THE

ACTIVE

OBTAINED

AS

OF

BE

DE GR A D E D

IS

i

CLEARLY

C A SE

INHIBITOR

THE

SHOW SERVE

THIS

THE

n

MAY

THE

FOR

ACTUALLY THAT

EARLIER.

FACTS

DISCUSSION OF

I

SUBSTRATE

WITHOUT

THEORETICAL

GROUP

THIS

PRINCIPLE,

ANALOGUE,

MlCHAELIS-MENTEN COMPETES

IN

IN

AND TO­ CON­ BOTH a ssa y

X U t ) .

THIS F OR

EXPERI­

CHECKING

TREATMENT

FOR J

97 r

i COMPETITIVE LY

ON

TWO

AT

THE

ED

IN

IN H IB ITIO N

SUBSTRATES

SAME THE

TIM E,

USUAL

THE

SUCH

INVESTIGATION

i f

form atio n duced LAST THE

of

the

because

RATE

OF

THE THE

POSSIBLE

ASSUMPTION IS

E) SHOWN

A ND

AT

IN

A

EXTRACT.

A

3.8

TYPICAL

IS

DIE-AWAY

PRESENTED

EARLIER

BEHAVIOUR

OF

THE

EMPLOY­

S HOWS

OBTAINED

involved

the

in is

process.

X

GRADUALLY

IN

IS

STEP

THE

DISQUALIFY

UP,

SEEMS

FIRST

IN

ACTIVATING

in tr o ­

USED

AND

IS

the

This

INTERPRETATION

N E O P Y R I T H I AM I N E

MIXTURE OF

PR

THE

9.

THESE ITS

PRODUCTS

OF

WH E N

CONTAINING PH

REACTION

AT

TO

BE

MISINTERPRETATION

REACTANT

NO

ADA M S^).

TO

ORDER IS

AS

REACTION

THE

FACTOR

WHICH

TIM E.

CURVE. AS

THE

DATA

REACTION

SIMULTANEOUSLY

REACTION

ANOTHER

THE

MAY

PRINCIPLE

THE

in

FALL

A ND

co m p licatio n

NECESSARILY

X

DETERMINATION

AND

a

AS

HOWEVER,

C LA M -N EO PYR ITH I AMINE PH

CLAM

fa c to r

up

THAT

THAT

NEOPYRITHI A M IN E .

THIAMINE

AT

used

SHOULD

NOT

SAME

DESTROYED

the

SIMULTANEOUS­

OBTAINED

TO

com plex,

is

REACTANT

BE

THE

ACTS

WHITTAKER

RATE-BETERMINING

DOES

THE

SO

SUJECT

SUGGEST

THE

TO

THE

X

ORDER).

SINCE

THAT

DEGRADED

a c tiv a te d

ALSO

HOWEFER,

BE

OBSERVATION

Y£T

IF

a c tu a lly

REACTION

PSEUDO-FIRST

KNOWN,

SEE

MAY

is

WOULD

(OR

NOT

TO

R eactant

STATEMENT

CONTRADICT

IS

X

ENZYME

DATA

BEHAVIOUR.

R eactant

AN

THO RN^j

KINETIC

MANNER

ENZYMATIC

since

WHICH

(SEE

TYPICAL AN

IN

TIME

RESULTS

CLEAVAGE

ACTIVE

TWO

pH

COURSE

DESTRUCTION

THE WERE

BE

WITH CLAM

CURVE

TOGETHER

AND

ALSO

PRESENT

DEPENDENCE

SHOWED

ITS

MAY

OF

OPTIMA, WAS WITH

ON E

A THOSE

REGENERATION PRESENTED

97a

D E M O N S T R A T I ON O F T H E S I M U L T A N E O U S D E S T R U C T I O N OF Th ia m in e and th e 2 , 4 , 6 - c o l l i d i n i u m An a l o g u e b y the clam Pr i n c i p l e .

Procedure: 4 ml of 0.1 GM O F T H E D E S S I C A T E D p H (3 .6 and 7 . 2 ) were u s e WERE

ADDED

TO

the d

extract,

POWDER,

2

. SAMPLE.

EACH

clam

CLAM

equivalent

ADJUSTED

TO

THE

Mc I l v a i n e T H I A M I N E AND 5 9

ml

of

standard

THEN

50

UG

T H E C O L L I D I N G U M A N A L O G U E W E R E A D D E D A ND T H E V O L U M E TO 2 0 M L W I T H W A T E R . T H E R E A C T I O N WAS RUN F O R TWO

30°C. THE

The

reaction

SAMPLES

was

DILUTED

for

An a l y s i s

TO

then

25

stopped

ML.

d estruction.-*

in

HEATED A 1 #0

S A M P L E WAS D I L U T E D T O 1 0 M L . T H E N 3 ML F E R R I C Y A N I D E R E A G E N T W E R E A D D E D AND T H E ATELY. DARK

The

OXIDIZED

OVERNIGHT

A ND

SAMPLE

THE

WAS

READING

THEN AGAIN

I S FOR T H I A M I N E AND T H E S E C O N D F O R ANALOGUE. T H E R E S U L T S S HOWN B E L O W pH 7 .2 • are s im ila r . were s p l i t . at

Incubated

1.

2.

L

the

BLANKS

ml

usual

to D E S I RED b u ffe r

UG

OF

M A D E UP HOURS AT manner

WERE

RUN.

aliq u o t

from

and

each

OF A 0 . 0 2 5 ^ A L K A L I N E READING TAKEN IM M EDI­

ALLOWED

TO

TAKEN.

THE

STAND FIRST

IN

THE

READING

T H I A M I N E AND C O L L I D I N I U M A R E F OR T H E R E A C T I O N R UN

T h e r e a d i n g s f o r t h e r e a c t i o n r u n a t pH 3 . 6 These r e s u lt s c le a r ly show t h a t b o t h a n a l o g u e s

Sample

Table

XI I I

F irst

Re a d in g

Sa m p l e w i t h a c t iv e CLAM E X T R A C T

27

Heated

50

Bl a n k

UNITS

Second

61

Rea ding

UNITS

1 1 0

J

98

r

at

the

116th

1949.

Septem ber, ed

M eeting

SIMILAR

OBSERVATIONS

DESTRUCTION

THE

PHOTOFLUORIMETRIC

MAY

BE

ASSAYED

F)

INCUBATED

WITH

ING

THAT

IT

DID

BUT

SLOWLY*

|N

THIAMINE

THIAMINE

0*1

CLAM

GM OF

REACTION

SHOWI NG

THAT

GROUP

RESULTED

L

IN

OR

REACT

Wh

THE

it e

ALSO

THAT



C ARP

report

­

PRINCIPLE,

MEASURED

PUBLICATION

FACT

HAS

THE

WAS

BY

T HE

N E O P Y R I T H I AMINE

of

20

OF

ML

PRESENCE

THE

OXYTHlAMINE

24

PRIOR

MAY

AT

TO

IS

NOT

FOR

INCUBATED

OR

THE

PH

3 .6

T HE

OF

WAS

BY

THE

REACTANT

WITH

IN

AND

WITH

IT

REACTION

200

ug

by

A TOTAL

pH 7 . 2

WITH

THE

SAME,

REACTION. OF

THE

A HYDROXYL METHLYLENE

X WAS

YEAST

ME A N ­

CONCENTRATION

SBOUT

THE

AS

EXPERIMENT,

REPLACEMENT

MOIETY

ADDI­

REACTED

from

30°C

AT

THAT

WAS

A C L A M - T H I AMINE

T HE

A DEACTIVATION AFFINITY

X,

INH IB IT

THAT

IT

INTERPRETED

destroyed

BOTH

MENTIONED

TO

INHIBITIO N

OXYTHlAMINE

DID

BE

15 TIMES

TO

HOURS

PYRIMIDINE

IS

HOURS

REACTANT

ADDED

OF

THEREFORE THE

EVEN I F

COMPETIVE

TWO

B EE N

REACTION

thiam ine

IN

ALREADY

THIS

WITH

WERE

POWDER

IN

IT

0 X Y T H I AM I N E ( 1 0

EITHER

ELSE

THE

OF

C ity ,

l a n t ic

and

BEING

THEIR

X !).

OXYTHlAMINE

INDICATION

4-AMINO

(TABLE

amount

THE

Sealock

EXTRACT

A TYPICAL

MIXTURE

OR V/1 TH OUT

CLAM

PRESENT)

The

|F

TO

At

at

ACTION

METHOD*

IN HIBIT

NOT

OF

m ixture*

C ARBON

NOT THE

OF

The

THE

S*

N E O P Y R IT H I AMINE

REFERENCE

DID

AMOUNTS

C.

PHOTO F L U O R I M E T R I C A L L Y .

TION

LARGE

ON

OXYTHI A M IN E * -

OXYTHlAMINE

OF

OF

PUBLISHED

a*

Subsequently,

THE

FIRST

the

of

IN

GROUP BRIDGE

LOST.

THE

PRESENCE J

99 r

i OF

THE

PYRIMIDYL

ALCOHOL,

THIAMINE

PRESUMABLY

GROUP

OXYTHlAMINE,

IN

THIAZOLE EXTRACT WHEN IT

MOIETY* AND

THE

BE

RESULTS

SHOWED

SIMILAR

TO

3.6

NE

FROM

OF

and

A

200

UG

PYRIMIDYL

DETERMINED THIAZOLE THROUGH

BY

DURING

THAT

hours

at

I . E . ,

WAS

WAS

USED

WITH

OUT

A

CL AM

FORMED

AND

SPLIT

THE

NEGLIGIBLE

126

SPLIT AS

IS

THE

CONDITIONS

ABOUT

about

THE

PRINCIPLE

IS

TO

REGENERATION,

(UNDER

THERE

30°C,

PRESENT,

THE

IT

4-AMINO

THIAMINE

C L AM

PERIOD

OXYTHlAMINE,

THE

DISPLACE

CARRIED

X II)

CONVERT

INCUBATED

THE

WAS

HOUR

TABLE

YIELDS

Ab o v e

pH

INDUCTIVE THE

MOIETIES

CHECKED

DECALSO

THIAZOLE. IN

THE

1 8 fo ug

AS

AT

of

BOTH

oxythiam i -

SHOWN

BY

PRESERVATIVE

IN

AND

EFFECT

THE

TESTING

THE

SPLIT

LIBERATION

PASSING

THIAZOLE

w ith

o x y t h ia m in e

4-am ino-HC1

the

4-AMINO

THE

BY

IN

THE THE

WAS

C A N N OT OF

REACTION FILTRATE

OBT A I NED

BE

THE MIXTURE

FOR

IN T H E

RE­ EXPECT­

FILTRATE.

results

5,

OBTAINED

REGENERATION.

WAS

ACTIVATED

These

L

ADDED

IS

OF

CANNOT

THAT

TOLUENE

YEAST

MOIETY

GENERATION

OF

YEAST

SHOWS

ORIGINALLY

STUDY.

ABSENCE

TO

EXPERIMENTS.

T he

ED

96

ABLE

TEST

ON

OF

NOT

IS

A 24

SHOWN

In

THE

EXPERIMENT

IN

SPLIT

7.2

REGENERATION THE

THAT

THOSE

T HE

INDICATION AN

IS

QXTHI AMINE

ALCOHOL

SUCH

OF

I.E ., NOW

f

A CLEAR

OXYTHlAMINE.

EVIDENCE

BECAUSE

A SUBSEQUENT

PYRIMIDYL

WOUL D

pH

I

YEAST

OF

GROUP

THE IN

is

are

already

4-HYDROXYL THIAMINE

q u ite

n e u t r a l iz e d

GROUP

WOULD

in t e r e s t in g

BE

IN

;

the

OXYTHIAMINE

A B OU T

.

AND

THE. S A M E .

-J

In

any

case,

the

e ffe c t

on

OF

THE

METHYLENE

C A RBON

WOULD

4 -

am ino

group

in

t h ia m in e

AC T E D

ON L Y

(

the

II)

THE

M ETHYLENE-BRIDGE-CAR8 GN,

(ALSO

T HE

COMPARABLE PYRIMIDYL ER

RATE

INDUCTIVE IS

EXPECT

THAT

A

EFFECT

THE

STRONGLY

THAT

MERE

LAB I L I Z AT I ON TO

BE

BINDING

OF

THE

is

our

AN

Mechanism

E

is

REALLY

LIKELY APPEARS AND

THE BUT

OF L

T HE

CANNOT

LIKELY,

THE

AND

PRIMARY

THE

THE

THAT

Hence

BE

GROUP

THIS

GROUP

R OL E

OR IT

AS

SO

the

AND

ANSWE RED AS

SUCH THE

QUATERNARY

WITNESS

FOUND WITH

THE

ENTER

THAT

RESPECT

FACT THE

TO

THE

AND

TO

A

THUS IN

THE

MECHANISM

CLAM

E

in an

PRIN­

EXERCISED

NITROGEN

IS

AS

IT

YET.

THAT

WAT ER

REACTION.

A STERIC TO

STRONG

BEY ON D

whether

A BINDING,

EXPERIMENTALLY

APPARENTLY

GROUP

BY

THE

postulated

o b servations,

A

PR OC E E D

CENTER

ACCORDING e ffe c t

ON

AT

INDICATES

ROL E

ACTIVE

I

LABILIZING

REASONABLE

IS

A

Gr o u p

HYDROXYL

WOULD

N OT

in

A MUCH

REGION

AN

steric

DURING

GoODLAND1 0

AMI NO

IS

THE

THI AMINE

P OL E

THE

If

SPLIT

EXERTS

PLAYS

(WHICH

the

by

GROUP

ACID

BE

POSITIVE

METHYLENE-BRIDGE-CARBON OCCUR

AND

S H OU L D

WHICH

IN

METHYLENE-BRIBGE-CARBON,

PR I N C I P L E

HOWEVER,

THIOGLYCOLLIC

II)

ALKALINE

4-AMINO

OF

AND

ADDITION,

THE

4-AMINO

SEALOCM

THE

indicated

CIPLE

T H ROUGH

IN

In

EFFECT

OXYTHlAMINE

AMINO

FACT

X ).

