The Microbiological Activity of Lysozyme

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PU R D U E UNIVERSITY

T H IS I S TO C E R T IF Y T H A T T H E T H E S IS P R E P A R E D U N D E R MY S U P E R V IS I O N

ALBIN N. SMOLELIS

e n title d

THE MICROBIOLOGICAL ACTIVITY OF LYSOZYME

C O M P L IE S W IT H T H E U N IV E R S IT Y R E G U L A T IO N S O N G R A D U A T IO N T H E S E S

A N D IS A P P R O V E D B Y M E A S F U L F IL L IN G T H IS P A R T O F T H E R E Q U IR E M E N T S

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

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TO T H E L IB R A R IA N :---I S NX 0 ^ T H IS T H E S IS K> N O T TO B E R E G A R D E D A S C O N F ID E N T IA L .

P n O F E S S O R T N CHAH G E

G RA B. S C H O O L FO R M 0 —3 - 4 9 —1M

THE MICROBIOLOGICAL ACTIVITY OF LYSOZYME A T h e s is S u b m itted t o th e F a c u lty of Purdue U n iv e r s it y by A lb in He S m o le lis In P a r t i a l F u lf illm e n t o f th e R equirem ents f o r th e D egree of D octor o f P h ilo so p h y A u g u st, 1950

ProQuest Number: 27714169

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TABLE OF CONTENTS Pag© i

ABSTRACT........................................

INTRODUCTION.............................................................................................................. 1 HISTORICAL...................................................................................................................4 EXPERIMENTAL..................................................................................

19

M ic r o b io lo g ic a l A ssa y o f Lysozyme• • » • » • • • • • • • * • • • • • • *19 P r e p a r a tio n o f C e lls f o r Use in th e A s s a y * ***** *!9 E f f e c t o f Time and Tem perature o f In c u b a tio n .......... • • • • • • • • • • . 3 3

on th e Standard Curve B u ffe r and D i l u e n t .

.......... . . . . . . * . * . . . * * . * * * * 3 6

Standard Method f o r th e A ssa y o f Lysozym e* .* .* .* 3 8 R e l i a b i l i t y and A p p lic a tio n o f th e A ssa y M eth od ..* * • . 4 8 F a c to r s A f f e c t in g Lysozyme A c t i v i t y . * E f f e c t o f H eat on Lys o

z

y

............ . . . . . * . * 4 7 m

E f f e c t o f Hydrogen Io n C o n c e n tr a tio n * E f f e c t o f Ion s and S a l t s

e

. 47

******52

.......... • • • • • ............ 58

Occurence o f Lys ozyme In B ird Egg A lbum in.................. • • « • 7 0 I n h i b it io n o f Growth and B a c t e r i o l y s i s by Lysozym e••* 7 5 DISCUSSION................................................................................................................81 SUMMARY............................ BIBLIOGRAPHY

89

............................................................................................. 91

L i s t s o f T a b le s an d F i g u r e s L i s t o f T ab le s T a b le 1.

Page T u r b i d i m e t r i c R esp o n se o f M. l y s o d e i k t i c u s and .................. Lys ozyme D i l u t i o n s

*21

2.

E f f e c t o f C e l l S u s p e n s i o n D e n s i t y on B a c t e r i o l y s i s * .* 2 4

3*

A C o m p ariso n o f t h e B a c t e r i o l y t i c R e sp o n se s o f K i l l e d C e l l s .................................................................................... 27

4*

A C o m pariso n o f th e S u s c e p t i b i l i t y o f F r e s h and S to re d U l t r a v i o l e t T re a te d C e l l s * • . . . . • • . . * • • • * # 3 1

5*

A C o m pariso n o f th e S u s c e p t i b i l i t y o f B r o t h - o r S l a n t Grown C e l l s ................ *

* .* *33

6*

E f f e c t o f I n c u b a t i o n Time on L y s i s

* * * * *34

7*

E f f e c t o f T e m p e ra tu re on L y s i s

8*

A ssay R e s u l t s o f R a t K idney *.....................................

9*

Lys ozyme T i t e r s o f F r e s h Hen* s Egg A lb u m in .................. . . 4 3

.....................• • • • • • • * • • 3 6 .* .4 0

10*

Lys ozyme T i t e r s o f D r i e d Egg A lb u m in ..................

