The binding of organic ions by proteins: The effect of structural isomerism

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NAME AND ADDRESS

LATE

NORTHWESTERN UNIVERSITY

THE BINDING OP ORGANIC IONS BY PROTEINS: THE EFFECT OF STRUCTURAL ISOMERISM

A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS for the degree DOCTOR OF PHILOSOPHY FIELD OF CHEMISTRY

By RAYMOND KENNETH BURKHARD

EVANSTON, ILLINOIS June 1950

ProQuest Number: 10101230

All rights re se rv ed INFO RM ATIO N TO ALL USERS The q u a lity o f this re p ro d u c tio n is d e p e n d e n t u p o n th e q u a lity o f th e c o p y s u b m itte d . In th e unlikely e v e n t th a t th e a u th o r d id n o t sen d a c o m p le te m anuscript a n d th e re a re missing p a g e s , th e s e will b e n o te d . Also, if m a te ria l h a d to b e r e m o v e d , a n o te will in d ic a te th e d e le tio n .

P roQ uest 10101230 Published by P roQ uest LLC (2016). C o p y rig h t o f th e Dissertation is h e ld by th e A uthor. All rights reserved. This work is p ro te c te d a g a in s t u n a u th o rize d c o p y in g u n d e r Title 17, U nited States C o d e M icroform Edition © P ro Q u est LLC. P roQ uest LLC. 789 East Eisenhow er P arkw ay P.O. Box 1346 A n n Arbor, Ml 48106 - 1346

ACKNOWLEDGMENT The author wishes to acknowledge the invaluable assistance of Dr* Irving M* Klotz through whose constant encouragement and advice this research was realized. The author also wishes to acknowledge the National Institutes of Health for their support of this research in the form of a fellowship*

TABLE OP CONTENTS

INTRODUCTION

1

STATEMENT OP THE PROBLEM

3

EXPERIMENTAL Preparation and Sources of Azo Dyes

8

Preparation and Sources of Proteins

22

Preparation of Buffer Solutions

25

Techniques

26

TREATMENT OP EXPERIMENTAL DATA

35

RESULTS AND INTERPRETATIONS Spectra of Frotein-Dye Complexes

37

Free Energy Changes on Complex Formation

47

Complexes Involving Modified Proteins

51

CONCLUSIONS

55

BIBLIOGRAPHY

57

VITA

60

APPENDIX

61

INTRODUCTION

i

Many of the reactions that comprise the phenomena termed life can he reduced to chemical reactions which involve enzymes, and hence proteins, as reaction sites* Thus if one desires to make a fundamental study of the nature of biological reactions, it behooves one to make a study of the nature of the reaction sites on protein molecules. Many aspects of the nature of the protein molecule have been well established.

The chemical basis for these

complex molecules was laid In the nineteenth century by the organic chemists who were able to isolate and identify degradation products from some of the more common proteins. The twentieth century and the development of new physico­ chemical tools resulted in a shift from this organic ap­ proach to the problem of protein structure. X-ray diffraction studies, for example, have supplied us with bond distances that might occur in proteins and also a physical picture of some of the simpler proteins. Determinations involving osmotic pressure, diffusion, x-rays, light scattering and the ultracentrifuge give us molecular weights for these complex molecules.

Their

size and shape may be determined from measurements in­ volving diffusion, viscosity, double refraction of flow and dielectric dispersion*

But while the gross nature of

the protein molecule has been established by these tech­ niques,

35 these data give us little indication as to the

2

nature of the reaction sites on the protein. Several techniques have been exploited to study the nature of these reaction sites. tion of proteins.4#35

One has been the titra­

Another has been the reaction of

proteins with specific reagents such as dinitroflourobenzene and identification of the subsequent hydrolytic products.33 The interaction of small particles with proteins pro­ vides us with still another method of studying the nature of protein reaction sites.

One phase of such studies is

concerned with enzyme-substrate interactions which can be carried out in living systems.3^

Another phase is concerned

with in vitro studies with crystalline proteins and small particles.