OF

DOES

THE

ADDITIONAL

ACTIVATION

I

THE

C L AM

Reactant

THEN

SAME*

analogues

INDUCTIVE

SINCE

ABSENT.

RATE.

APPEARS

T H AN

ELECTRON-RICHNESS

THE

other

4-A M IN O -H C l) ,

SPLIT

VERY

5

OF

A BOUT

the

ITS

GROUP PH

(THE

ALREADY

SLOWER

IN

A B OVE

MOIETY

GROUP,

AT

THROUGH

ANALOGUES

BE

and

AND

degree

THE

DISPOSITION QUATERNARY J

101 r

i NITROGEN BITORS

IN

THE

THEY

USED

(SEE

PAGE

C ARP

PRINCIPLE

THE

)

ACTIVE

GROUP TORY

IN

ACTIVE

THE

he

(

CENTER

THIAMINE

such

AND

as

THEIR

ON

THIAMINE,

PRESUMABLY

T HROUGH

IN

THE

IS

CARP

Th I S

BY

A NITRO

OBSERVATION

THE

IMPORTANCE

OF

FOR

THE

BINDING

THE

presented

AND

THE

INVOLVED

exa m ple

BEHAVIOUR

does

THE

WHEREIN

,

the

WITH

OF

not

QUATERNARY IN

of

THE

T HE

CLAM

OF

S

THE

BLOCKING

GROUP,

OF

AMI NO NO

IN H IB I­

SEALOCK

4-AMINO

course

NITROGEN

of

A T HE

OF

IN H I­

C OMPOUND

ACTION

I f

AND

GROUP

imply

ARE

AS

RING

Se a l o c k

PRINCIPLE

SOL E A

IS

STUDY

VARIED Go o d l a n d )

and

WOULD

that

THE

PROCESS.

PYRIMIDINE S

THE

FISH-PR IN C IP L E •

ACTIVATION

Compound THE

THE

PRINCIPLE.

REPLACED

IN

REACTION.

INHIBITIO N

ANALOGUES

for

IMPORTANT

TO

GR OU P

GROUPS

WAS

C A R P - T H I AM I N E

OBSERVED.

POINTS

MOIETY

S T R ON G

S

evid e n c e

DECISIVE OF

ON

IS

4-AMINO

THE

CAUSED

COMPOUND

EFFECT

T

FOR

CENTERS

GOODLAND^ AN

THIAZOLIUM

BE

OF

INTEREST.

It THAT ON

would

THE

THE

SPECIFICITY

PRESENCE

SUITABLY

L

appear

PLACED

IN

from OF

THE

B ET WE EN

the

THE

observations

REACTION

SUBSTRATE TWO

AMINO

OF

in

th is

APPARENTLY AN

ELECTRON

S ection

DEPENDS POOR

ONL Y

CARBON

GROUPS.

J

102

P A R T M l.

CONCENTRATI ON

T WO

OF THE T H 1 A M I N E - O E S T R O Y I NG P R I N C I P L E

IN CLAM. -

ONLY THE

OUR

INVESTIGATIONS

ONE

FACTOR

THE

ARE

FACT

STUDY THE

(PRESUMED

DESTRUCTION

FACTS

OF

FOR

PRODUCTS

LARGELY

BY

THE

NATURE

APPARENTLY

CHARACTERISTICS OF

THE

STRONGLY

PRESENTED

ONLY ON

STRENGTHENED

THE

ALLY,

b

)

c)

TO

THERE

AN

STATED

DETERMINATIONS OF L

DIFFERENT

FIND

OUT

TWO X

can

be

OF

OF

OF

OF

AS

NOT

OF

THE

KINO,

OF

THE

THIAMINE

AND

WELL

UNDER

THE

AS

OF

DIFFERENT

CLAM

THE

IN

OBSERVED

REACTANT;

DIFFERENCES

AND

THE

THIAMINE

REACTION

THIAMINE

OBSERVED

THE

STUDIES

WH E T H E R

ARE

OR

SYSTEM

PH 3 , 6

AT

RESULTS

ANALOGUES

AND

JUST

H A VE

from

"PYRIMIDINE

ON

MAY

INVOLVED

BE

FORMED

ARE

PRINCIPLES;

a c t iv a t in g

COMPLEs"

PRODUCTS

THIAMINE

an

FACTOR

INTERPRETATIONS

CONCENTRATES

SPECIFIC­

NOTJ-

A SE COND

REACTION

DESIRABLE.

THIAMINE-SPLITTING

separated

OF

THE

EARLIER, OF

THE

INVOLVED

SYSTEM,

A COMPETING

PROPERTIES

DISTINCT

INDICATION

BREAKDOWN

AS

DEGREE

IS

THAT

INDICATION#

ARE

Re a c t a n t IF

THE

CONCENTRATION

WE W I S H

A)

THE

BEHAVIOUR

THIS

HOWEVER,

AND

THAT

OF

OF

DESTRUCTION

INDICATE

ARE

IS

ASSUMPTION

X) ,

REACTANT CL AM

CONDITIONS,

7 ,2

BE

THE

THE

REACTION

PR

TO

FAVOUR

BY

WAT ER

THE

FAR

THIAMINE

EXPLAINED THAT

SO

RUN

CAN

BY

IN

BE

MADE

fac tor

;

THE

OBTAINED.

ONLY

THE

IF

ACTION

CONCURRENTLY J

103 r WITH

-i ACTIVITY

There

has

OF

c entra tion

CASES

CITED,

FORMED. ABLE

DETERMINATIONS.

THE NO

AGREN

TO

been

19

OBTAIN

WITH

FULL

ACTIVITY

FROM

AN

AT

PR

BY

BY OF

A QU E OU S

USING

THE

THE THE

TREATMENT

CIPITATIO N FURTHER

work

MADE

PRECIPITATE SULFATE

WAS

DISSOLVED

ASSAY

WITH

FRESH

EXTRACT. OF

DIFFICULTIES

AND

CENTRATION

(60-100^

SATURATION).

CIPLE

THAT

WAS

OBTAINED

t r if u g a t io n

FOUND

BY

DUCIBLE CATION STUDIES L

A HIGH

and

AGREN EV EN

IN

OF

TO

BE

IDE

HAS

NOT

BEEN

OF

THE

PH

d ia l y s is

INAPPLICABLE ITSELF.

AGREN OF

.

THE

ME A S U R E D

THE ALSO

ALCOHOL

AGREN

DUE

THE

P R OG R E S S MADE

NO

BECAUSE

IDE-PRIN-

was

THIS

MENTION

I D E - T H I AM I N E

CON­

AGREN^

SELDOM ON

OF

ULTRACEN-

method

AND

BUT

TO

R ANGE

PAPER, OF

PRE­

EXTRACTS

DIALYSIS, T he

REGAIN­

ISOELECTRIC

LATER

TO C A R P

FURTHER

REPORTED.

DEPENDENCE

OF

WAS

AL.

A WIDE

PURIFICATION

A COMBINATION

c ataph o r etic

A

REGAINED

PREPARED

WAS

BY

WAS

SATURA­

PRINCIPALLY

OV ER

IN

BUT

ACTIVE

YIELDS

ALL

PRODUCTS

FULL

AND

REPEATED

PRECIPITATED

DEGREE

BY

ACETONE

IN

­

SULFATE

ACTIVITY ET

con

VISCERA,

DIALYSATE

YIELDED

LOW

WAS

BY

ACTIVE

A B ANDONED

MATERIAL

IDE

PRECIPITATE

SULFATE

AND

AMMONI UM

THE

SUCCESSFUL.

E X P E R I M E N T A T I 0 N WAS

OF

S E A LO Ck "*5

SULFATE

AMMONI UM

BY

the

REACTION

OBTAINED

G L U T ATM I O N E .

NOT

THE

SLIGHTLY

IN

MET HOD

AMMONI UM

HE

on

PRINCIPLE

EXTRACTS

ACTIVE

REPORTED

reported

ON

PURIFICATION

A QU E OU S

WAS

WITH

EXPERIMENTAL THE

,

WHEN

FRACTIONATION

THE

WERE

UNTREATED

ACTIVITY ED

STUDIES

AMMONI UM

7.4

l it t l e

THIAM INE-SPLITTING

10-FOLD

PRECIPITATION. TION

very

however

REPRO­ PU R IFI­ OF

REACTION

NOR J

104

OF

THE

I

REACTION

n

our

SEPARATELY ITY. Ma

N

and

AND

AT

itrogen

unless

orating drying

THE

T HE

THE

Z uazaga

basis,

F ORME D

concentration

USED

cedures

PRODUCTS

work

RESULTS

END

OF

.

The

dry

o therw ise

SOLUTION

TO

AT

v is io n a lly

DEFINED

AS

THAT

DESTROY

CHLORIDE-HYDROCHLORIDE 2

ML

OF

MClLVAlNE

TERMINED

IN

SUGGESTED

THE

SEALOCK**5

BY

A TEMPERATURE

C LAM

37.5°

HAVE

The

FOR by

THE

10

ML

OF

ET

AL.

AT

PH 7 . 4

on

a

pro

CLAR­ of

dialyzed by

evap­

is

PRINCIPLE

pro­

WHI CH

UG

OF

THIAMINE-

SOLUTION

AT

PH 7 . 2

THE

DESTRUCTION

DESCRIBED.

USING IS

842

UG

NOT

SUITABLE

USING

BEING

(THE

OF

­

SUBSEQUENTLY

OF A C T I V I T Y

FROM 1 0 0

PREVIOUSLY

AND

OF

method

determ ined

UG

30°,

the

are

70-30°

OF

SAKE

always

50

AT

THE

the

ASSEMBLED

are

UNIT

A MOUN T

BEEN

,

DE­

UNIT

THIAMINE

AND

FOR T H E

PR I N C I P L E . )

CASE THE

OF

IN

BUFFER

MANNER

w eights

DESTRUCTION.'

p r e se n te d

made

DRYNESS

100°.

WILL

were

THIAMINE

be

SECTION

AND

at

H OU R S

to

stated,

residue

TWO

THE

OBTAINED

THIS

the

IN

now

d e t e r m in a t io n s

46

IN

I.

a c id

PRELIMINARY

CLAM

POWDER

TISSUE MINE

ALSO

OF

PRODUCTS CRUDE

EXPERIMENTS WH OL E

SUSPENSIONS

BUT

EFFECT

OR

Pu r i f i c a t i o n . -

IN

CLAM

NOT

THE

FORMED.

TISSUE

SUSPENSION

OR

AN

TISSUE

MERELY

AND

ACID

SUSPENSIONS FROM

THAT

REACTION

ACCELERATORS

THE

I t

IN

IN

BEHAVED THE

no ted

OF

THE

SIMILARLY

AS

CRUDE

SPLIT

THIA­

TO

CHARACTERISTICS, THE

NATURE MADE

EXTRACT

OF BY

PROPERTIES,

THE

REACTION

TREATING

EXTRACT OF

in

EXTRACTS

ABILITY

CENTRIFUGED

A QU E OU S

alr e a d y

AQU EOU S

EXTRACT, OR

was

THE

T HE

FROM

CLAM

THIS

POWDER

105 r

WITH OFF TO

pH

3

THE

M cIlvaine

INACTIVE

BEHAVE

IN

A

POINT OF

WORK

FOR

T HE

FULL

THE

TYPICAL

g iven

STABLE

AT

ia l y s is

OF

WHOL E TION

.

The

ACTIVE

from

grude

I I .

GA V E

GM OF

3.6

0.04 AND

ME A S U R E D

TO

ac id

7.2 BY

THE

THE

THE

WHICH

On e

THE

BUFFER

PROCESS

BUT I S

KEPT

|F

made

IN

THE

RAPIDLY

THE

and

COLD.

a d d it io n

ACID

EX­

IS

IMPROVED

by

g r in d in g

DIRECTLY.

PERHAPS

IS

BECAUSE

the

INACTIVA­ OF

THE

EX­

OCCURS.

BE

gram

Powder

MADE

of

INTACT

from

FROMTHE .

The

AND

d e t a il s

THE THE

REGENERATION

the

acid

E x tra c t. -

AC t D E X T R A C T ,

fresh clam

W H I T E P O WD E R .

YEAST

SUPERNATANT.

a c t iv it y

be

OPERATION,

completely

STABILITY

cannot

ACID

s u s p e n s io n

A

of

IF

REACTIVATION.

A Dessicated CAN

is

FOR

AMOUNT

EXTRACT

TEMPERATURE

loss

7 .2 ,

SIMPLE

ACID

BUFFER

A STARTING

LARGE

OPALESCENT

extract

ROOM

to

extract

REMAINED T HE

NO

PH

IN

t is s u e

Pr o c e d u r e

leads

CAUSES

POWDER

BY T H E

1

F OUND

SUITABLE

AS A

WAS

TH I S

NOT

BECAUSE

HOURS S T A N D I N G E V E N

DURING

I I .

THE

a c id

OUT,

ATTRACTIVE

WORK

IN

WAS

.

cen trifug in g

RESPECTS.

EXTRACT)

PREPARING

T he

then

SEPARATES

R EMOVE D

H OU R S AT

24

FOAMING

CASE

FOR

I.

8

TISSUE

OC C U RS

WAS

REMAINING

ADJUSTED

CLAM

TENSIVE

AN

ON

AC I D

and

ALL

PARTICULARLY

PROCEDURE

LEAST

IN

PURIFICATION

in v a r ia b l y

IS

somewhat

WAS

PRECIPITATE

ACCELERATORS

TRACT

CALLED

Pro cedure

INACTIVATED D

BUT

ACTIVITY

in

WHICH

SI M l L A R F A SH I ON

FURTHER

INACTIVE

buffer

PRECIPITATE

EXTRACT(HENCEF0RTH ROUTINE

standard

are

g iven

(

wet

ACTIVITY

AT

t is s u e

REACTION MET HOD

PREPARED in

b a s is

)

BOTH

PH

PRODUCTS

AS

WERE

THE

SAME.

106 r FURTHER

STUDIES

WERE

N OT

MADE

ON

THE

IMPROVEMENT

OF

THE

1

METHOD*

CASE TATION ACID

III.