*44

11*

Lysozyme T i t e r s o f R at K id n ey E x t r a c t • • • • • • • • • • • • • • • • 4 5

12.

Lys ozyme T i t e r s o f R e n 's Egg A lbum in E x t r a c t ................... 45

13*

E f f e c t o f E x p o su re

of Lys ozyme t o 8 0 ° C............................. . .4 8

14.

E f f e c t o f E x p o su re

o f Lysozyme t o 10 0 ° C . . . . . . . . . . . . . 4 8

1 5.

E f f e c t o f E x p o su re

o f Lys ozyme t o 1 2 1 ° C. • ................... . . 5 0

16.

E f f e c t o f H e a t i n g Lys ozyme a t V a r io u s pH V a l u e s ........... 50

17 *

E f f e c t o f V e r o n a l B u f f e r on V i s i b l e Lys i s . . . . . . . . . . . * 5 4

18.

E f f e c t o f P h t h a l a t e B u f f e r on V i s i b l e L y s i s • • • • • * • * * * 5 4

19.

E f f e c t o f P h o sp h a te B u f f e r on V i s i b l e L y s i s . . ..............

20.

I n f l u e n c e o f V a r io u s S a l t s on B a c t e r i o l y s i s . . . . . . . . . . 6 6

21.

Lys ozyme T i t e r s o f F r o z e n Egg A lb u m in s ........................... . * . 7 2

56

L is t o f T a b les ( c o n t d , ) 22.

Zones o f I n h i b it io n and L y s is Produced by th e Albumin o f T u r tle E g g s.............................................................74

23.

E f f e c t o f Lys ozyme on th e Growth o f B a c t e r i a . . .............. 77

24.

S u s c e p t i b i l i t y o f S . lu t e a t o

Lys o z y m e ......... ................80

L i s t of F ig u re s 1.

T u r b i d i m e t r i c R espo nse o f M. l y s o d e i k t i c u s and Lys ozyme D i l u t i o n s ......................................................................22

2.

E f f e c t o f C e l l S u s p e n s i o n D e n s i t y on B a c t e r i o l y s i s . . . 2 5

3*

A C om parison o f t h e B a c t e r i o l y t i c R e sp o n se s o f K i l l e d C e l l s ....................................................................................28

4.

A C o m p ariso n o f t h e S u s c e p t i b i l i t y o f F r e s h and S to re d U l t r a v i o l e t T re a te d C e l l s . . . . . . . . . . . . . . . . 3 2

5.

A C o m pariso n o f th e S u s c e p t i b i l i t y o f B r o t h - o r S la n t- G ro w n C e l l s ...................................................

.3 2

6.

E f f e c t o f I n c u b a t i o n Time on L y s i s .............................................37

7.

E f f e c t o f T e m p e ra tu re on L y s i s .....................

8.

A ssay R e s u l t s o f R at K idn ey E x t r a c t . ...................................* .4 1

9.

E f f e c t o f E x p o su re o f Lys ozyme t o 8 0 ° C . .

.37

............

49

10.

E f f e c t o f E x p o su re of Lys ozyme t o 1 0 0 ° C................. . . . . . . 4 9

11.

E f f e c t o f E x p o su re of Lys ozyme t o 12 1 ° C

12.

E f f e c t o f H e a tin g Lysozyme a t V a r io u s pH V a l u e s . . . . . . 5 1

13.

E f f e c t o f V e r o n a l B u f f e r on V i s i b l e L y s i s ............................ 55

14.

E f f e c t o f P h t h a l a t e B u f f e r on V i s i b l e L y s i s ................. ... .5 5

15*

E f f e c t o f P h o s p h a te B u f f e r on V i s i b l e L y s i s . ................. . .57

16.

E f f e c t o f P h o s p h a te B u f f e r on V i s i b l e L y s i s . . . .................57

17.

The E f f e c t o f S a l t s on L y s i s ................................................

18.

The E f f e c t o f P o ta s s iu m S a l t s on L y s i s ...................................6 1

................ 51

.6 1

L i s t o f F ig u re s

( c o n t d •)

19.

The E f f e c t o f C alciu m an d Magnesium S a l t s on L y s i s . . . 63

20*

The E f f e c t o f O rg a n ic S a l t s on L y s i s . . ....................

21.

The E f f e c t o f I o n i c S t r e n g t h on L y s i s . . . . .............................65

22.