Such an approach to the problem of protein

reaction sites has been exploited by Klotz and many others 13,16,20,23,24,31,3B

The use of azo dyes in this respect is of particular interest for here one is dealing with highly colored com­ pounds and from spectral data one can obtain valuable infor­ mation in regard to the nature of the protein-dye complex, and hence the nature of the reaction sites on the protein. This is a report of investigations involving azo dye and protein interactions with special emphasis on the ef­ fect that structural modifications in the azo dye have on these interactions.

An interpretation of these data in

terms of protein reaction sites and the configuration of the protein surface is given*

STATEMENT OP THE PROBLEM

3

The nature of a particular protein-dye complex de­ pends on both the nature of the protein and of the dye.-**® For example, it has been shown that the isomers and analogs of methyl orange behave differently with the two proteins bovine serum albumin (BSA) and human serum albumin (HSA) depending upon the structure of the azo dye*3.6

If one

examines these two proteins it is soon apparent that they are very similar as far as molecular weight, size, shape and amino acid content are concerned (Table No. 1).

However, the

fact that they behave differently toward these azo dyes suggests a quite subtle difference in protein structure. In the case of the BSA complexes, it was observed that the interactions with ortho, met a and para methyl oranges were all of the same nature at pH's 5.8, 6.8 and 9.2,

CH N -N

Ortho -methyl orange

Meta-methyl orange

Table No, 1

Item

Bovine Serum Albumin

Mol* Wt*2 Shape^S

69,000 prolate spheroid

69,000 prolate spheroid a » 38 A; b * 150 A

Size**8 Amino Acids glycine alanine valine leucine lsoleuclne proline phenyl alanine cystdime Half-cystine methionine tryptophan arginine histidine lysine aspartic acid glutamic acid glutamine serine threonine tyrosine

Human Serum Albumin

Residues/mole 18 39 73 15 34 26 6 32 4 2 25 17 59 56 37 43 30 38 21

Resid 15 -46 64 9 31 33 4 32 6 1 25 16 59 55 39 44 25 29 18

e

4

/N- ^

^ — N= N — ^

^ — SO3 Na

Ch*3

Par a-Methyl Orange

The absorption spectrum of this type of binding is char­ acterized by a decrease in intensity and a shift toward shorter wave lengths * However, in the case of the HSA complexes the absorp­ tion spectra depend upon both the pH of the solution and the structure of the azo dye*

With the ortho-methyl orange-

HSA complex the absorption spectrum is almost identical to that for the BSA type complexes*

But with the meta and

para-methyl orange-HSA complexes the absorption spectra depend on the pH and are quite different from those of the BSA complexes especially at high p H ’s* Selected data from these studies are shown on Figures Ho* 1-6* The phosphonic and arsonic analogs of methyl orange were observed to give similar results*

These data are

shown on Figures Ho* 7-9* Thus it appeared that these phenomena are due to the structure of the azo dye and not to the nature of the acidic group on that molecule*

FIGURE NO. 1

20,000 HSA

BSA

15 000

420

460

440 Wavelength

480

500

mmu

Spectra of ortho-methyl orange and lta complexes with bovine serum albumin and human serum albumin at pH 7.6.

FIGURE HO. 2

DYE

20,000

HSA BSA

15,000 ..

10,000

..

5,000 ..

420

440 Wavelength

460

480

mmu

Spectra of ortho-methyl orange and its complexes with bovine serum albumin and human serum albumin at pH 9.0.

FIGURE NO. 3

BSA

DYE HSA

20,000

15,000

10,000

5,000

420

460

440 Wavelength

480

500

mmu

Spectra of meta-methyl orange and its complexes with bovine serum albumin and human serum albumin at pH 6.8.

FIGURE NO. 4

HSA 30,000.

BSA

DYE

440

460

25,000..

20,000..

15,000..

10,000

5 ,0 0 0 ..

420

Wavelength

500

mmu

Spectra of meta-methyl orange and its complexes with bovine serum albumin and human serum albumin at pH 9.1*

FIGURE NO. 5

DYE

HSA 25,000

BSA

20,000

15,000

10,000

5,000

420

440 Wavelength

500 mmu

Spectra of methyl orange and its complexes with bovine serum albumin and human serum albumin at pH 6*

FIGURE HO. 6

HSA 35,000 _

30,000 DYE

20,000

5, 000.'

440 Wavelength

460

480

500

mmu

Spectra of methyl orange and Its complexes with bovine serum albumin and human serum albumin at pH 9.