BY

ALCOHOL

EXTRACT,

SOLVENT

WAS

ACETONE WHILE

ON

CASE

However,

ing

THE

2

TO

CENTRIFUGE 60

TO

70%

REGAINED

A

PREPARED

FROM

WAS

60%

using

DOWN

WERE

t r a c t

BUT

A

ACID

fu rth e r

CARRIED

A QU E OU S

THE

SUCCESSFUL AS

AS

HIGH

ON

STANDING

IMMEDIATE

PRECIPI­

EXTRACT

EV E N

PRECIPITATES

8 0 fb

L OS S

THE

OF

OR

THE

WH E N

AND

OBTAINED

WITH

THE

WITH

SOLVENT

ACTIVITY

SLIGHTLY

OU T

AT

THE

OF

CAUSES

IS

ALSO

ACID

EXTRACT

AT

BOTH

EXTRACT

IS

REGENERATION

WAS

MADE.

ROOM T E M P E R A T U R E .

YIELDS,

TO

OBTAINED

AT

A

3.6

STARTING AND

7.2

ADDED.

THI 0 -

THE

of

A CTIVITY.

the

acid

ex­

PRECIPITATION ACTIVE

stand­

I.E .,

DlALYSATE

THE THE

of

WHEN

OF

s a t u r a t i o n

comes

DIFFICULT

AS

PH

pre­

p e r io d s

GOOD

THE A C T I V I T Y

f u l l

SULFATE

FOR

not

Be l o w

m aterial

long

PRECIPITATES

ACTIVE

A QU E OU S

s tu d ie s ,

AMMONIUM

THE

is

active

REQUIRED

PRECIPITATE

USING

MEASURE

the

S u lfa te .-

clam

in

s atu ratio n ,

ARE

FILTER.

ALSO

Ammonium

p rin cip le

60-70%

THE

FRESH

w ith

satu ratio n ,

(4-8^)

OR

LARGE

WITH

THE

ALCOHOL,

SATURATION

GLYCOLLIC

NOT

RAPIDLY

active

WEEKS.

MATERIAL

For

the

ICEBOX 4

USING

CONCENTRATION

-1 0 °.

OF

Above

down.

FROM

WERE

P recip itatio n

saturation,

in

IN

F R A CT I O N A T I O N . -

ALCOHOL

PRECIPITATION*

IV.

c ip ita te d .

5.7

WAS

CASE

AND

ACETONE,

ACTIVITY

THE

OC C U R R E D

PH

U SE D

LOSE

IN

AT

OR

PRESENT

TEMPERATURE

50%

ACETONE

MATERIAL

BUT

PRECIPITATED ENOUGH ING

TO

PRECIPITATED

BUT

of

t h e

D U CE D

BY

FEW

TION

OF

WA T E R IN

PH

HERE

SUGGESTED THEY

IN

PRESENCE CRUDE

AGO,

AND

FRESH OF

THEY

ONE

PH 3 . 6

ACETATE

THE

THE

BEING

AN

|N

MUSSELS

PREPARED WHOLE

A N OT H E R

OBSERVED AND

BY

ARE

AFTER

PH 6 . 5 .

WAS

­

AL LOWED THOSE

PRO

X V I1)•

(TABLE

T HE

FACTORS OF

FRESH

SIGNIFICANCE PRESENTED ET

REDDI

A L,2^

PH 6 . 5 )

AND

THE

BY

OF

WHICH PRIN­ THE

ENZYMES. AN

T H ROUGH

A

SEPARA­

IN

THEREFORE

FILTERING

6 .5 ,

PHOSPHATE

r e

INDICATIVE

BE

MUSSELS

pH

The

THIAMINE

PREPARATION AT

XVI.



THIAMINE-SPLITTING

THAT THE

BUFFER(0.2M)

MIGHT

degree

A L U M I N A C QaMMA#*

PAPER,

OF

L A RGE

The

SAME A S

ON

( PH 3 . 6

DESTRUCTION

A

WAS

.

REPORTED

ARE

EARLIER

pH O P T I M A

MACERATED

AND

REPORTED

THE

REGAINED

EXTRACT.

GlRI

OF

BUT

THE

20

STAND­

PRECIPITATE

T able

ADSORPTION AND

DETAILS

CONCENTRATE

A QU E OU S

BY

DISTINCT

F OUND

THE

LONGER

d i a l y s a t e

on

OBSERVATIONS

TWO

EXTRACTS

OF

shown

RESULTS

WA T E R

TWO

NIGHT,

ACTIVITY

IN

T.2

WAS

THE

THIAMINE-DESTROYING

THEIR

THE

IV.

ESSENTIALLY

REDDI

DETAIL.

TISSUE

AT

THE

THAT

SUSPENSION

OF

THE

fresh

WHEN

WERE

DISTINCT

OBSERVED

CIPLE

is

STANDING

ACTIVITY.

AND

a d d in g

OBTAINED,

ACTION

SOME

3.6

o b t a in e d

MONT HS

WORK

IN

on

HOURS

MATERIAL.

AT

MUSSELS.

OUR

THE

ACTIVE

PUR IFIC A TIO N

TWO

OF

PROCEDURE

THIAMINE,

CASE V . A

INACTIVE

a c t i v i t y

THE

MOST

FOUR

IN

PRODUCTS

DESTROY

WELL.

GIVEN

p u r i f i c a t i o n

a c t io n

TO

ARE

SLIGHTLY

pa rt

BUT

PRECIPITATE

PROCEDURE

of

S L OWL Y

SHOWED

USING

A QU E OU S CLOTH TWO

OVER­

PH

SHARP D E C R E A S E

OPTIMA IN

ACETIC-ACID-SODIUM

BUFFER(M /l5)

WERE

USED

108 r IN

THE

DETERMINATION

OBSERVATIONS a

p

H

6

°

T he

p

H

ALSO

3 .6 -

THE

PH

DEPENDENCE.

THE

FOLLOWING

MADEJ-

t h ia m in a s e

. 5-th tam in ase( 30

for

8

)

WERE

OF

Heating

is

the

more

stable

buffered

to

heat

than

suspensions

at

the

70°

m in u tes)*

b) for

The p H - 3 . 6 - t h ia m in a s e is more s t a b l e 1 5 d a y s t h a n t h e pH 6 . 5 - t h i a m i n a s e .

on

standing

at

c ) T h e pH 3 . 6 - t h i a m i n a s e r e q u i r e s no c o - e n z y m a t i c p rin ­ c i p l e w h i l e t h e pH 6 . 5 - t h i a m i n a s e lo s t 50^ of it s a c t i v i t y ON

D IA LY S IS .

d ) A l u m i n a C q amma a d s o r p t i o n : - b o t h t h i a m i n a s e s w e r e a d ­ s o r b e d b u t u p o n e l u t i o n w i t h p H 7 . 2 o r pH 6 * 5 M / l 5 p h o s ­ p h a t e b u f f e r , o n l y t h e pH 6 . 5 - t h i a m i n a s e w a s e l u t e d .

In THE

other

a l l

TWO

THIAMINASES

0.2M

USING

THE

ACID

AFFECTED BY

BY

ON

A

ION.

TO

TISSUE

3-4

NO

STUDIES

UCTS

THE TO

THEIR ML

MG

being

LOW

THE

ON

THE

HYDROLYTIC

assumed

to

be

GM

OF

WERE

ACCELERATED

WATER AND

FRESH A

ML

OF

NATURE

OF

THE

e f f e c t

WE R E

SIMILARLY

S H O WE D

OF

FIVE-FOLD

WERE

20

IN

s tic s ,

THIAMINASES

CLAM

MECHANISM

in

GAVE

FRESH

TO

EXTRACT

THIAM INE

REPORTED

THE

1*0

TO

FILTERED OF

OF

BOTH

i

PURIFICATION

BUFFER

THIAMINASES AND

character

ACID

BOTH

COMPARED

0.8

DATA,

OF

TWO

ACTIVITY

BE

LEVEL

WERE

PRODUCED,

Wo o l l e y

BASIS.

INHIBITORS

ACCORDING

20

WEIGHT

ACETATE

INORGANIC

APPEARS

AT

DRY

reaction

SIM ILARLY*

THE

PRINCIPLE

TIMUM

BEHAVED

as

EXTRACT.

MANGANOUS

IN

such

ACETIC -AC ID -SO D IU M

PURIFICATION IN

respects,

MUSSEL

CARP,SINCE MUSSEL

SUBSTRATE REACTION

MIXTURE.

REACTION

KRAMPITZ

OP­

PROD­

AND

. Q

In

the

L T I O N A T I ON u

second STUDIES

paper, LEADING

Reddi TO

AN

and

Gi r i

ACTUAL

published SEPARATION

OF

fra c THE

I

r TWQ

THIAMINASES

AMMONIUM pH

6 .5

SULFATE

WITH

T R A F I L T R A T I ON

N OT

ON

of

ADDED

the

LOSS

ACHIEVED

ADDING

AN

EQUAL

ACETATE

BUFFER

TO

THE

w ith

PHOSPHATE

THE

C gamma*

A lum ina

BUFFER

pH 3 . 6

ELUTION

OF

PURIFICATION MENTION (WHICH

WAS

OF

WAS

F OR

Wang

T he c a tio n

w ith

REQUIRED

THE

WITH

OF

UNDER

OF

20

and

and

H a rris

thiochrom e

THE

CL AM

2

eddi

a l

PRINCIPLE

.

OF

pH

WAS

TREATED

7.2

M /l5

COMPLETELY,

SUBSEQUENT

A 60-FOLD

TRIED.

NO

PH 6 . 5 - T H I AM I N A SE THE

PRINCIPLE)

THE

DIALYSATE.

AMOUNT

OF

CONDITIONS THIAMINE

the

TWO

. 5 - T H I AM I N A S E .

FRESH

WAS

ACID-SODlUM

1 UG OF T H I A M I N E

pH 6 . 5 ,

et

6

THE

"THAT

UG

pH 3 . 6

of

OF

OPTIMUM

a t

M E T H OD

or

THE

NOT

PH

ADDED

DESTRUCTION 37°

6.5

T HE

RE­

EXTRACT,

EXTRACT,

CO-ENZYMATIC

AS

AT

ACID

T H I AM I N A S E

WAS

THE

DEFINED

3.6-A C TIVITY OF

UL-

WAS

BUT

assay

IN

being

THE

ENZYME

AFTER

ONE

OF

SUB­

20

ML

made

OF by

m ethod".

was

BUT

a p p lie d

THE

to

RESULTS

the

WERE

p u r i f i

NOT

AT

d e te r­

MENTIONED.

ACETIC

BEHIND.

ELUTION A

pH

pH 6 . 5

THI AMINASE

CONCENTRATION( I . E . ,

s o lu tio n ) the

E lu tio n

AFTER

WAS

Q.2M

OF

FILTERED

DETERMINED

HYDROLYSIS

STRATE

IF

ACTIVITY

REQUIRED HOUR

MADE

THE

W IfH

ACTIVITY

A c tiv ity

SEPARATION

FOLLOWS:

FOR

THE

NOT

pH

THE

AQUEOUS

OBTAINED

APPARENTLY

ACTIVITY UNIT

WAS

FILTRATE.

THE

OF

a c t iv it y .

WERE

SATURATION

PH 6 . 5 - A C T I V I T Y

THE

VOLUME

FULL

50j b

OF

DIALYSaTE

REMAINING

PH 3 . 6

THE

3.6

pH

AS

REMOVED

T H I AMINASE

LOSS

DISTINCT

A

BY

MUSSELS.

A

OF

THE

BACK

FINALLY

WAS

IN

FRESH

CAUSED

AFFECTED.

WATER

RESULTED

ADDING

THIAMINASES MADE

FRESH

2 5 f0 l o s s

and

m inations

GAINED

IN

­

GOOD.

no r T he

p u r if ic a t io n

BUFFER OF

DID

NOT

INACTIVE

C GAMMa

A L..

MATERIAL.

BUFFER, The

RECOVERED

THAT

IN

THE

THE

and

5

acetic

CAUSED

ACCORDING

TO

THE

WAS

DETERMINATION,

EXTRACT

MEASURED

WAS

AT

PH

BOTH

GIVEN

REMOVtL ALUMINA

7.2

7.2

PROVIDED

DIALYSaTE

WITHOUT

THE

7 .2

AND

M /l5

REDDI

AND

LOW),

FRESH

3.6

acetate

BY

PH 3 . 6

WAS

ADDED.

pH

w ith

BOTH

RECOVERY

sqdium

S U B J E C T E D TO

METHOD

AT

-

NEGLIGIBLE

e lu tio n

MEASURED THE

a c id

ONLY

EXTRACT

subsequent

A CTIVITY

A C TIV ITIES

H

THIS

(ALTHOUGH

AQUEOUS

p

AND

A C TIVITIES

WERE

UNTREATED

the

WORKWELL

ad sorptio n

PHOSPHATE ET

w ith

F ROM

DIALYSATE,

WE R E

INAPPRE­

CIABLE.

A (THE

MODIFICATION DETAILS

OF

M O DIFICATIO N, P O WD E R INOUS

WAS

c ip le

C OUL D

PHATE

L

ARE

GIVEN

THE

BE

WI TH

BUFFER, GA V E THE

CONTAINS

CAREFULLY

SATURATION. POWDERY

OUT

WITH

AND

OF

THEN THE

MASS

PH

AC I D

ELUATE,

TREATED ACTIVE

WITH

WAS

THIS

BUFFER

AND

THE

OPALESCENT WITH

OR

PH

pH

BUFFERS IS IT

WAS

COMPLETELY

7.2 DID

NOT WAS

ACID

p rin ­

STANDARD

8

M /l5

VOLUM­

ALUMINA

active

AMMONI UM

MATERIAL

IN

CLAM

The

WHICH

A C TIVITY.

'/) *

MADE,

THE

TREATED

7.4

WAS

FROM

THE

RECOVERY.

THE

WHICH

EXTRACT

occurred.

WITH

HIGH

7.4

PROCEDURE

OFF.

LOW R E C O V E R Y . pH

PROCEDURE

3 M clLVAlNE

adsorption

MOST

AND

pH

PH 5 . 2

TO

IN

AQUEOUS

CENTRIFUGED

ELUTED

ACTIVITY.