S u s c e p t i b i l i t y o f S . l u t e a t o Lys ozyme............................ . . S O

.6 3

ACKNOWLEDGEMENT The w r it e r i s in d e b te d and in d eed g r a t e f u l t o P r o f e s s o r S . E . H arts e l l under whose d i r e c t i o n t h i s work was d o n e , f o r h i s u n f a i l i n g i n t e r e s t , g u id a n ce and s u g g e s t io n s d u r in g th e c o u r s e o f t h i s i n v e s t i g a t i o n . G r a te fu l acknowledgem ent i s e x p r e sse d t o th e Purdue R esea rch F o u n d ation f o r th e g ra n t t h a t made t h i s work p o s s i b l e .

i

ABSTRACT Lysozym e, w h ic h was f i r s t d e s c r i b e d by F lem in g i n 1 9 2 2 , h a s b e e n t h e s u b j e c t o f a c o n s i d e r a b l e amount o f r e ­ search ,

Much o f t h e d a t a i n t h e l i t e r a t u r e i s in c o m p le te

o r c o n f u s i n g due t o t h e u n a v a i l a b i l i t y o f a h i g h l y p u r i ­ f i e d p r e p a r a t i o n o f t h i s enzyme.

T h is s t u d y was c a r r i e d o u t

i n an a t t e m p t t o c o n f ir m and e x t e n d some o f t h e g e n e r a l know ledge o f ly s o z y m e , A r a p i d and a c c u r a t e m ethod o f a s s a y f o r lysozym e fo u n d i n n a t u r a l m a t e r i a l s i s d e s c r i b e d .

The method was

b a s e d on t h e i n c r e a s e i n l i g h t t r a n s m i s s i o n o f a s u s p e n s i o n o f M ic ro c o c eu s l y s ode i k t i c u s a f t e r e x p o s u r e t o v a r i o u s co n ­ c e n t r a t i o n s o f ly s o z y m e .

A h ig h ly p u r ifie d c r y s ta lli n e

p r e p a r a t i o n o f lysozym e was u s e d as a s t a n d a r d f o r c o m p a r is o n . M. l y s o d e i k t i c u s was grown i n l a r g e q u a n t i t i e s and t r e a t e d w i t h u l t r a v i o l e t l i g h t , f o llo w e d b y l y o p h i l i z a t i o n . T h is p r o v id e d a l a r g e number o f c e l l s t h a t c o u ld be s t o r e d i n t h e i c e box f o r u s e i n s u b s e q u e n t a s s a y s . The a s s a y m ethod was u s e d t o d e t e r m in e t h e lysozym e c o n t e n t i n f r e s h h e n s f eg g a l b u m in , d r i e d egg a lb u m in , a b e n t o n i t e p y r i d i n e e x t r a c t o f f r e s h egg a lb u m in and a r a t tis su e e x tra c t.

I n th e ca se o f th e k id n e y e x t r a c t th e e r r o r

was l e s s t h a n 7 p e r c e n t , w h ic h r e s u l t i n d i c a t e d t h e r e l i a b i l ­ i t y of th e t e s t .

The s e n s i t i v i t y of th e a s s a y p r o c e d u r e i n

d e t e c t i n g s m a l l o r r a t h e r l a r g e q u a n t i t i e s o f lysozym e i n

n a t u r a l m a t e r i a l s was a n i n t e r e s t i n g f e a t u r e o f t h i s t e s t . U sin g t h e m ethod o f a s s a y i t was p o s s i b l e t o s t u d y q u a n t i t a t i v e l y some o f th e f a c t o r s a f f e c t i n g ly so zym e a c t i v i ­ ty .

V i s i b l e l y s i s o f s u s c e p t i b l e c e l l s h a s b e e n u s e d as a n

i n d i c a t i o n o f lysozym e a c t i v i t y .

In a s e r ie s of d e te rm in ­

a t i o n s i t was shown t h a t c e r t a i n o p tim a l c o n d i t i o n s w i l l p e r ­ m it an i n c r e a s e i n v i s i b l e l y s i s .

The optimum h y d r o g e n i o n

c o n c e n t r a t i o n o f t h e medium f o r maximum v i s i b l e l y s i s was f c u n d t o be pH 6 . 6 ; h o w e v e r, a c t i v i t y was o b s e rv e d b e tw e e n pH 4 . 6 t o 9 . 0 .