FIGURE HO. 7

HSA 25,000.

BSA

20,000..

DYE

5 ,0 0 0 .

10,000

5,000 ..

420

440

460

480

500

Wavelength mmu Spectra of meta-pho sphome thy 1 orange and its complexes with bovine serum albumin and human serum albumin at pH 9.2.

FIGURE NO. 8

HSA DYE

20,000

15,000

10,000

5,000

420

460

480

Wavelength mmu Spectra of para-pho sphome thy1 orange and Its complexes with hovine serum albumin and human serum albumin at pH 9.1.

FIGURE NO. 9

HSA

2Q,000

DYE BSA

15,000

10,000

440

460

Wavelength mmu Spectra of para-arsonomethy1 orange and its complexes with bovine serum albumin and human serum albumin at p H 9.0.

5

If one is to be able to interpret these data in terms of the nature of the p rote in-dye complex, then one should examine absorption spectra of an azo dye itself under var­ ious conditions which are definitely understood in terms of molecular changes.

Comparison of these spectra with those

obtained above might reveal the nature of the interaction observed* Figure Ho* 10 shows the spectra for methyl orange in alcohol,^ o.l N sodium hydroxide and 0*1 N hydrochloric acid.39

it is apparent that the spectra of the BSA com­

plexes and the ortho-methyl orange-HSA complex resemble that of methyl orange in a non-Ionizing solvent such as alcohol.

The spectra of the met a and par a-methyl orange-

HSA complexes resemble that of methyl orange in an acidic solution when the pH is sufficiently high.

Thus the type

of binding in these two cases appears to be Qulbe different, and could involve different forms of the azo dye. It has been shown that the basis of the BSA type of binding is the electrostatic interaction between the charged acidic group on the dye and a positively charged amino group in the protein.23

Also the similarity of the BSA type

spectra and the spectrum of an azo dye in a non-ionizing sol­ vent suggests an electrostatic interaction since In a non­ ionizing solvent the cation and the anion of the dye would probably be associated. Thus, in the BSA complexes and the ortho-methyl orangeHSA complex It could very well be that only the positive

FIGURE NO. 10

5 0 ,0 0 0 ..

40,000 ..

30,000 . ALCOHOL

0.1 N / BASE

20,000 ..

440

480

530

Wavelength mmu Spectra or methyl orange in various solutions•

6

amino group of the protein and the negative acidic group on the dye are involved in the interaction observed.

In the

case of the met a* and para-methyl orange-HSA complexes the acidic type spectra at high pH*s suggests a different type of binding, one in which the

acid form of the dye is

involved.

The acid form of an azo

dye Is characterized by

thead­

dition of a hydrogen ion to one of the nitrogen atoms of the dye.

Thus, it appears that in the meta and para-methyl

orange-HSA complexes* not only is the charged acidic group of the dye involved in the complex but also one of the ni­ trogen atoms*

If one could determine which of these nitro­

gens is Involved It would then be possible to determine the distance between the two sites on the protein surface. Evidence has been accumulated indicating that the phenolic group of the amino aeid tyrosine is responsible for the acidic type spectra of the HSA complexes.16 group could possibly share a

Such a

hydrogen atom with a basic

nitrogen by means of a hydrogen bond.

Iodlnation

of

the

protein should destroy the acidic type spectrum for the HSA complexes due to the fact that the phenolic group in the protein would now lose its hydrogen and become negatively charged.15, figure Ho* 11 shows that this is actually the case. Thus while the nature of the possible protein binding sites had been determined, the nature of the binding sites on the azo dye and the effect of structural isomerism in

FIGURE NO. 11

4 0 ,0 0 0

HSA

3 5 ,0 0 0

50°/« IO D O -H S A ' 3 0 ,0 0 0 DYE 2 5 ,0 0 0

100°/. ^

IODO-HSA

€ 1 5 ,0 0 0

5 , 0 0 0 ..

440

460 480 Wavelength mmu

500

Spectra of methyl orange and its complexes with human serum albumin and iodinated human serum albumins at pH 9. S.

7

the azo dye had not been fully elucidated. It was thus desired to determine first whether the above phenomena could be extended to the carboylic analogs of methyl orange.