WHITE

TURBID

BROUGHT

BUFFER

CLKAR,

A

WHICH

Com plete

MclLVAlNE

FULL

ADSORPTION

TREATED

WAS

Cqamma*

5.6

THE

PRECIPITATE

EXTRACT

THE

OF

PHOS­ N OT

ELUTE

A L MOS T

WaTER-

B ROUGHT SULFATE

TO TO

PRECIPITATED SOLUBLE

IN

PH

AS

Wa T E R . J

m

At

each

step

in

change

in

the

pH

3.6

to

that

AS

IN

THE

THIAMINE

the

ra tio

OF

BOTH

CONCENTRATES

of

the

destroyed

RATIO AT

procedure,

THE

PH

there

amount pH

at

(TABLEXIX).

7.2

POWDER

IS

ADDED.

IN

THE

ABSENCE

FERENT

fractions

are

but

s lig h tly

p rin c ip le .

The

38-fo ld (b ased from

the

We

have

Reactant

the

X

above)

X

seen

nor

a

above.

to

There The

(Mechanism

PYRIMIDYL

ALCOHOL

resu lts

o ffer

pressed

EARLIER

THAT

DO

N OT

PRODUCTS

REPRESENT

is

E ).

no

THE

active

REASONABLY

DISTINCT

of

AND

does

en ter

the

T h iACTIVE

about made

not

con

­

in to

HENCE,

d iffe re n t

change

o p e r

observed

te n ta tiv e Icthiam ine

for

OPTIMA AND

DIF­

in

reaction to

the

opinion

ex­

CONSTANT.

support PH

THE is

THE

THE

pH.

THIAMINE.

the

OF

XX.

may

WITH

ra tio

FRESH

being

DIALYSATE

w hich

OF

obtained

Table

complement

TWO

DIFFERENT

both

calcu latio n on

w ell

EXTRACT

at

at

FROM

DIALYSaTE,

throughout

(ICTHIAM INE

TWO

THE

s ig n ific a n t

strong

THE

REACTIVATION

the

The

as

F ORMED

A QU E OU S

OF

REACTION

remain

STAYS

The

END

t h a t

su b s ta n c e

resu lts

mechanism

TINCT

shown

DISPLACEMENT

appears

system.

as

and

X Y 1 11)

OF

1

distroyed

DETERMINATION,

p u rific a tio n

w eight

dry

a lr ea d y

N UC L E O P H I L I C

ations

on

of

THE

thiam ine

ACTION

ORIGINAL

CAUSES

degree

eluate

Re a c t a n t

t a in

AL SO

AN

s ig n ific a n t

PRODUCTS

EACH

PREPARED

ACID

FROM

IN

no

(Table

THE

BY

DIALYSATE

OGLYCOLLIC

of

PYRIMIDINE

VALUES

is

THE

the

AND

THE

TWO

PYRIMIDYL

SEPARATE

D IS­ ALCOHOL)

REACTIONS

­

112

r ACT I V a T E D FACTS

ARE

THIAMINE WITH

A

OF

EXPLAINED BASE

of

AND

PRINCIPLES.

LARGELY

THE

FACT

COMPLEX

No

THIAMINE.

OBSERVED

IN

I C T H I AMINE

REMAINED

L

SEPARATE

RELATIVELY

CLEAVAGE WAS

BY

qUlTE

THE

FORMED

BY THAT

THE

MOLECULE, INDICATION

THE

TOTAL

THE

D t-A C IDIC

WAT ER

CONCENTRATION TO

RATHER,

OBSERVED

NATURE

APPARENTLY

REACTANT OF

WORK

X,

THIAMINE

THE

COMPETES IN

A S E C ON D ABOVEJ

OF

“1

THE

THE

F A CT OR RATIO

DESTROYED

CONSTANT#

Jf

113

PROCEDURE D E S S I C AT ED

POWDER

MET HOD

TO

GIVE

44-55';b

OF

TOTAL

TRACT,

4

SLOWLY

AND

ML

IMMERSED BECAUSE

The

A

32

OF

THE

FOAMING

addition at

TURE.

CORRESPONDS

TO

RESULTS OBTAINED

OF

3

BUT

a

c a t io n .- 1 .0

W/ . T ER

FOR

The

TWO

DILUTED

TO

IS 32

IS

ARE

GIVEN

IN

TaBLE

XIV.

IS

GIVEN

IN

TABLE

XIVa*

OF

buffer

IN

AT

DISCARDED. TYPICAL

EX­

added

THE

is ROOM

THIS

P OWD E R .

ABOUT

A

FUNNEL

UNDESIRABLE

DECANTED ML.

THE

THROUGH IS

the

USUAL

is

m ixture

MINUTES

ORIGINAL

A C TIVITY,

ML

LOSS

of

EXTRACT,

STIRRING

SUPERNATANT

THE

16

PREFERABLY

RESULTANT

gm

BY T H E

AQU EOU S

lv a in e

m inute.

RPM

GM OF

LITTLE

Mc I

f i

To

VIGOROUS

WITH

FINALLY 0.5

WITH

STIRRING

about 1000

t

DISSOLVING.

standard

OCCURS

d

CENTRIFUGED

OPALESCENT AND

HAS

ML

EXTRACT.

A B OU T

T HE

EXTRACTED

GENTLE

took

CHEESECLOTH

WHICH

H

Aci

by

IS

SOLIDS p

WITH

IN

trifu g ed

L

P u rific a tio n

t.

A C TIVITY.

then

cen­

TEMPER-

T H RO UG H VOL U ME THE

RESIDUE,

TYPICAL

PURIFICATION

J

113a

r Table ACTIVITY

OF

XIV

DIFFERENT

CLAM

P r e p a r a t IONS

PREPN

Vo l . E x t . E Q U I V . TO 0 . 1 GM OF POWDER

Clam POWDER Aq u e o u s Ex tr a c t A C 1D Extract

32 ML

Total SOLIDS

Ash p e r W_n p e r g m GRAM—E Q U 1 V * E Q U 1 V OF OF POWDER EPOWDER

Ac t i V I t y PER G M E Q U I V . OF POWDER

1 .0

0 . 0 4 6 2 gm : * - 0 . 0 8 2 4 gm

10 U N I T S

GM

0 .5 1 2 **

*0 .0 3 0 5

0.0335*

9 .5 -1 0

EL

0.1773

64

:-0 .0 2 1 5

0.002 *

8-9

¥

ft*

The ash is g r e e n is h b l u e . T h e c o l c RED S U B S T A N C E I S w ater-so lu b le. T h e w h i t e r e s i d u e r EM A IN IN G INSOLUBLE IN w ater i s s o l u b l e i n HC1 AND s u l f u r i C A C I D . NO F U R T H E R 1 D E N T I F 1 CA T 1 ON M A D E . * * U N D 1A L Y 2 E D B A S I S , I . E . , T H E AMOUNT 0 F T O T A L S O L I D S 1 N T H E A QU E OU S E X T R A C T Wa S D E T E R M I N E D W I T H OUT A P R E V I O U S E X H A U S T I V E D I A L Y S I S OF T H E E X T R A C T AG A I N S T D I S T I L L E D Ho 0 . .

T able Degree

Prepn

UN l T S

OF ACT!VITY PER GRAM TOTAL S O LI DS

Crude C lam Susp .

De s s i c . Powder

6

.

eL

X IV a

of

Pu r if ic a t io n

by

A c id

Ob t a i n e d

Pu r if ic a t io n

U N I T S OF Ac t i v i t y PER GRAM N 1 T R OGE N

N .8*

1 GM C L AM POWDER E Q U I V . TO 7 . 1 5 GM w h o l e : t i s s u e . T h is amount o f WHOLE T I S S U E ON A V E R A G E CONTAINS 1 . 6 1 GM T O T A L S O L I D S ( U N D 1ALY2ED)

. c. 5

1 0 . 0

1 2 1

18.55

306

50.9

425

aqueous

Ex tr a c t AC 1 D Extract

■Li

114 r

i

PROCEDURE

1 1* SuspensION. 100

THE

ML

USUAL

OF

CRUDE

MANNER FOR

ATURE*

TURBID

The

OF

standard

OBTAINED

AVOID

IMMEDIATELY* OF

ICE-COLD

VALS

OF

MIXTURE IN

A

IS

SLOW

EXTRACT FIRST

ML

N OT

VICE

A TOTAL

OF

THE

PRECIPITATED

IN

THE

COLD,

ED

WITH

OF

AND

SUCTION m a te r ia l

IS

OF

is

ML

OF

WAS

THE

DRIED

IN

ADDED

4 -5 °,

the

20

T WITH

N a OH C A R E F U L L Y

IN

IN

25

THE

TO

4-5°

ML

PORTIONS

TO

ADD

IS

AVOID

ALL

POWDERY

VACUO

OVER

water

150 AT

INTER­

IS

THE

A DDE D TO

OBTAINED

THE

WITH

THE

NECESSARY

INACTIVATION

THE

ACETONE FIVE

MATERIAL

WAS

MINUTES WAS

PHOSPHORUS

so lu b le

USED

ML

ACETONE

FOUND

FOR

IS

ADDITION,

THE

TO

STAND

ACID

PORTION

PRECIPITATE

TO

AND

EACH

2 r -ML

AFTER

THE

EXTRACT(tV)

UURI NG

ML

extract

I.

ACETONE

completely

AT

acid

WAS

WHITE

ROOM T E M P E R ­

NOW

IT

AL L OWE D

AT

WAS

T HROUGH

BUT

MATERIAL.

THEN AND

ACETONE,

TISSUE)

FILTERED

and

IMPORTANT A

IN

PROCEDURE

EACH* WHILE

Tissue

WAS

COOL ED

ADDED

RPM

of

PREPARED

GM WHOLE

NEUTRALIZED

VERSA.

150

MIXTURE

PH

I V WAS

WERE

IT

ADD

THE

THE

GENTLY

PORTION

ADDED,

TO

HALF-MINUTE

STREAM.

25-ML

dry

OF

IS

buffer IN

Extraot

1000

(it)

E X T -1I

SHOWN

EXCESS*

STIRRED

AND

OF

B R OU GH T

TO

The

ML

1.0

A B OU T

SUPERNATANT

ACETONE

ABOUT

AT

acid

SUSPENSI ON( I )

CONTAINING

MINUTES

MANNER

25

from

TISSUE

M cIlvain e

IS

LOCAL

ML

60

3

THE

(It I)

EXTRACT TO

TO pH

IN

15

Powder

CL AM

(4

CENTRIFUGED

CHEESECLOTH.

L

acetone

FILTER­

PENTOXlDE.

*

J

115

1.0 GAVE p

GM OF

0.04

H 3.6

and

N O TE:-

WH OL E

GM OF 7.2

THE were

ATTEMPTS

C L AM

TISSUE

WHITE in t a c t

TO

( WET

P O WD E R .

AND

THE

DRAINED

ACTIVITIES

BASIS) AT

BOTH

.

DIALYZE

OUT

I N 0 R G ANi I C

SALTS

BEFORE

ACET ONE P R E C I P I T A T I O N I N V A R I A B L Y CAUSED I N A C T I V A T I O N . ADDI­ T I O N OF F R E S H D I A L Y S A T E A N d / o R T H I O G L Y C 0 L L I C A C I D TO SUCH D I A L Y Z E D B U F F E R E X T R A C T S D I D N O T CAUSE ANY R E A C T I V A T I O N . L

-J

r RROc a)

IV *

eo u re

Ammonium

E x tra c t-!.

tracted

IN

Five

THE

grams

USUAL

Pr e c i pi t a t i o n . -

Su l f a t e

of

MANNER

the

dessicated

WITH

WATER

TO

powder

GIVE

were

ex­

100

ML

OF

ml

of

PH 3

EXTRACT. 2 b)

acid

Me

ILVAINE

ATED The

ML

EXT-1

E x tra c t.-

IS

E x T -ll

of

c)

Ammonium

is

now

to

p re c ip ita te

The

p r e c ip it a t e

g r a v it y

,

FILTER

TILL

ING

H OU R S

INACTIVE

WAS

to

TION.

t h is

F OUR HOU R S

et

was

CHANGES

Ex

be

WAS

WAS

OF

ONE

THE

COLD.

DIALYSIS

WAS

60 ML

ML

50

- II

I.

LITER THE SO

Ex OF

t

TO

—f I I

WAT ER

FINAL

ML

OF

GIVE was

w e ll.

The

VOLUME

OF

g e la ti­ m ixture

tem perature. a

t im e s

fluted

through

FILTRATE A

ON

SMALL

IS

EQUIV.

TO

DISTELLED AM

AMOUNT

WATER.

OPALESCENT d ia ly ze d

FOR

GM

AFTER

P O WD E R .

|T

SOLU­

a g ainst

A PERIOD

E X T-1I I

0.1

the

STAND­

THAT,

ExT-ll!

paper

PRECIPI TATE(A)

now

EACH

satura­

w hite

DISCARDED).

IN

sulfate

The

DEPOSITED

The

N a 2 HP 0 4 *

f u ll

through

several

RPM.

E x t - II.

ammonium to

SEPAR­

1000

0.2 M

room

CLEAR.(THE

COMPLETELY t

at

f il t e r e d

m ix tu r e

WHICH

but

hours

TEMPERATURE

AND

IN

2 .4

four

then

the

slow ly

be

using

im m ediately.

out

WHICH

A B OU T

th is

PH 5.6

to

Extract

REDISSOLVED

EASILY

L

5.6

for

MATERIAL

DISSOLVED

Let

AT

above,

ROOM

IMMEDIATELY

PRECIPITATE

MINUTES

o ff.

20

added

pH

FILTRATE

AT

is

S olid

stand

THE

GM POWDER

P re c ip ita tio n .

settled

passing

E xt-I

TWO

appeared

(A)

0.1

VOLUMINOUS

adjusted

the

C loudiness

allow ed

OF

now

TO

of

decanted

S u lfate

added

24

THE

is

is

was

by

ml

C E N T R I FUGED O F F FOR

pH

nous

EQUIV.

50

To

BUFFER.

supernatant

tio n .

IS

OF

24

117

i The

determ i na t i on

THE

USUAL

THE

SAME

METHOD. TIME

OPERATIONS

L

30

WOULD

of

a c t i v i t y

ALL THAT NOT

shown

SAMPLES ANY

WERE

on

TESTED

INACTIVATION

VITIATE

THE

T a b le

XV

FOR

was

made

by

ACTIVITY

AT

OCCURRING

RESULTS

TOO

DURING

THE

GREATLY.