I t was fo u n d t h a t i o n c o n c e n t r a t i o n and ty p e

o f c a t i o n p r e s e n t w ere t h e c o n t r o l l i n g f a c t o r s i n l y s i s . S odium , p o ta s s iu m o r ammonium w ere n e c e s s a r y f o r maximum l y s i s ; c a lc i u m and magnesium c a u s e d an i n h i b i t i o n o f l y s i s , a s d i d b i l e s a l t s and u r e a . A s t u d y o f t h e h e a t s t a b i l i t y o f lysozym e showed t h a t h e a t i n a c t i v a t i o n c a n be c o r r e l a t e d w i t h t h e pH o f t h e medium.

At a l k a l i n e pH v a l u e s , lysozym e was r e a d i l y d e n a t u r e d

by h e a t .

The s t a b i l i t y of lysozym e i n a c i d m edia c a n be con­

s i d e r e d one o f i t s

id e n tify in g c h a r a c te r i s tic s .

A s u r v e y o f th e alb u m in s o f a number of b i r d eggs i n d i c a t e d t h e w id e d i s t r i b u t i o n o f h i g h c o n c e n t r a t i o n s o f lysozym e i n b i r d and t u r t l e eg g a l b u m in s . I n an a t t e m p t t o e s t a b l i s h th e s u s c e p t i b i l i t y o f o rg a n ism s to ly sozym e a s u r v e y o f a l a r g e number o f s p e c i e s was c a r r i e d o u t .

S a r c i n a l u t e a was fo un d t o be e x t r e m e l y

s e n s i t i v e t o ly s o z y m e ; i t s o f M. ly s ode i k t i c u s .

s u s c e p t i b i l i t y was e q u a l t o t h a t

I n a d d i t i o n , tw o new s p e c i e s w ere r e -

ill

p o r t e d t o be a f f e c t e d b y ly s o z y m e , S e r r a t i a r u b i d a and B a c illu s b u ty r jc u s »

I t i s o f i n t e r e s t t o n o te t h a t of t h e s e

tw o o rg a n ism s one i s Gram n e g a t i v e and t h e o t h e r i s Gram v a r la b le $

T h is s u s c e p t i b i l i t y o f a Gram n e g a t i v e o rg a n is m may

i n d i c a t e a w id e r d i s t r i b u t i o n o f t h e lysozym e s u b s t r a t e , w h ic h i s now c o n s i d e r e d t o be l i m i t e d p r i m a r i l y t o Gram p o s­ i t i v e o rg a n ism s*

Reprinted from J o u r n a l o f B a c t e r i o l o g y Vol. 58, No. 6, December, 1949

THE DETERM INATION OF LYSOZYME1 A.

N.

SMOLELIS

a n d

S. E. HARTSELL

Laboratories of Bacteriology, Department of Biological Sciences, Purdue University, Lafayette, Indiana Received for publication September 8, 1949

Since the discovery of lysozyme by Fleming (1922) several authors have pro­ posed different methods for determining the activity of various preparations con­ taining this enzyme. Most of these methods were based upon the clearing of dense suspensions of a susceptible organism without concern for accurate quan­ titative results. The isolation of highly purified crystalline lysozyme by Alderton and Fevold (1946) suggested the possibility of a method for assaying lysozyme. The procedure described here provides for the rapid and accurate microbiological assay of materials that show lytic activity considered to be due to lysozyme. Fleming (1922), in his original work, observed the lytic activity of lysozyme either as clear zones on agar plates seeded with Micrococcus lysodeikticus or as a clearing of a suspension of the same organism. Sandow (1926) used serial dilutions of egg white in meat infusion broth inoculated with various species of organisms. After incubation these mixtures were observed for growth, as evidenced by the turbidity of the tubes. This procedure was applied to a study of the different organisms affected by lysozyme, the dilution of egg white capable of producing inhibition or sterilization being noted. Goldsworthy and Florey (1930) devised a scheme of assay which consisted of washing an 18-hour culture of M. lysodeikticus with saline and adjusting the opacity of that suspension to that of Brown’s barium sulfate standard no. 4. Lysozyme was serially diluted so that each succeeding dilution contained only half as much of the enzyme as the one previous. Then 0.5 ml of each dilution was mixed with an equal quantity of cell suspension. The mixtures were allowed to incubate at 38 C for 1 hour. A unit was defined as the least amount of lysozyme necessary to produce complete lysis. Rosenthal and Lieberman (1931) in determining the lysozyme content of infant stools mixed a susceptible sarcina with stool extracts. Visual observations of the mixture were made under the microscope. A disappearance of the sarcina cells indicated lysozyme activity. Boasson (1938) developed a technique involving the use of optical meas­ urements of turbidity. A phenol-killed suspension of the test organism was mixed with various dilutions of lysozyme. The amount and rate of clearing was carefully measured in a Moll extinctometer and correlated with the con­ centration of lysozyme. Since the activity for the known concentration could be observed accurately, it was possible to compare the extent of activity of an unknown and in this manner to determine the amount of lysozyme present. 1 Grateful acknowledgment is expressed to the Purdue Research Foundation for the grant that made much of this work possible. 731