Having settled this question the next

step would be to obtain quantitative binding data for the three Isomeric azo dyes in order to determine the effect that structural isomerism had on the binding process.

The

nature of the second binding site in the HSA complexes should then be Investigated and an explanation for that phenomenon advanced.

To complete the picture it was desired

to extend the work of previous Investigators wherein the effect of protein modifications were studied. From all these data then it was hoped that a clearer picture of the protein-azo dye complex could be obtained; and hence a clearer picture of the reaction sites on these proteins*

EXPERIMENTAL

8

Preparation and Sources of Azo Dyes

Methyl red was obtained from Merck and Co.

CH, V y

It was

N =N — '

CHCOOH purified by extraction with toluene in a Soxhlet extractor followed by crystallization first from toluene and then from a pyridine-water mixture until its melting point was constant.

The dye was dried at 110* C. m.p. observed (uncorrected) m.p. literature^

178-17$f C 181-182*0

Meta and para methyl reds were prepared according

CH N =N CH.

COOH

to the procedure for methyl red appearing in Organic

9

Syntheses.9 8*5 g. of met a or para-amlnobenzoic acid were dissolved in 300 ml. water containing 16 ml. concentrated hydro­ chloric acid. ml. water. 0-10° C.

4.5 g. sodium nitrite were dissolved in 10

Both of these solutions were then cooled to

The sodium nitrite solution was then added to the

acid solution using a buret with the tip beneath the surface of the liquid.

11 ml. of dimethyl aniline were added

dropwise followed by 6 ml. of a solution of 8.5 g. of sodium acetate in 15 ml. water.

This mixture was then

stirred for several hours and allowed to stand overnight in a cold room.

After that time the remainder of the

sodium acetate solution was added followed by 15 ml. of 40$ sodium hydroxide.

After standing for an additional 24

hours the solution was filtered, the crystals washed with water, 10$ acetic acid, again with water and then dried. Having obtained the crude methyl reds, they were each suspended in 100 ml. boiling methyl alcohol and on cooling again filtered, washed and dried.

The dyes were then

extracted with toluene in a Soxhlet extractor and crystal­ lized from toluene and then a pyridine-water mixture until constant melting points were obtained.

The dyes were dried

at 110° C. Compound

m.p. observed (uncorrected)

m-methyl red p-methyl red

199-200* C 271-273 C

m.p. literature40 (corrected) _ 210* C ---

10

The samples were analyzed for nitrogen content. Compound__________ Calc__________ Pound m-methyl red p-methyl red

15.61 15*61

15.88 15.85

As a further check on the purity of dyes prepared in this manner, met a-methyl red was subjected to chromatographic analysis.

The column used was 60-200 mesh alumina that had

been washed with hydrochloric acid, water and then activated. The dye was placed on the column and eluted with three sol­ vent s-dilutb hydrochloric acid, dilute potassium hydroxide, and basic potassium sulfanilate.

In all cases only one

component was obtained. *Para-aminoazobenzene was obtained from Eastman Kodak

and was Eastman Grade. Para-azobenzenesulfonic acid was prepared by S. Preis

Chromatography was done by D* Zaukelies of Northwestern University.

it

11

of Northwestern University by sulfonation of azobenzene,

On

recrystallization from water the trihydrate was obtained as established by titration of the dye. Mol, Wt, Gale,

Mol, Wt, Found

316

316

4-dimethyl amlno-2» 2* -dimethylazobenzene-4 -sulfonic acid was prepared by coupling diazotized 2-aminotolu@ne-5-

CH N = N

CH sulfonic acid with N, N-dlmethyl-meta-toluidine, The amine half of this dye was prepared by methylation of meta-toluidine according to the procedure of Evans and Williams,6

The acid half of the dye was prepared by sulfona­

tion of ortho-toluidine according to the procedure of Schultz and Lucas,^6

The coupling reaction was carried out according

to the procedure outlined by Fieser,8 To 85 ml, of freshly distilled meta-toluidine were added 159 ml, of methyl sulfate in 15 ml, lots; each addition fol­ lowed by enough 30/C sodium hydroxide to neutralize the methyl sulfuric acid formed.

The reaction was stirred vigorously

and the temperature kept below 30 C during this addition,

A

pinch of dry phenolphthalein was added to aid in the neutral­ ization process.