J

/

117a

Table XV The a c t i v i t y o f D i f f e r e n t E x t r a c t s Obtained inthe C o ncentration By A m m o n i u m S u l f a t e P r e c i p i t a t i o n

PH

Sam ple

D ia ly s a te

THia m in e FROM 1 0 0

De s t r o y e d UG P R E S E N T

Ex t r a c t (1 .5 ml)

3.6 7.2

E x tra c t 1t* (2 .5 ml)

3.6 7 .2

added

37 34

us

added

Ex t r a c t I I I * * (3.6 ml)

3.6 7 .2

NONE NONE

10 17

UG

3.6 7.2

ADDED

40 42

ug

ADDED

7.2

ADDED

0

UG

H'

| Ex ’ (h

tr a c t ea ted

- 11 I )

35 35

added added

ug ug

ug

. *

Vo lume

used

e q u iv a le n t

to

0,75

gm

clam

f * V O L U M E U S E D E Q U I V A L E N T TO 1 . 5 GM CL AM A C T I V I T Y M I G H T BE D U R I N G T H E D I A L Y S I S

Table The by

l

id s

1 .0 GM 0 . 5 6 gm 0.146

*

As s u m e d

all

**

Un

d ia l y z e d

per

POWDER

Clam Powder* Ex tr a c t- I EXTRACT-I 1I

that

.

P O WD E R . THE OPERATION.

LOSSIIN

X VI

D e g r e e o f Pu r i f i c a t i o n Ob t a i n e d Am m o n i u m S u l f a t e P r e c i p i t a t i o n

Total so l GM.EQUIV.

Sa m p l e

powder

a c t iv it y

Un i t s a c t i v i t y PE R GM E Q U 1 V . POWDER 9.33 9.33 5.6

extracted

Ac t i v i t y per GRAM T O T A L SOL 1DS 9.3 16.7 38.4

UNITS

by

w ater

.

.

j

n?B T a b l e XV11 T h e Pr o d u c t s Ob t a i n e d b y t h e Am m o n i u m S u l f a t e C o n c e n t r a t e

the

Extract

p

i

tio n

on

Th

RegenI? dines

of

ia m in e

S U L F I T E - I N S E N S 1T I V E PYR.FRACTN

Extract w

I

3.6 7.2

181 161

UG

12 24

UG

Ex t r a c t

111

3.6 7.2

165 130

ug

12 30

UG

n

*

Va lu e s are OR I G I AN A L L Y

Ge n e r a l TO

0.15

GM

PREPARED

t h ia m in e

Pr o c e d u r # OF

4

V OL UME

AND

THE

MIXTURE

F or

com parative

was

run

using

the

t im e

t io n

OF

THIAMINE

IN

used

as

a

p

3.6

OF

WAS

200

from

complete

and

7.2

To

a

ROOM

ADDED

ug

t h ia m in e

n

the

two

20

ML

ML

for

the

sample

.

T

AND WITH

(E

FRESHLY 200

using

UG

Wa T E R

FOR

ONE

WEEK.

m ixture

xtract

-1 )•

complete

l i t .

he

e q u iva len t

OF

in c u b a tio n

EXTRACT

d a ys

5

e xtr a c t

required

I

- I I t

TEMPERATURE

aqueous

OF

t

BUFFER

TO

s im il a r

C A SE

in

WAS

Ex

ml

MclLVAlNE

AT

,

.

3.6

ADJUSTED

was

THE

p r e s e r v a t iv e

H

ML

o r ig in a l

reaction

both

:-

POWDER,

p u r po ses

of

at

e d

INCUBATED

week

was

Us

ORIGINAL

THE

str u c tio n

regenerated

1 1

PRESENT.

DIALYSATE,

THIAMINE.

L

T otal P yri m

H

Ac

destruc

TOLUENE Extract

reaction

On e

was

­

WAS I,

de

­

run



J

r

1

PROCEDURE

V.

The ON

Am m o n i u m

l f a t e

a

Let

Su

Aqueous

)

POWDER

WAS

t h is

B> WERE

Ex

be

IN D A YS

AT

THE

DIALYSATE

WAS

TWO

The

residue

MC

EXTRACT

The

volume

Let

BAG

GIVE

by

WITH

dessicated 125

0,1

ML

gm

OF

clam

EXTRACT.

powder

DESSICATED

50

ML

AGAINST

THE

CLAM

100

AND

BY

ML

UNDER

OF

WAS

THE

POWDER AND

THEN

DISTILLED

TOLUENE*

OBSERVING

EXTRACT.

C L AM

SUSPENSION

DIALYSATE

P u rific a tio n . -

THE

ML)

ADJUSTMENT

.

MAKE

OF

buffer

AFTER

BUFFER

THE

w a ter

TO

THE WAS

IN

to

ITS

WAT ER

THE

TESTED

CLEAR

BEFORE

EFFECT

ON

AN

A M B E R - C 0 L ORED

MANNER.

B R OU GH T

BEING

MADE

CAREFULLY

t h is

extract

be

Ex

was

tr a c t

then

-111.

TO

E x t-I,

20

AT

ml

THE

made

MINUTES,

ABOUT

1000

WAS

POURE D

of

p

to

LOCAL 120

of

THE RPM OFF.

H 3.6

USING

AVOID up

2

ml

w ith

COMBINED

PH 5 . 2 TO

30

c are fu lly

EXTRACT

w ith

THEN

of

STOOD

OFF

ACID

WAS

the

HAD

C E N T I FUGED

SAME

ml

added

MIXTURE

extracted

THE

75

were

OPALESCENT

fu rth er

was

of

the

to

THE

EXPERIMENTS

PRECIPITATE

(103

of

.

e q u iv

GM OF

standard

MINUTES.

ILVAINE

P rin cip le

ACTIVE.

STIRRING.

FOR

Clam

PRECIPITATION

TO

ROOM T E M P E R A T U R E

A cip

VOLUMINOUS

gm

WAT ER

is

i

5*0

DlALYZED

M cIlvaine

GENTLE

-

WAT ER

ENSUING

c) 3

t

EFFICIENCY

EXHAUSTIVELY

6*25

WITH

A CELLOPHANE

REACTIVATION

IN

IN

the

-I

Ex

ml

SUSPENDED

TWO

USE

E x tra c t*-

tract

of

C „ A U U i i ANO GAMMA

. -

DlALYSATE. -

DIALYZED FOR

ALUMINA

EXTRACTED

2

pH

C oncentration

ADSORPTION

ACID

0.3

M NA 2 HPO4 ,

EXCESS. ml

w ith

3 .2

)

d

120

ML

OF

MIXTURE

TEN

MINUTES,

TEN-MINUTE RPM

WAS

IN

THEN

SHAKING

PERIOD,

FOR

Al u m

in a

equiv.

2

C ARE

THE

A L L OWE D

THE

BEING

TO

3.47

.

e

E

in

75

AT

ROOM

TO

TIM E.

ml

THREE

ml

l u t io n

of

MIXTURE

.-

2

ML

f) EXTRACT-V AMMONIUM

OF

AN

HOUR,

A BOUT

WAS 7 5

WAS

AL L OWE D

The

w h ite

TO

the

C q amma)

WITHOUT

TO

TIME.

WAS

T HE

UNDUE

STIRRING.

TEMPERATURE AFTER

CENTRIFUGED SEPARATE

AT

FOR

T HE

A B OU T

OUT

WELL.

T H E VOLUME

LET

OF T H E

equiv.

was

now

to

The

MIXTURE T HE

LABELLED

gm

powder

redispersed

SHAKING

RPM.

0.1

m ixture

carefully

was

CAREFULLY

WAS

FROM

CENTRIFUGE

ELUATE,

EXTRACT-V.

WHICH THE

kept TIME

FOR IS

NOW

V OL UME

OF

ML. EXT-Y

WAS

STAND

m a ter ia l

of

SUSPEND

TIME

HOUR,

1000

WAS

ADJUSTED

SULFATE

TO

ROOM

b u ffer.

THE

IS

P rec ip itatio n WAS

ALUMINA

EXTRAC-1V.

alumina

FOR

WATER-CLEAR, ELUATE

powder

ml

AT

WAS

is

M cIlvain e

ONE

AT

15

TAKEN

ALUMINA

E x t -IV

The

TEMPERATURE AFTER

To

OF

BUT

gm

ML.

of

pH 7 . 4

MINUTES

ALMOST

130

0.1

STAND

FROM

M I N U T E S . THE

WAS

1 5 0 MG

MIXTURE,

GENTLY

to

C GAM m A # -

O P A L E S C E N T S U P E R N A T A N T BE

SUPERNATANT

THE

is

E X T -M l,

THOROUGHLY

The

THE

—11 I

SUSPENSION (CONTAINING

ALUMINA

1000

t

A d s o r p t i o n on

ALUMINA ADDED

ml Ex

EQUIV.

w ith

CAREFULLY

ADDED FOR

w hich

TO

FOUR

TO 0 . 1 GM

ammonium TO

FULL HOURS

separated

POWDER

PH 5 . 6



THEN

SATURATION. AT out

35

S u lfa te .-

ROOM was

ml

of

SOLID

THE

MIXTURE

TEMPERATURE. f il t e r e d

off

120

USING MORE ING

A

FLUTED

PAPER*

T HROUGH THE P AP ER , FOR

AT E D

o u t

TWO

DAYS

*)

T he

REDISSOLVED THE

FILTER

AID

OF

IN A

Th

s olution

500

ML

OF

DRY

WEIGHT

ML

FINE

COMPLETELY

ROOM

w h ite

35

WE N T is

AT

IT

was

DISTILLED

)*

Ex

HAD

TIONS,

A SEPARATE FROM

E x tra c t-y i.

volume

OF

of

THE

in

a

20

was

not

EXPERIMENTS

the

DONE

WITH

a g ainst

(FOR

THE

DlALYZED

A MOUNT

LATER

to

a vo id

OF

SALT

DETERMINA­

SHOWED*

a fte r

SEPAR­

SOLUTION*

employed

PRECIPITATE

extract

STAND­

im m e d ia te ly

bag

WAS

SMALL

THE

ONCE

PRECIPITATE

HOURS*

SAMPLE

ON

SULFATE

EASILY

WHITE

RELATIVELY EFFECT

was

A WATER-CLEAR

FOR

LEFT

MATERIAL

cellophane

d ia l y s is

AMMONI UM

were

Extracts of

WHICH

TIME

TURE.

T he

t

-YI

1 M ,[V ,

the

De

and

THE

ICE-BOX

OF

the

V (

THE

FROM

of

results

WERE

Extr act

START

gm

THE WAS

DIALYZED

LABELLEO

d ialysis

was

powder

The

a c t iv it ie s

method

determined

D IA LYSIS,

THE

*-

usual

were

EXTRACTS

a c t iv it ie s

0.1

to

t e r m in a t io n

by

e xp e r im e n ts

ALL

equiv*

is

measured

CONCLUSION THE

Ex

a c t iv it y

start

L

ml

)

extracts

IN

SET

WHEN

MORE

BEING

GIVING

4-8°

THE

AND

FILTRATE

ML •

g

THE

THIS

A SEPARATE

SUBSEQUENT

THE

The

2*29

of

NO

NO

T HE

p r e c ip it a t e

WATER,

AT

h a u s tiv e

REMAINING

40

OF

WA T E R

INACTIVATION.

SOLUTION

WATER,

d ia l y z e d

EXTENSIVE

AS

powdery

DETERMINATIONS,

e x h a u s t iv e l y

W A T E R —C L E A R

SOLUTION

now

PASSING

TEMPERATURE,

STREAM

INTO

WAS

and

OF

(AFTER

TO

shown

.

T

10

hours

T

on

able

STANDING

AT

I

were

and

THE THE

YI

TWO END

he

the

various

a c t iv it ie s

after

X V III)

the

during

ROOM T E M P E R A ­ determ ined

EXTRACTS OF

of

THE

BEING

at

KEPT

DIALYSIS

OF j

121 p

"I

Extract- V I . PERIOD

OF

(Ex t - | )

CASES

TO

DESTROY

STANDING,

was

DIALYSATE

Su b seq u ently,

not

WAS

in

Table

H)

ADDED.

50

ABOUT

The

OF

a l l

AMOUNT

OF

OF

THE

t h a t

ORIGINAL

a c t i v i t y

ASSAYS EACH

FROM

100

d u r in g

RUN

EXTRACT

PURIFICATION

EXTRACT

d e t e r m i n a t i o n s

WE RE

UG

t h i s

IN

WHICH

UNDER

THE

,

ALL WO U L D

ASSAY

OBTAINED

IS

SHOWN

Ac t i o n

of

the

XI X.

Re a c t i o n

Products

WE RE

DETERMINED

SATE

WAS

ADDED

TO

on

Table

XX.

L

I n

THIAMINE

DEGREE

DIFFERENT

shown

.

shown

PRELIMINARY

THE

UG

was

ACTIVITY

i f f e c t e d

DETERMINE

CONDITIONS.

THE

i t

IN

EXTRACTS THE EACH

Produced ON

USUAL

in

the

THIAMINE. MANNER.

INCUBATION

I n

SAMPLE.