732

A. N. SMOLELIS AND S. E. HARTSELL

[VOL.

58

The method herein reported is somewhat similar to the Boasson technique and to the one employed by Goldsworthy and Florey (1930). Meyer and Hahnel (1946) developed a viscosimetric method for measuring the mucolytic activity of lysozyme. A mucopolysaccharide was used as a sub­ strate for the enzyme. The test was based on the depolymerization of this material, which thereby effected a change in the viscosity of the substrateenzyme mixture. The change in viscosity could be measured and correlated with the lysozyme concentration. The preparation of this mucopolysaccharide fraction is a very involved and laborious procedure, which does not lend itself to a rapid method of assay. Meyer, Hahnel, and Steinberg (1946), in reporting on lysozyme of plant origin, used the viscosimetric method to measure mucolytic activity and an optical method to measure the bacteriolytic activity. The optical method was not very different from those used by other investigators. M . lysodeikticus cells were suspended in a m / 1 5 phosphate solution and mixed with lysozyme dilutions. After 1 hour of incubation at 37 C, 2 drops of normal sodium hydroxide were added and the clearing was read visually. A unit was expressed as the highest dilution giving complete visible clearing under the conditions specified. Hartsell (1948) used a suspension of M . lysodeikticus in phosphate buffer, pH 6.2, mixed with lysozyme contained in rehydrated, spray-dried, whole egg powder. An incubation temperature of 52 C was used, and clearing of the sus­ pension was observed visually. The lysozyme concentration in a sample was expressed as the reciprocal of the highest dilution causing clearing. None of the tests described above appear to be capable of providing accurate results with a minimum expenditure of time. The desire for an accurate and rapid method of assay prompted this study. METHOD

The method for lysozyme assay described here is based on a comparison of light transmissions of crystalline lysozyme dilutions with the values for the substance being tested, after the addition of susceptible cells and incubation. A purified chloride salt of lysozyme2 is used as the standard. This material was chosen because of its availability and constancy of activity. M. lysodeikticus Fleming, ATCC 4698, is sub cultured on yeast water, veal infusion agar3 with 0.2 per cent glucose every 24 hours for 3 days. After the final subculturing a suspension of the organism is made in phosphate buffer,. pH 6.2, and a large number of Roux bottles containing the same medium are inoculated. A heavy suspension as inoculum with a minimum amount of residual moisture on the surface of the agar will give the highest yield of cells. After 18 hours at 37 C the cells are harvested in phosphate buffer. The suspension is then exposed to ultraviolet light in the following manner: A “mediquartz” 2 The crystalline lysozyme was supplied by the Western Regional Research Laboratory, U .S.D .A ., Albany, California, and The Armour Laboratories, Chicago 9, Illinois. 3 Ten per cent veal infusion, 10 per cent yeast water, 0.5 per cent salt, 0.5 per cent pep­ tone, and 2 per cent agar.