After the reaction was complete several

hundred ml, of 30$ sodium hydroxide were added and the amine

12

allowed to oil out.

The amine was separated from the alka­

line solution and an ether extract of the aqueous layer was added to the amine.

After drying the ether solution with

sodium hydroxide the ether was evaporated Whd the amine treated with acetic anhydride until heat was no longer evolved.

The resulting solution was then distilled under

vacuum and the fraction boiling at 65-66° c/15 mm. was col­ lected.

Mtrosation of this product by the procedure of

Wdrster and Riedel4**- gave 2-nitro so-5-dimethyl amino toluene which melted at 91-92° C. (m.p. literature 92°C). The sulfonic acid was prepared from ortho-toluidine and fuming sulfuric acid.

80 ml* freshly distilled ortho-to-

luidine were placed in a three necked flask in an ice bath. To this was slowly added with stirring 100 ml. 20$ fuming sulfuric acid.

After the addition, the ice bath was removed

and the mixture was heated in an oil bath for 10 hours at 180° G.

The molten

mass obtainedwaspoured into cool water

and the crude acidprecipitated.

It was then decolorized

and recrystallized twice from water and then dried.

Titra­

tion of the product showed that it was mono-sulfonated. Mol* Wt. Gale. 187

Mol. Wt. Found 184

To achieve the coupling reaction 9.4 g. of the 2aminotoluene-5-sulfonic acid and 2.65 g. of sodium carbonate were mixed in 100 ml. water and heated.

On cooling 3*7 g*

sodium nitrite were added and the solution poured on 50 g« cracked ice and 10 ml. concentrated hydrochloric acid.

The

13

diftgonium salt precipitated at this point.

To this suspen­

sion of the diazonium salt was added a mixture of 6.3 ml. N>N-dimethyl-meta-toluidine and 3 ml. glacial acetic acid o keeping the temperature near 0 C. After a few hours 35 ml. of 20$ sodium hydroxide were added to precipitate the redorange dye.

To purify the crude dye it was placed in a

Soxhlet extractor and extracted with water; the aqueous solution was evaporated and the powder was then put back into the Soxhlet extractor and the operation repeated using toluene as a solvent.

After this extraction the cup was removed from

the Soxhlet and dried to remove the toluene.

It was then

placed in the Soxhlet again and the dye extracted with water. The dye was then recrystallized from an acidic alcohol water mixture several times and finally dried at 110 C.

The dye

was titrated to establish its neutral equivalent* H.E. Calc. N.E. Found 333 3 33i 6 To determine whether the coupling might have taken place in an unexpected position In the N, N- dimethyl -metatoluidine the dye was reduced in a neutral sodium hydrosulfite solution followed by ether extraction of the amine and Identification by acetylation. m.p. derivative 158-158.5° C

m.p. literature^ 158° C

Since only one derivative of this amine could be found in the literature, the amine was prepared in another manner and acetylated.

Witrosation of N,N-dimethyl-meta-toluidine

gave the nitroso derivative mentioned earlier*

Reduction of

this with acid stannous chloride gave the amine desired. Aeetylation with aqueous acetic anhydride gave the derivative desired*

The melting points of the acetyl derivatives ob­

tained were identical and on mixing equal parts of the two there was no change in melting points*

It was thus concluded

that the coupling had taken place in the position para to the dimethyl amino nitrogen atom* Methyl orange was obtained from Merck and Co*

It was Merck Reagent Grade* Ethyl orange was prepared by coupling diazotized sul-

fanllic acid with diethylaniline according to the procedure described for 4#-dimethyl amino-2,2* -dimethyl azobenzene-4 sulfonic acid*

The dye was analyzed for nitrogen content. 1 N Calc*

11.8

%

H Found 11*3

15

Propyl orange and butyl orange were prepared by coupling

V “ 0

>“ N = N " ^ 0 >_S03H

C3 H7

V QjHg

f

O

/

_ N = N "

^

} '---

_ S 0 ^

sulfanilic acid to the proper amine according to the procedure of Hickenbottom and Lambert*^ was obtained from Eastman Kodak* acetic anhydride*