THE

REACTION

ALL

CASES,

THE

PRODUCTS

DIALY­

RESULTS

ARE

J

121a

T able

T he

Ac

in

t i

the by

Ad

v ity Pu r i f

Ex

tract o l

tr a c t

3 D i

.

v. Powder

0.15

- 1

GM

Ex t r 5.2

a c t

0.15

- 1V

GM

-V

0.15

-VI

0 . 2 2

GM

ML

tr a c t

I

GM

ML

tr a c t

3

0.15

- ! 11

ML

5

ML

n

all

c a s e s

incu b a tio n

* * T H I AMINE

,

w ith

3

ml

of

d ia l y s a t e

t h ia m in e

DESTROYED

FROM

ed e

w ith

pH Rea c tn

q u i

ML act

*

e c ip it a t io n

alysate

Ex t r 4.8

Ex

)

on

ML

3

Ex

Gm - E C lam

*

used

ic a t io n

Pr

E x t r a c t s Ob t a i n of the C l a m Pr i n c i p l A l u m i n a C GAMy . w i t h

d if f e r e n t

so rptio n

Su b s e q u e n t

Ex ( v

of

X V I 1I

Th ia m in e FROM 1 0 0

destroyed

3.6 T.2

56 52

3.6 7.2

0

3.6 7.2

58 50

3.6 7.2

0

3.6 7.2

47 45

ug

3.6 7 .2

47 42

ug

ug

0

ug 1

0

were

added

prior

to

UNDER

USUAL

ASSAY

CON

* 100

UG

DITlONS*

L

**

MICROGMS

-I

121b

XIX

Table T he De g r e e Concentration on

a l u m in a

of of

Pu r i f i c a t i o n Ob t a i n e d C lam Pr i n c i p l e by

th e

C qamma

and

Am m o n i u m

w it h

P r e p a r a t i ONS

T o tal

S o lid s*

# to P OWD E R

e q u i v

GM

Ex

tract

Extr

0*1846

1 * 0

gm

- 1 11

Un

th e

Pr e c ip i t a t io n

Su l f a t e

it s

a c t iv it y

PER G M - E Q U I V * OF P OWD E R

6*93

UNITS

6 * 8

Un

it s

TOTAL

37.5 ♦





*

o

o

-1 V

0*1196

gm

0 * 0

Ex

tract

-V

0.0228

gm

6 . 0

263*2

Ex

tract

-VI

0*021

GM

3.81 s

180.6



tract

a

a c t iv it y

P E R GM SOLIDS

Ex

*

All

a)

Assuming t h a t a l l a c t i v i t y in t h e pow der was e x t r a c t e d in t h e p r e p a r a t i o n o f E x t r a c t - ! (aqueous e x t r a c t ) , the f i g u r e s f o r E x t r a c t - V s h o w an a p p a r e n t 3 8 - f o l d p u r ifi­ c a t i o n ON D R Y - W E I f c H T B A S I S FROM T H E C L A M POWDER# THE F I G U R E S FOR T O T A L S O L I D S OF WHOLE C L A M T I S S U E USED I N P R E P A R I N G T H I S POWDER ARE NOT A V A I L A B L E , BUT U S I N G T H E V A L U E OF 1 * 6 1 GM T O T A L S O L I D S FOR T H E C R U D E C L AM T I S S U E E Q U I V A L E N T TO 1 # 0 GM OF D E S S I C A T E D POWDER ( T A B L E X I V A , P * / J 3 A ) F O R C O M P A R I S O N , T H I S WOU L D G I V E AN A P P A R E N T 6 1 - F O L D P U R I F I C A T I O N ON A D R Y - W E I G H T B A S I S FROM T H E C R U D E CLAM T I S S U E #

b

L

act

-1

Subsequent

in

Ad s o r p t i o n

)

d e t e r m i n a t i o n s

on

e x h a u s t i v e l y

d i a l y z e d

b a s i s

#

T he f i g u r e s show t h a t in a c t iv a t io n q c c u r r e d ( or L OS S OF A C T I V I T Y ) I N THE AMMONI UM S U L F A T E P R E C I P I T A T I O N # T h e R E A S O N FOR t h i s w a s n o t f u r t h e r investigated#

_J

121c Table

XX

T he Re a c t i o n Products Produced in thie De s t r u c t i o n * Th ia m in e by the Ac t i o n of the D if f e r e n t Extracts

of

Ob t a i n e d by

in

Al u m i n a

th e

F R T F a R A T 1 ON of

EXTRACT-1

Ex

tr a c t

8

5

-V

a d s o r p t io n

Volume Extract USED

5

E®TRACT-I 1\

Concentration

C o a M, , a G AMMA

ML

ML

ML

p

H

of and

the

C lam

Salt

T o t a l Regv P Y R I M 1 D 1NE F R A C T 1 ON

3•b

156

7.2

146

3 .6

1

7.2

142

3 .6

Pr i n c i p l e

Pr e c i p i t a t i o n

SULFITE-INSEN-A S I T I VE P Y R 1 M 1 D 1 NE F R A C T I O N

UG

18 38

UG

60 u g

2 0

ug

162 138

36 ug

4 36

UG

3.6 7.2

160 u g

18 26

UG

ft*

2 0 0

0

UG

7*2 b

Ex

tract

He a t e d

*

10

-VI

ML

Bl a n k

166 ug

I n a l l c a s e s , t h e amount o f t h e e x t r a c t g iv e n above was i n c u b a t e d w i t h 2 0 0 ug t h i a m i n e w i t h t h e a d d i t i o n o f 5 ML of fresh d ialysate# I n c u b a t i o n was c a r r i e d o u t f o r two DAYS

AT

sample

ROOM u s in g

THIAMINE A )T H I AMINE

WAS

TEMPERATURE. IN ALL C ASES, EXCEPT WITH THE Extract-VI at pH 7 * 2 , th e d e s t r u c t io n of COMPLETE AFTER THE TWO-DAY I N C U B A T I O N *

REGENERATED

FROM

200

UG

OF

THIAMINE

ORIGINALLY

PRESENT*

T H E S A M P L E I N C U B A T I O N AT PH 7 # 2 , 6 3 UG OF T H I A M I N E R E ­ M A I N E D U N D E S T R O Y E D F RDM T H E 2 0 0 UG O R I G I N A L L Y P R E S E N T I N T H E TWO DAY P E R I O D . T H I S P R O B A B L Y E X P L A I N S T H E SOMEWHAT H I G H E R R E A D I N G S O B T A I N E D AT PH 7 . 2 U S I N G T H E A M M O N I U M SULFATE CONCENTRATE, E X T - V I S I N C E THE R EG E N E R A T I O N D E ­ T E R M I N A T I O N WAS MADE A F T E R T H E T W O - D A Y P E R I O D . IN THE E A R L I E R E X P E R I M E N T D E S C R I B E D ( AMMON I UM S A L T P R E C I P I T A T I O N , PAGE I0 Q ) T H E O B S E R V A T I O N WAS ALSO MADE T H A T A LONG T I M E OF I N C U B A T I O N WAS R E Q U I R E D FOR T H E C O M P L E T E D E S T R U C T I O N O F 2 0 0 UG OF T H I A M I N E BY A VOL U ME OF T H E A MMON I U M S U L F A T E C O N C E N T R A T E E Q U I V A L E N T TO 0 . 1 5 GM OF T H E P O W D E R . THE REA­ SON I S P R O B A B L Y T H E I N S T A B I L I T Y OF T H E E X T R A C T S I N C E T H E R A T E D E T E R M I N A T I O N ( A S S A Y OF A C T I V I T Y ) SHOWS T H A T T H E E X ­ T R A C T I S Q U I T E A C T I V E , D E S T R O Y I N G A B O U T O N E - H A L F OF 1 0 0 UG T H I A M I N E I N A T W O - H O U R P E R I O D AT 3 0 * ^

B ) IN

L

122

NOTES ON THE METHOD G I V E N

1*

The

PROCEDURE

OF

slu rry(about ALIQUOT TILLED

The

WATER

AT

2.

MCtLVAlNE

TWICE

used

PE R

in

TO

ML

the

GIVE

AND

WAS

hydroxide.

For

F I V E VOLUMES

AS

A

an

EACH

OF

DIS­

FROM

AMMONIA.

FREE

IN

WA T ER

TO

MAKE

SUSPENSION.

THE about

ADSORPTION

the

use,

UNTIL

is

to

STORED

OR

COMPLETE

OF

A SUSPEN­ AMOUNT

OF

1^

t im e s

THE

ACTI­

5 .2 .

C Ga m m a

separates

out

very

w e ll

during

OPERATIONS.

RECOVERY

BY

ELUTION

A B OU T

THE

RECOVERY

IS

ONLY

The

ammonium

sulfate

FROM

pH

W IT H ’

90$$

WITH

A B OU T

45$.

used

WATER,

in

THE

8

OR

pH 7 . 2

the

PH 7 . 4 M /i 5

STAND-

PHOSPHATE

p u r if ic a t io n

C .P.

GRADE

was

BEING

USED

MATERIAL.

TEMPERATURE

VIGOUROUS

OPERATIONS

OF

according

p u r if ic a t io n

IS

LOW

ESSARY.

BE

MG

RECRYSTALLIZED

4.

L

10

BUFFER

STARTING

WITH

RESUSPENDED

Alum ina

fo

TIMES

KRAUT^

CENTRI FtOUT I O N ,

THEN

PH OF

A

T he

4.

TO

is

4 fo

FIVE

CENTRIFUGATION

BUFFER,

AS

BY

FOUND

The

3. ARB

ammonium

C Ga m m a

AMOUNT

VITY

THE

Q.1M)

CONTAINING

m in a

THE

A ND

in

PROCEDURE V .

prepared

WlLLSTATTER

WASHED

ALUMINA

SION Al u

IS

C gamma. w a s

Alum ina

IN

EASILY

OPERATIONS

SHAKING LEADS

TO

DURING

DO

T HE

NOT

APPEAR

ADSORPTION

INACTIVATION

AND

IS

TO AND

BE

NEC­

ELUTION

THEREFORE

AVOIDED.

Jl

123 r CONCLUDING

The BY

ITS

WITH

enzym atic

nature

PROPERTIES,

THIAMINE

ORGANIC

THE

AN

IN

THE

ACCELERATORS

AND

INHIBITORS.

BY

THE

C L AM

AS

YET

PRINCIPLE,

CLAM X

exh austion

OF

TISSUE. may

ITS

THE

CL AM

T HE

BEHAVIOUR

OUT

THE

TO L

ACT

THE

DIFFERENT FOR

EXPERIMENTAL

Sp

DID

OF

THE

PRINCIPLE

BY

An

SYSTEM

EVIDENCE

ON L Y

ACTION,

AN

GN

a

concept

BE

OF

CALLED

SINCE

IT

ANY

ISOLATION

OF

s t u d ie s

ANALOGUES

OF

BUT

IN

A

IS

DECISION

X

IN

THAT the

w ith

THE

THE

AT

LEAST

THE

FINAL

CONCENTRATION CHANGE

THIS

X

IS

THAT CAN

THE FROM

BE

the

inconsist­

X REMAINED

OBVIOUS

that

THIAMINE

TO

REACTANT

REACTANT show

not

OPERATIONS.

THAT

FINAL

;

OF

OBSERVABLE

REGCTANT

IT

PRESENT

PROTEINBEHAVICUR),

NOT

ITSELF,

it s e l f

is

50-FOLD

HYPOTHESIS

X)

LIMITATION

OF

SUPPLIED

assoc ia ted

(which

IN­

PRODUCT

INDICATES

A BIOCATLYST,

FRACTIONATION

AND

DESTRUCTION

(REACTANT

APPROXIMATELY

SYSTEM.

REACTION

F R A G ME N T

CONTRIBUTES

RESULT

indicated

A REACTION

A

be

CONSEQUENT

ITS

THE

p r in c ip l e

to

is

ORGANIC

EVIDENCE

clam

THEORIES

APPROACH,

e c if ic it y

CONTAINS

appears

THE

CANNOT

CHANGES.

X

|N

HOWEVER,

OVER-ALL

OF

TOWARDS

REACTANT

a c t iv e

such

ESTABLISHED

VITRO

ENERGY

C L AM

From

PRINCIPLE IN

the

A C T I VAT I ONAND

WITH

CLAM

THE

Re a c t a n t

of

d estru ctio n . ent

be

WHICH

COMPLEX

U N K N O WN

p rin cip le

CHARACTERISTICS BEHAVIOUR

Re a c t a n t

LOSS

clam

ITS

( IC T H I AMINE) I S OBTAINED BY

the

BY

THIAMINE

AND

BY

of

remarks

IN

OF IN

THROUGH­ IS

ST RONG

ACTIVE THIS

REACHED

ITSELF. the

clam

WHICH

p r in c ip l e

THE

is

able

SUBSTITUENTS -I

1E4 r

t

ON

THE

THIAZOLIUM

THIAZOLIUM WHOSE TO

COMPETE

N OT

AS

WITH

AN

INATION

OF

ENZYME

BY

X.

PRINCIPLE THE

AT

PH

DESTROYING

AS

EXPLAINED THAT

A

SHOW \ VE L

APPEAR

THAT

MAY

THE

HALF. THE

MENTION

TWO

THE

TO

BE

THE

OF

REACTIONS.

ONL Y

THE OF

MOLECULE

OF

REACTION

QUANTITATIVE

AT NOT

SEPARATE

CHAR­

ACID OF

BY

AND

KINDJ

THI AMINE-

DIFFERENCES

AND

THIAMINE

PRODUCTS,

(SIMULTANEOUS

THEMSELVES)

OF T H I A M I N E

PH 3 . 6

AT

R E A C T li O N

AND

OBSERVED

BY

DIFFERENCES,

PRINCIPLE

TWO

RE­

clam

(ONE

THE

IN

THE

MECHANISM

the

THESE

CONCURRENT

EVIDENCE

OF

SUPPLIED

PYRIMIDINE

TWO

ATTACKS

EXPERIMENTAL

by

D E GR E E

CHARACTERISTICS

PROPERTIES

THE

OF

EVIDENCE.

DESTRUCTION

OPTIMA

ALCOHOL.

EXISTENCE

NOR

STUDIES

A N0 N -E N Z Y M A T IC

DISTINCT

VARIATIONS

CL AM

INHIBITIO N

FRAGMENT

PH

IN

DETERM­

t h i a m i n e

TWO

PROPERTIES

REACTION

COMPLEX

THtaZGLE

WITH

A

AP P E A R

THE

OF

FAVOURS

of

FOR

THE

GROUP

REACTANT

AVAILABLE

MODE

WHICH

ANALOGUES

ME T H OD S

THE

IN

PYRIDINIUM

LIMITING

THE

BY

PYRIMIDYL

PRINCIPLES

BY

OF

OR

THESE

CONTAINS

AND

A

COMPETITIVE

PRINCIPLE

INDICATE

THE

BY

BASIS

9}

T HE

INDEPENDENT)

WELL

PH

THE

AND

THE

o b s t r u c t io n

TOGETHER

NOT

THE

CLAM

AT

ACTERISTICS

DO

ACTION

BY

KINETIC

CHARACTERIZED

AND

ALKALINE

OF

MODIFIED

VARY.