1949]

DETERMINATION OF LYSOZYME

733

germicidal lamp is placed about 4 inches above an inclined piece of Venetian glass. The cell suspension is allowed to flow slowly down the rough side of the glass. The procedure is repeated. In this manner the cells are twice exposed to approximately 2,700 microwatts per cm2,4 which provides a cell suspension with but few living cells. The organisms are then collected, shell-frozen, and dried in a vacuum. In this way it is possible to obtain approximately 0.15 g of dried cells per 100 ml of medium. The cells are stored at 4 C until needed. The cell suspension is prepared from the dried cells in Sprenson’s phosphate buffer, pH 6.2, prior to the assay. Rehydration is easily accomplished since lyophilized cells can be readily resuspended. The turbidity of this suspension is adjusted to show 10 per cent light transmission in a Coleman spectrophotometer equipped with a PC-4 filter, at a wave length of 540 mju, and to show 100 per cent transmission with a distilled water blank. From a 1:10,000 stock solution of crystalline lysozyme, dilutions are pre­ pared in Sdrenson’s phosphate buffer, pH 6.2. Twofold dilutions, starting with 1:200,000 and progressing to 1:6,400,000, are made, thus giving a range of 0.0007 to 0.025 mg of lysozyme per 5 ml of dilution used. Prior to the test the unknown should be checked for its activity to determine what dilutions are needed to give the same level of activity as the crystalline control. Before the assay is begun a sufficient number of test tubes are matched so that all tubes will show the same light transmission value with distilled water as a reference. At measured intervals a 5-ml quantity of the lysozyme dilution is mixed with 5 ml of the cell suspension. The same procedure is used for the dilutions of the material being tested. All mixtures are made in duplicate. After 20 minutes’ incubation at room temperature, the light transmissions for the various mixtures are recorded and the concentration of the unknown is deter­ mined. The results of a typical test are shown in table 1. With the transmission values for the crystalline lysozyme mixtures, a standard curve is prepared by plotting the transmission against concentration. A log scale is used on the abscissa of the standard curve. The transmission values for the unknown dilutions are located on the ordinate and projected to the standard curve. By projection to the abscissa from these points, the concen­ tration of lysozyme in each dilution is determined. Multiplication by the dilution factor results in the concentration of lysozyme per ml of undiluted extract. Figure 1 represents the curve for the values in table 1. Among the criteria of adequacy of a test of this nature are the accuracy and reliability of the results. The effectiveness of this test was established in a series of assays using materials that showed lytic activity and were capable of main­ taining this power when stored. The first preparation tested was powdered egg albumin, which upon rehydration in phosphate buffer showed high lytic activity. The results of the assay of this material are given in table 2. It is to be noted that the titers are consistent and that the error does not exceed 7 per cent,6 which is not considered excessive for this type of assay. Some of the 4 All wave lengths are less than 3,400 A. BComputed from the A .S .T M . Manual on Presentation of Data, Supplement A., A.S.T.M ., July, 1947, the data indicate 99 per cent certainty that the error does not exceed 7 per cent.

734

[VOL.

A. N. SMOLELIS AND S. E. HABTSELL

58

differences in the values may be attributed to the difficulty of uniform rehydra­ tion of the albumin. TABLE 1

A ssay results for rat kidney extract LYSOZYME MG PE R ML AVG

DILUTION

2-LOG % T

%T

Diluted* extract

Undilutedt extract

0.0021 0.0013 0.0006 0.0004

0.021 0.026 0.024 0.024

R at kidney extract 0.1627 0.2518 0.387 0.469

68.7 56 41 34

1:10 1:20 1:40 1:60

Crystalline control 1:200,000 1:400,000 1:800,000 1:1,600,000 1:3,200,000 1:6,400,000

0.0848 0.1322 0.2596 0.395 0.505 0.569

82.3 73.7 55 40.3 31.3 27

* Value obtained from figure 1. f Value obtained by m ultiplying the amount obtained from figure 1 by the dilution factor.

.1 0 0 -

& KIDNEY EXTRACT

O

LYSOZYME CONTROL

.200-

* o

o .400-

.500-

.60 0

0.00015 LY S O Z Y M E

0 .0 0 0 3

0 .0 0 0 6

0.0012

CO NCENTRATION MG P E R

0 .0 0 2 5 ML

Figure 1. The result of a typical assay with rat kidney extract.

Other preparations with a lower lysozyme concentration were tested by the same method. A rat kidney extract and a hen's egg albumin extract6 provided 6 Alderton, Ward, and Fevold (1945) bentonite pyridine extraction, as modified by H. Feldmann, Purdue University; personal communication.