The dipropyl aniline used It failed to react with

The dibutylaniline was prepared from

butylanlline and butyl bromide according to the procedure of Hickenbottom and Lambert*^ 73 g* n-butylaniline were mixed with 103 ml* n-butyl bromide and refluxed for about 40 hours* used did not give a carbylamine reaction*

The butylanlline The mixture was

then treated with aqueous sodium hydroxide, the amine layer taken off and dried with sodium hydroxide*

The butyl bro­

mide was distilled off and the remaining liquid was treated with acetic anhydride until no further heat was evolved* The resulting mixture was then fractionated to remove all the low boiling liquids (slight amount of butyl bromide with

acetic anhydride and acetic acid),

This product was dis­

tilled under vacuum and the fraction boiling at 88-92°G/2 mm* was collected. dride.

The product did not react with acetic anhy­

The picrate was prepared to give a derivative that

melted at 123-124°C (m.p, literature 125°C)32 The diazotization and coupling was carried out as follows:

11.4 g. dipropylaniline (13.2 g. dibutyl anil ine)

were dissolved in 50 ml. glacial acetic acid and the mixture stirred at 0-10°0.

11.1 g. of sulfanilic acid were diazo-

tized by the previous procedure and the diazonium salt iso­ lated by filtration.

The diazonium salt was then added to

the amine with stirring and after half an hour 24.0 g. sodium acetate was added.

After another 2 hours 16.5 g.

sodium acetate was added followed by an ice cold solution of 71 g. sodium hydroxide in 250 ml. of water. ing mass was filtered and air dried.

The result­

The crude dye was then

purified in the same manner as with 4-dimethyl amino-2,2* dimethylazobenzene-4 -sulfonic acid.

The final crystalliza­

tions were done in acidic alcohol-water mixtures to isolate the free acid form of the dyes.

The melting point of propyl

orange and the decomposition point of butyl orange were obtained. Compound

m.p. or d.p. ob served

m.p. or d.p. literature

propyl orange

224° a

225° C37

butyl orange

204° C

198° C?? above 200 C^2

17

Jlt&tyl brfiiig’© Wftfl titrated to 6&ta.felifl]!i its notltpal ©equivalent. N.E.galc.__________ H.B. Found 389

389*6

Ortho-(para-hydroxyphenylazojbensoio acid was prepared

HO-

by S. Preis of Northwestern University by coupling diazotized anthranilic acid with phenol.

The compound melted at 202-

205*0. Para- (para-hydroxyphenylazoflbenzblc acjdr was prepared

by S. Preis of Northwestern University by coupling paraaminobenzoic acid with phenol.

The compound melted (with

decomposition) at 273°C. Sodium ortho-(par a-hydroxyphenyl azo) benzene sulfonate was prepared by S. Preis of Northwestern University by coupling diazotized orthanilic acid with phenol.

Reduction

18

HO

©f the compound gave gara-ami nophenol as one of the fragments* Sodium para-(para-hydroxyphenylazo)benzene sulfonate was prepared toy S* Preis of Northwestern University toy

— N = N --

‘SOj Na.

coupling diazotized sulfanilic acid with phenol* Ortho-toluidine orange was prepared toy coupling diazotized sulfanilic acid with N, N- dimethyl* ortho -tolui dine *

— N=N

The amine half of this dye was prepared toy methylation of ortho-toluidine according to the procedure of Evans and Williams6 described earlier*

The product was distilled

under vacuum and the fraction tooiling at 70 G /20mm* was

19

collected*

The product did not react with acetic anyhydide

and its picrate melted at 115-117®C (lit. value 116-117°C) The coupling reaction was rather difficult to carry out by the usual procedures due to the greatly reduced reactivity of the amine prepared above*

The procedure that seemed to

give the best results was to prepare and isolate the diazon­ ium salt of sulfanilic acid and then carry out the coupling in glacial acetic acid* 10*5 g. of sulfanilic acid were diazotized and isolated according to the procedure described earlier.

This salt was

then placed in 100 ml. of glacial acetic acid and cooled to 0-10° G*

6*3 ml. of the amine were added and the reaction

mixture allowed

to stand overnight in a cold room.

The

following day the salt form of the dye was prepared by adding concentrated sodium hydroxide to the solution.