FOR

JCTHIAMINE

OTHER

HOWEVER,

ALSO

SE

PREVIOUSLY,

The

IS

ICTHIAMINE

MAY

ON

THE

I.E .,

Reactant

AND

REPLACED

BE

APPLICATION

MENTIONED

ACTION,

THEY

MAY

THIAMINE

POSSIBLE

THIAMINE

AND

MAY

SUBSTITUENTS

TISSUE.

IS

MOIETY

MOIETY

BY

MAY

THE

(AS BE

FACT

DISPLACING

THE

HAS

BEEN

PRESENTED

INVOLVES

SUCH

A DISPLACEMENT.

RECOVERY

OF

THE

TO

THIAZOLE _i

125 r

1

HALF

WH E N

THE

DITIONSAND A

REACTION

ALSO

NUCLEOPHILIC

The BEEN

data

and

BY

THE

E ).

THE

ENUMERATED

IN

ADDITION,

IS

GREATER

ABLY

OF

THAN

THE

IS

ALSO

IS

IN

THE

THAT

OF

EXPLAINED

LINE

IN

OF

WITH

TENTATIVE

THIAMINE A

THEKNOWN

|N

A

SERVICE

IT

ACTS

EXPERIMENTS

Wh

il e

ENZYMATIC

AND

the

MADE

USUAL

THAT

METHODS

PRINCIPLE

BE

*

OR

THEN,

AS

IS

A

DETERMING

BY

ONLY THE

AND

OF

FROM

AN

MECHANISM

ONE).

(PART

I C T HI A M I N E AND

WHY

STAYS

REACTION

THE

REASON­

BY

THE

CONDITIONS.

CLAM

PRINCIPLE

PROCESS

PROPERTIES A

TOOL

IN

THE

THE

THE

ISOLATION

a

DETERMINATION

OF

FUTURE.

non

MPi Y

OF

FURTHER

THE

RESULTS

CLAM

THIAMINE.

VARIOUS

®F

ACTION

WHICH

IMPORTANT

THE

favours

OF

INTERPRETATIONS

OF

a ll

FRACTIONATION

BETWEEN

ENZYME

NATURE BY

RESULTS

P E R F O R M S AN

h er ein

have

explains

EXPERIMENTS

EVIDENT

THE

WORKI NG

ACTIVATION

AS

IT

t h e s is

ALCOHOL

THE

BRIDGE

DEDUCTIONS

ELUCIDATED

ITSELF,

A ND

SERVED

reported

IT

F OR

THE

ANALOGUES

HAS

t h is

I C TH I AMIN E

IN

CONTEMPLATED

INDISCRIMINATELY.

PERHAPS,

TO

OF

REACTION.

YIELD

DIFFERENT

FACTS

SENSE

evid en c e

REACTION,

INVESTIGATIONS THE

THE

THE

HYPOTHETICAL

MECHANISM

THAT

WHY

PYRIMIDYL

THE

in

OF

CON-

PARTICIPATION

TENTATIVE

SUMMARY

VARIATIONS

BY

THE

DIFFERENT

s a tis fa c to rily

ALCOHOL

INVESTIGATION. IN

A

ALSO THE

OF

UNDER

reported

OF

THE

EXPLAINS

WITH

DESTRUCTION

OUT

DISPLACEMENT

mechanism

THROUGHOUT

A ND

THE

USE

PYRIMIDYL

CONSTANT

OPERATIONS The

The

IT

IN

o bser va tio ns

(Mechanism POINTS

CARRIED

DEMONSTRATION

REAGENT

CORRELATED

RATIO

L

THE

IS

-

OF

OUR

BASED NOT

BE

PRINCIPLE THE

ON

MAY,

ACTIVE

TTS -I

F PHYSI0L0GICAL THE

CLAM

FUNCTION,

PRINCIPLE

MAY

SINCE BE

IT

IS

INVOLVED

POSSIBLE I N AN

THAT

IN

ENZYMATIC

VIVO,

1

TRANSFER

SYSTEM.

The IN ♦

not

THE

clam

p

H

o p t im a

p o s s i b l e

m e n t a l

p r i n c i p l e

to

s a y

d i f f e r e n c e s

FI SH-PRINCIPLES

L

and

ARE

,

d i f f e r s

in

the

h o w e v e r

s i n c e

from

th a t

from

of

,

t h e s e

c o m p a r a t iv e

data

so urces

I c t h ia m in e .

fo r m a t io n

whether

other

It

r e p r e s e n t

r e g a r d in g

is

f u n d a

­

other

LACKING*

-I

r

S U P P L E M E N T

L

.J

THE

The

FLUORESCENCE

compounds

Group

I*

) b)

a

c)

Gr o u p

BEHAVI OUR

w hich

were

s t u d i e d

were

ANALOGUES

the

f o l l o w in g

: *

N e o p y r i t h i a m i n e - b r o m i d e - h y d r o b r o m i de 2 , 4 , 6 - c o l l i DINIUM analo g u e ’ 2 , 6 - L U T I D I N I UM a n a l o g u e

11 * a)

b)

All

the

OXIDATION

2 ,4 2 ,5 -

d im e th y l

th ia zo liu m

analogue

d im e th y l

t h ia z o l iu m

analogue

above

WI TH

analogues

ALKALINE

PHOTOFLUORIMETRICALLY, DIRECTLY

PROPORTIONAL

(Table

a

ed

Compounds

for

and

OF T H I A M I N E

)*

The

Todd^ .

g i v e

FERRICYANIDE SINCE

TO

THE

d e v e l o p m e n t

I b ,

They

f l u o r e s c e n t

I c

MAY

the

I I a t h a t

BE

FLUORESCENT

CONCENTRATION of

and

repo rted

THE

AND

products

in

t h e

of

IN

AQUEOUS

the

c o m p o u n d

ASSAYED

INTENSITY

flu o r es c en c e

view

upon

,

is

IS MEDIUM

u n e x p e c t

work

of

Be r g e l

(Compound

d

) ,

BELOW

gave

no

t h io c h r o m e

ACCEPTED MENTIONED THE

MECHANI SM THAT

IN

CONDENSATION

OF THE

OF

ME TH Y L - P Y R I M I D I N E

*

t e s t

,

as

was

THI OCHROME SYNTHESIS

4-AMINO

to

be

FORMATION. OF

COMPOUND

THE

APPROPRIATE

at

end

of

See

f l y

FORMULAS.

le a f

It

0,

from

the

MAY

BE

INVOLVING

5 - T H I 0 ACETAM I D 0 - M E T H Y L - 2 -

AND

A ND

e x p e c t e d

BROMOKETONE,

supplement

for

IWE.,

chemical

­

names

11

+

»■ — CvfWofr

THE

YIELD

b e i n g

TIME

of

Gr o u p

IN

CONTACT

R OOM

TEMPERATURE

CANT

FLUORESCENCE

D. EVELOPEMENT

IS

IN

THE

UPON

MIXING

WITH

COMPOUNDS,

EXCEPT

THE

CENCE WISE

A

of

GIVEN

analogue, ARE BE

ADDED.

WAS

THE

BULK

OF

THE

PRODUCT

OF

Tn

THE

BY

S,

AND

FULL

OXIDANT

SHOW

SECONDS#

HEATING.

THE

NO

AT

S IG N IFI­

THE

RATE

MEMBERS

FLUORESCENCE

OXIDANT.

only

ALL

OF

OF

IMMEDI­ THE

ABOVE

ANALOGUE( I B ) ,

DEVELOP

FLUORESCENT

THIAMINE. THE

f lu o r e s c e n c e

ALKALINE

TWENT Y

COLLIDINIUM

|N

TESTING

PROCEDURE

INTENSITIES THE

USED,

FLUORES­

UNLESS

OTHER­

FOLLOWS:

F lu o rescen ce:

ml

TABLE

ALKALINE

COMPOUND, AS

fu l l

THE

DEVELOP

THE

THAN

ANALOGUE

3

ON

AMOUNTS

LESS

GIVEN

STATED

Test OF

A

WITH

FIRST

THIAMINE,

EQUIVALENT

OF

POOR,

de ve lo p

ACCELERATED

ATELY

SIGNIFICANTLY

I

S H OWN

I I ,

IN

LIKE

AS

GROUP

USED

VERY

E.

members

S OME

D WA S

CPD

Compound

The AFTER

OF

CONTAINING

A 0 .0 2 ^

TAKEN I M M E DI AT E LY

T o 10

CASE BUT

ml

of

THE

FOR

GROUP GR OU P

aqueous

DESIRE0

FERRICYANIUE

OF

an

it I

UG

OF

SOLUTION

solution THE IN

1 5;o N a OH

COMPOUNDS,

READINGS

ANALOGUES,

THE

MA Y

OXIDIZED

SOLUTIONS HOU R S

ARE

B E F OR E

Table

3

AL LOWED

TO

READINGS

shows

STAND

MAY

the

BE

SAMPLES

FLUORESCENCE

IN

ALL

CASES

FLUORESCENCE

WAS

STUDIED

THIAMINE

AND

THE

THE

WAS

DIRECTLY

RATE

BEING

MADE

EVERY

DEVELOPED) INDICATE

The USED T HE

AND

ON L Y

NO

OF

AN

FROM

FOR

IS

THIS

FOR

THAT

READINGS

FOR

THE

READINGS

The

2 , 4 , 6 —C O L L I D I N I UM N E O P Y R I T H I A M I NE

THE IS

READINGS DUE

CYANIDE SOME

TO

THE

AND

FURTHER

(WHICH

GIVES

COULD TIME

ONE

AL SO

AL SO

REACTION THE

OF

THE

AND

OF

AS

THE

LIGHTEST

AN

OF

NEOPYRI-

[N

BOTH

CASES,

WHICH

WAS

MIGHT

FLUORESCENT

DOES

NOT OF

TABLE 11

PRINCIPAL

S H O WS ,

C OMPOUNDS

SHOWN BUT

EFFECT

BE

OF

IN

THE

TO

A

IN

YELLOW

UNKNOWN

SHADE

T HE

FLOURESCENT

DUE

TO

WHILE

TABLE)

AND

CASE

COLOR

OF

NATURE.

AFFECTED THE

THE

THE

THE

AND

LESSER

THE

REASON

AFFECTS

ARE

2 , 6 - L U T I D I N IUM

T HE TO

THE

C

INTENSITY

NECESSARILY GIVE A

CONCENTRATION

AFFECTED

BELIEVED

C A SE

LAG.

THE

OF

RATE

(MEASUREMENTS

DETECTED

GROUP

THIAMINE.

.

THE

THE

FLUORESCENCE

ANALOGUES.

FOR

t i m e

MINUTES.

THE

TIME

(MAXIMUM)

OF

INTERFERENCE

IS

IN

FULL

BE

ANALOGUES ( N O T

ARE

FOR

TILL

GREATLY

MEMBERS

FOUR

w i t h

DARK.

TO

F E R R I C X A N I DE

THIAMINE

LESS.

DETAIL

THE

PROPORTIONAL

DIFFERENT

FOR

IN

20

ANALOGUE.

THE

LEAST

f l u o r e s c e n c e

AFTER

STABLE

MEASUREMENT

THE

of

READ

IN

INTENSITIES

THE

AT

- C 0LLIDINIUM

B R EAK

READINGS

MUCH

IS

EMPIRICAL

COMPARATIVE

PRODUCTS

6

BE

HALF-MINUTE

A REASON

MET HOD

IS

2 ,4 ,

MAY

DARK

TAKEN.

d e v e l o p m e n t

N E O P Y R I T H I A M ! NE

OF

THE

IN

FOR

DE GR E E OF

T H AN

THIAMIN

FERRI —

INCIDENCE

OF

M E 0 P Y R I T H I AM I N E

FLUORESCENCE

OF

ALL

THE

rv r

i c o m p o u n d s

)

AFFECTED

MOSTLY

THE

and

th e

BY

F E R R I CYAN I D E .

LEAST

UP

TO

REAGENT

ON

THE

THIAZOLIUM

OF

THE

OXIDATION

BLUE-VIOLET

The ONLY

WH E N

EXTENT

THE .

I n

THE

TEST

CONCENTRATION

DIZING

MIXTURE

USE

THE

GIVE

UNDER

THE

IS

ARE

HAS

BECAUSE

THESE

(AT

LITTLE

THE

ANALOGUES

OF

FOUND

EACH

ANALOGUE

EFFECT

FLUORESENCE IS

OF

WH E N

USUALLY

THE

TO

THUS

WHEN

A

DARK

LEAST

DETERMINING BY

THE

n a t u r e

,

MADE

IN

WHICH

WO U L D

TABLES BETWEEN

CONDITIONS.

e x a m p l e

THE

FIND

THE

A

THE

OXI­

SUITABLE

II,

OF

OF

IN

SECTION

(CONSULT

CLAM

INTERFERENCE.

GENERALLY

SAMPLE

ARE THE

FOR

TO

FERRICYANIDE BE

SERVES

BLANKS

CONCENTRATIONS

READING

EXPERIMENTAL

AND

LATTER

0.03s / b OF

GALVANOMETER

WITH

IS

GIVE

ABOVE

ANALOGUES

THE

of

REPORTED

TAKENFROM

OTHER

THE

DIFFERENT

TO

BEEN

EACH

DESIRED

PROCEDURE

WOUL D

0.25

HAS

OF A

R UN

G U I fo )

DESCRIBED

REAGENT

EXPERIMENTS

AMOUNT

WO U L D

OF

PERHAPS

FROM

WHOLE

WITH

OF

ALWAYS

FERRI C YA NI DE

e x p e r i m e n t s

CONCENTRATION

THAT

OF

FERRICYANIDE

ALIQUOT

OF

YELLOW

VALUES

WHICH

AN

COLOR

THE

MEASUREMENT

CONCENTRATION

IN

be

THE

COLOR

YELLOW

to

OF

DESTRUCTION

PRELIMINARY

ALKALINE

ap pe ar

INTERFERENCE

PRODUCTS

OF

THROUGH

p r i n c i p l e

an a lo g u e s

THE

ANALOGUES

RELATIVE

OF

p y r i d i n i u m

SHADE*

METHOD

CARRIED

A

o ther

TWO•

PART

A

F OR

CONTAIN AND

8)

WHICH

TOO A N D 1 5 0

BLANKS

ARE

1 5 ^ N a OH . I

Further ASSAY l BUT

METHOD

THE

s t u d i e s

(SUCH

FOLLOWING

on

AS

the

p o s s i b l e

ISOBUTANOL

OBSERVATIONS

m o d i f i c a t i o n

EXTRACTION)

MA Y

BE

NOTED I

WE RE

of

the

NOT

MADE j

,

r 1.