The crude dye

was subjected to the usual extraction procedures using the Soxhlet extractor as described earlier. This dye had been prepared earlier by H&ntzch^-O noted that it showed peculiar spectral properties.

This

phenomenon has since been attributed to an inhibition of resonance caused by the methyl group ortho to the dimethylsmino group.

Such a substituent would prevent the molecule

from being planar and thus inhibit the resonance. The spectra observed agree with H&ntzsch’s observations and thus the dye was not used for further studies. Attempted synthesis of g-dimethylamino-^-methyl-azoi)enzeno—4 .-carboxylic acid.

To prepare this dye the coupling

20

of para-aminobenzoic acid with N, N-dimethyl-para-tolui dine

COOH n -c h 3 was undertaken with the *n896

Run No• 3 1 la

1.21 1.70

0.49

14.7

27.4

0.536

-4. 917

2 2a

2.22 2.90

0.68

20.4

27.4

0.745

-4. 654

3 3a 4 4a

3.50 4.60 4.20 6.00

1.10

33.0

27.4

1.20

-4. 456

1.80

54.0

27.4

1.97

-4. 377

Binding Methyl Orange-HSA pH 9.2 Albumins

American Gyanamid

Tube

Cone* (A) c M/lxlO"°

Amount Gone* Diff. _ Bound M/lxlO-5 MxlO-8

Moles Protein xl0~8

1 la

0.83 1 *28

0.45

13.5

25.7

0.526

-5.081

2 2a

1*76 2.53

0*77

23.1

25.7

0.900

-4.754

5 3a

2*65 3.88

1.23

36.9

25.7

1.44

-4.576

4 4a

3*80 5.25

1*45

43.5

25.7

1.69

-4.432

r

log A

Binding Butyl Orange-HSA pH 9.2 Albumin:

American Cyanamid

Tube Gone • (A) M/lxlO- 5

Amount Bound M/lxlO-5 MxlO”8

1 la

0*80 0.91

0.11

3.30

25.1

0.132 -5.096

2 2a

0.39 0.42

0.03

0.90

25.1

0.036 -5.408

3 3a

2.25 2.55

0.30

9.00

25.1

0.359 -4.648

4 4a

3.15 3.35

0.20

6.00

25.1

0.239 -4.501

Cone •

Moles Protein xlO-8

r

log A

Binding Para Methyl Red-HSA pH 9,2 Albumin*

Tube

American Cyanamid

Gone* (A)

„

Cone. Diff.

Amount Bound

Moles Protein

r

log A

1 la

1*23 1.69

0.46

11.5

12.2

0.943

-4.910

2 2a

1*42 1.96

0.54

10.8

12.2

0.885

-4.707

3 3a

3.05 4.10

1.05

21.0

12.2

1.72

-4.515

Binding Para Methyl Red-Acetyl HSA pH 9.2 Albumin:

Acetylated HSA as prepared above. cular weight as 72,300.

Tube

Cone. (A) M/lxl0-5

Cone. Diff. M/lxlO-5

Amount Bound MxlO"8

Moles Protein xlO-8

1 la

1.13 1.25

0.12

3.00

2 2a

1.35 1.43

0.08

3 3a

2.75 3.13

4 4a

4.30 4.90

Assume mole­ r

log A

12.7

0.236

-4.946

1.60

12.7

0.126

-4.870

0.38

7.60

12.7

0.598

0.60

12.0

12.7

0.945

-4.560 -4.366

VITA

Name#

Raymond Kenneth Burfchard

Borns

August 6, 1924, Tempe, Arizona

Education: Elementary and Secondary Public Schools, Tempe, Arizona, 1930-41 Arizona State College, Tempe, Arizona, 1941-43; 1946-47 University of* Washington, Seattle, Washington, 1943 University of California, Los Angeles, California, 1943-44 Northwestern University, Evanston, Illinois, 1947-50 Degrees: Professional Degree in Physics-Meteorology, University of California, 1944 Am

B. in Education, Arizona State College, 1947

Positions Held: U* S* Army 1943-46 Teaching Assistant, Arizona State College, 1946-47 Teaching Assistant, Northwestern University, 1947-48 U* S* Public Health Service Research Fellow, 1948-50 Affiliations: American Chemical Society

Sigma XI phi Lambda Upsilon Kappa Delta Pi