T he

REAGENT

concentration

of

IS

NOT

IMPORTANT,

2.

All

the

compounds

a lo n e

.

Th

made

strongly

UTES

BEFORE

GREAT

u s

if

THE

CASE

DUE

TO

OF

THE

SECONDARY

3}

THE

THE

GROUP OF

REACTIONS.

tN

OXIDIZED

IN

ALALOGUES ALL

CASES,

SOLUTION

IS

THIS

R E A S ON

THE

IN

THE

alkali

are

f ir s t

a

MIN

few

THERE

IS

A

OBTAINED. VERY

WITH

CASE

IN

P R OB A B L Y

CONSEQUENT

OF

T HE

ABVIOUS.

ACIDIFIED THE

AND

IN

DISAPPEARS

REAPPEARS

S O L U T I ON

COMPLETELY

FLUORESCENCE

to

stand

IS

RING

FLUORESCENCE

MAKING

to

INTENSITY

ANALOGUES,

N OT

o x id iz in g

F E R R I CYAN I D E ,

THE

ARRESTS

OF

THE

the

s e n s it iv e

allowed

THIAZOLIUM

IS

in

analogues

THE

4.

VELOPMENT

then

OF

II

ON

C

these

FLUORESCENT

UNDIMINISHED VITAMIN

alkali

however

of

and

ADDITION

OPENING

PYRIDINIUM

are

so lutio n s

a lk a lin e

DECREASE

THE

AND

,

the

T HE

CASE

THE

IMMEDIATELY

ALKALINE

AND

IF

(pH

IMMEDIATELY

OF

THE

8

).

THE

DE

PYRIDINIUM

/

ALALOGUES in g s

.

T

h is

MECHANISM T he OF L

WITHOUT m ight

OF

THE

ik e w is e

,

in

there

the

analogues

I N AT I ON

OF

THE

of

.

use

n CHROMEn

FROM is

of

THE

THE

The

in

WITH

T HE

the

in v e s t ig a t io n

the

t im e

-

lag

PYRIDINIUM

of

known

in

about

fluorescent

observations

MECHANISM

the

READ**

of

the

development

ALALOGUES

2 , 6 - L U T I D I N I UM A ND

ACCEPTED

GALVANOMETER

PRODUCTION.

nothing

formation

2 , 4 - t h I A Z O L I UM, NIUM

be

s ig n if ic a n c e

FLUORESCENCE

volved

INTERFERING

the

THE

NOT

mechanism

products

c e r t a in l y

FOR

IS

from

KNOWN in

­

the

2 ,4 ,6-C 0LLID Im e r it

THIOCHROME

A

re

-

exam

FORMATION

VI sable

a

T ‘ He

Comparative

Fluorescence

of

Th ia m in e

An al o g u e s

Ga l v ; n o m e t e r

UG T H I A M I N E E Q U I V . I N 1 0 ML A Q . SOLN of

4

2

COL OR

ading

b

8

94

118

T3

93

1 0

of

Chrome

1 2

An a l o g u e

2 , 4 - t h 1A Z O L 1u

m

( 1 I a)

24

43

69

2>5-

m

( 11b)

16

34

51

60

119

t h

2 ,4 ,6 -

IAZOLiu

c o l l id in

I

um

(IB) 2,6-L U T l Ne

Re

o pyr

d in iu m

ITH t

( I

a m in e

c

(1

) a

)

|

134

1 1 2

DARK-BLUE V Iolet

tt BL UE

46

99

38

TT

it 1 1 0

L I GHT GREEN

1SH

BL UE

110

T h 1AMINE

Note:

The in

L

reagent

15% Na OH,

used

vvas

fre s h ly

BL UE

0.025%

potassium

prepared.

fe r ri cyanide

vi

r

Table The

i

B

Ra t e o f Fl u o r e s c e n c e De v e l o p m e n t i n Ox i d a t i o n o f t h e T h i a m i n e A n a l o g u e s

iy

Compound

CXKjitft'v

0.5

1 . 0

MIN.

MIN.

n n te .

X 'n 'fe .r V * ?

flfh /in * 1 0

MIN.

A -j-ftA .

J i r r * * . y » * ! < /* - .

60

4

MIN.

HRS .

24

HRS

2 , 4 - T H 1A ZOL IUM

( i n )

43

43

43

41

2 , 5 - T H 1AZO LIU M

(llB )

34

34

34

34

2 ,4 ,6 -

(1

)

0

0

30

50

119

117

(1c)

0

0

20

46

46

46

(1A )

0

2

76

77

77

76

110

109

110

108

2

,

6

c o l l id in iu m

-lu tid in iu m

Ne o p y r i t h i a m i n e * Th i

*

a m i

ne

b

107

THE FULL FLUORESCENCE I S O B T A IN E D AFTER 1 0 M I N U T E S . IT I S FOUND B E S T TO T A K E R E A D I N G S A F T E R T WE N T Y M I N U T E S S I N C E T H E RATE OF D E V E L O P M E N T I S I N F L U E N C E D BY T H E C O N C E N T R A T I O N OF T H E F E R R I C Y A N I D E . T H E R E A G E N T USED I N T H I S I N S T A N C E WAS 0 . 0 2 5 ^ 3 F E R R I C Y A N I D E I N 1 5)3 S O D I U M H Y D R O X I D E .

A l l S A M P L E S E Q U I V A L E N T TO S A M P L E S WERE K E P T I N T H E T h e R E A G E N T U SE D FE R R IC YAN ID E IN

L

in

the

4 GAMMA T H I A M I N E , DARK E X C E P T D U R I N G

IN ALL CASES 1 5p NaOH*

WAS

0 . 0 2 5 fo

ALL O X I D I Z E D MEASUREMENTS.

POTASSIUM

J

l

VI I I

T able The in

Effect

o f

Fe r r ic y a n id e

Fluorescent

th e

Ox i d a t i o n

Products

*005

, 4 - t h i a z o l i um 6

l

in

0 * 0 1

0 . 0 2

0*03

0 * 1 0

73

73

73

72

(11b)

54

56

60

55

53

98

1 0 2

84

78

151

134

113

UG 1

a)

90

UG

amount

aqueous

the

60

1 1

T h 1 AMINE

t h e

of

An a l o g u e s

a)

(

N eopyrithiam ine( 1 5 UG

4

from

UG

2 , 5 —TH I A Z O L I UM 6

Concentration

I n t e n s it ie s

F erricyan id e Concentration 1 Sfo S o d i u m H y d r o x i d e

An a l o g u e

2

C

of

each

s o l u t i o n

*

compound

shown

pr e s e n t

in

10

ml

of

r

-1 R

Ob t a i n e d

th r ou gh

e

m

t h e

a

r

k

Che m ica l

s

c o u r t e s y

of

Me r c k

Co .

and

I nc.

O b ta in e d th ro u g h th e kin d n e s s o f Mr. G. B o n v ic in o . C a lc d . f o r t h e 2 , 4 , 6 - c o l l i d i n i um b r o m i d e , H-j g N . B r , H g O . : C, 4 9 .2 6 ; H, 6.1 5 Found

:

C,

4 8 .4 7 ;

H,

Ob t a i n e d thr ou gh t h e k i n d n e s s Ca l c d . for C 1 3 H1 8 N 4 B r 2 .

of

Fo und:

C,

3 9 .1 3 ;

H.4.41

k i n d n e s s

I b .

2 , 4 , 6 -T R im e thy l- N - 2 - methyl- 4 - a m ino- p y r im id y l ( 5 ) M E T H Y L - P Y R 1 D l N I U M - B R O M 1 0 E - H Y D R O B R O M 1 DE

Mr .

G.

C, C. Mr .

2 , 6 - 0 1 METHYL-N- 2 -M E T H Y L -4 -A M IN 0 -P Y R IM ID Y L ( 5 )M E T H Y L —P Y R 1 D 1 N 1 U M —B R O M 1 D E —H Y D R O B R O M I D E

*

3 4 .2 3 3 .8 2 * G.

2,4-DlM ETHYL-NMET HYL

H ,4.15 H .4.30

2-METHYL-4-AMINO-PYRIMlDYL

( 5 )-

Ca l c d .

for

th e

k i n d n e s s

of

3 4 .2 ; 3 3 .8 2 ;

Mr .

A .J.

2,5-DIMETHYL-N METHYL

H .4.15 H.4.01

124

UG

118.5

390.10

105.5

2-METHYL-4-AMIN0-PYRIMlDYL

(5 ) -

37 .2

118

396

118

I

- T H 1A Z O L 1 U M - B R O M I O E - H Y D R O B R O M I D E

A ,>trt

G.

396

‘fe--p C ,

H*oH-

Eu s e b i. 302

0X YTH 1AMINE-CHLORIDE

;

h ,3 .3 3

3 7 .5 2 ;

H .3 .2 8

N-

2 - M E T H Y L - 4 - A M I N O - P Y R 1 M 1D Y L - ( 5 ) - M E T H Y L THIAZ0LIUM-6R0MIDE-HYDR0BR0MIDE.

BENZ-

89 .5

er

Bo n v ic in o . l l l B .

Found:

L

Mr .

C, C,

EQ U I V A L E N T

Bo n v i c i n o .

C, C,

C ^ H .^ N .S B R o . C a lc d :

THIAMINE

- T H 1A Z O L 1 U M - B R O M 1D E - H Y D RO B R OM I D E

y— thr ou gh

UG

fil.'

1 11A .

Ob t a i n e d

ILOO

404.13

Bo n v ic in o .

l l B .

of

420

Ne o p y r i t h i a m i n e - s r o m id e - hydro bro mide

1t A.

of

Formula

Nam*

I a .

1C.

C a lc d : Found:

C ommon

Bo n v i c i n o . 1- 1,4. 61

Ob t a i n e d t h r o u g h t h e k i n d n e s s Ca lc d . for C i j H- j c N . S B R o .

the

G.

4 0 .0 ;

C a lc d : Found:

thr ou gh

Mr . C,

of

or

1 Mo l . V«' GT.

5.21

Ca lc d :

Ob t a i n e d thr ou gh t h e k i n d n e s s Ca l c d . for C T i^ g N ^ S B R g .

Ob t a i n e d

1

418

124

r

i

BI BLi OGRAP HY

1.

Green,

2.

S p i t z e r , E. H. , A. C o o m b e s , C . E l v e h j e m , and W. W i s n i c k y , P r o c . S o c . E x p t l . B i o l . Me d . , 4 8 , 3 7 6 ( 1 9 4 1 ) .

3.

g r e e n , R . G . , W. C a r l s o n 2 1 , 243 ( 1 9 4 1 ) .

4.

Gy o r g y ,

5.

W oolley,

6 .

wolf,

7.

De u t s c h , H . F . Me d . , 5 3 ,

and

8 .

L i e c k , H . a nd (1944).

Agren,

9.

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997

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G.,

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KRAMP I T Z , L . (1944).

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Elvehjem (1943).

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G.,

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Ac t a

Ph

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3d.

Soc.

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ysio l

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9_,

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203,

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t

Scand.,

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60, 268, — BlOCHEM., 1 4 , 8 3 , —

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Ex p t l .

Canada,^,

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and

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ARCH.

12.

H. 119

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Physiol.

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agren,

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63

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L 19.

92,

Bi o

66,

152,

l

.

(1945).

9,

Me d . ,

r

1

20.

Re

2 1 . a)

b)

,

d d i

K.

and

K.

Gi

r i

,

En z y m o l o g i a , £ 3 ,

M elnick, 30,

D . l . , M. H o c h b e r g 81 ( 1 9 4 5 ) .

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Handbook

of

1947.

b)

(1949).

H e n n e s s y , D . J . a nd S . W a r n e r , A b s t r a c t s , p . 2 0 b , A m . C h e m . S o c . , 1 0 9 t h Me e t i n g , At l a n t i c C i t y , N . J . (A p r il, 1946). Ba r n h u r s t , J . a n d He n n e s s y , D . J . Pr i v a t e C o m m u n i c a ­ tion - Un p u b l i s h e d d a t a .

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H e n n e s s y , D . J . and C e r e c e d o , 6£ , 179 ( 1 9 3 9 ) . H e n n e s s y , D . J . , C e r e a l Che m.

N utrttion,

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Am.

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2

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25.

O b e r m e y e r , H. (1945).

26.

v an

27.

S ch u ltz, A.L. Atkin A n a l . E d . , 14^,

28.

R e d d i , K . , K. (1943).

29.

W illiam s, 1856

30.

Sealock, 379

31.

Mc I

32.

Agren,

33.

Sealock,

34.

C l a r k e , H . T. a n d S . 1876 ( 1 9 3 5 ).

35.

O g s t o n , A . G . a n d R . A. P e t e r s , b i o c h e m . J . , 3 0 , 7 3 6 , (1936). Moggridge, R . C . G . a n d A.G. O g s t o n , i b i b , 2 9 , 8 6 6 , (1935).

a nd

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Ling

Chen,

Broquist, I n d . En g .

Giri

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oas,

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H.

Livermore,

,F. and (1944). G.,

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Fr o s t ,

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and C . N . F r e y , 35 ( 1 9 4 2 ) . and

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£,

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221

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238,

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66, —

57,

156, ------1317,

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987,

57, —

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37.

B e l o f f , R . L . a n d K . G. S t e r n , J . B i o l . C h e m . , 1 5 8 , 1 9 , ( 1 9 4 5 ) . --------------------------------------------------------------------------

38.

W i l l i a m s , R . R . , R. W a t e r m a n and J . Ch e m . S o c . , 5 7 , 5 3 6 ( 1 9 3 5 ) .

3 8 (a) .

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