1949]

735

DETERMINATION OF LYSOZYME

further evidence for the reliability of the test. These results are given in tables 3 and 4. The values are not absolute titers because the extracts were not prepared for quantitative determinations. The rat kidney and the albumin extract both show the same reliability of titers during storage in the icebox for a period of 4 weeks. TABLE 2

Lysozyme titers of dried egg albumin rehydrated in phosphate buffer, pH 6.2 ASSAY NO,

LYSOZYME

ASSAY NO,

mg per g

1 2 3 4 5 6 7 8

LYSOZYME

mg per g

20.3 19.2 19.8 20.1 20.4 21.1 21.1 20.9

9 10 11

12 13 14 15 16

22.1 20.2 19.1 16.6 18.8 16.0 17.7 21.9

TABLE 3

Lysozyme titers of hen’s egg albumin extract ASSAY NO.

LYSOZYME

ASSAY NO.

mg per m l

1 2 3 4

LYSOZYME

mg per ml

0.80 0.79 0.89 0.91

5 6 7 8

0.85 0.87 0.91 0.91

TABLE 4

Lysozyme titers of rat kidney extract ASSAY NO.

LYSOZYME

ASSAY NO.

mg per m l

1 2 3 4

LYSOZYME

mg per ml

0.023 0.024 0.025 0.022

5 6 7

0.026 0.023 0.025

To further demonstrate the practicability of this test several students con­ ducted the assay using the same source of unknown material. The results were similar and the differences were within the limits of experimental error. SUMMARY

The method of assay for lysozyme described has proved to be sufficiently accurate to recommend its use in testing various materials for lytic activity.

736

A. N . SMOLELIS AND S. E. HARTSELL

[VOL.

58

I t has been successfully applied to the examination of several preparations containing lysozyme (dried egg albumin, fresh hen’s egg albumin, and extracts from animal tissue). The method is rapid and thus makes possible the testing of large numbers of materials in a relatively short time. A method has been described for the preparation of a large number of Micro­ coccus lysodeikticus cells for use in the turbidimetric assay of lysozyme. I t is possible to store these cells at icebox temperatures without any appreciable reduction in their sensitivity when used in assaying lysozyme. Consistent titers for lysozyme activity were obtained in replicate tests. The method has been shown to be adaptable for both high and low concen­ trations of the enzyme from natural materials, and gives reproducible results in each instance. R EFE R E N C E S L. H . 1946 D irect crystallization o f lysozym e f r o m e g g white and some crystalline salts of lysozym e. J. B iol. Chem., 164, 1-5. A l d e r t o n , G., W a r d , W. H ., a n d F e v o l d , L. H . 1945 Isolation of lysozym e from egg w hite. J. Biol. Chem., 157, 43-58. B o a s s o n , E. H. 1938 On the bacteriolysis by lysozym e. J. Immunol., 34, 281-293. F l e m i n g , A. 1922 On a remarkable bacteriolytic element found in tissue and secretions. Proc. R oy. Soc. (London), B, 93, 306-317. G o l d s w o r t h y , N . E ., a n d F l o r e y , H . 1930 Some properties of mucus, with special reference to its anti-bacterial functions. Brit. J. E xptl. P ath ., 11, 192-208. H a r t s e l l , S. E . 1948 T he lysozym e activ ity of rehydrated spray-dried whole-egg pow­ der. Food Research, 13, 136-142. M e y e r , K . , a n d H a h n e l , E . 1946 The estim ation of lysozym e by a viscosim etric method. J. B iol. Chem., 163, 723-732. M e y e r , K ., H a h n e l , E ., a n d S t e i n b e r g , A. 1946 Lysozym e of p l a n t o r ig in . J. Biol. Chem., 163, 733-740. R o s e n t h a l , L ., a n d L i e b e r m a n , H . 1931 The role of lysozym e in the development of the intestinal flora of the new born infant. J. Infectious D iseases, 48, 226-235. S a n d o w , A. 1926 The antibacterial a ctivity of egg w hite. Proc. Soc. E xptl. Biol. Med., 24, 172-175. A l d e b t o n , G ., a n d F e v o l d ,

1

THE MICROBIOLOGICAL ACTIVITY OP LYSOZYME INTRODUCTION In r e c e n t y ea r s many ad van ces have b een made In s t u d ie s on a n t i b i o t i c and a n t i b a c t e r i a l r e la t io n s h ip s in n a tu r e •

A lthough much o f th e em phasis has b een p la c e d on

th e r a p y and p r a c t ic a l a p p l i c a t i o n , i n t e r e s t i n some o f th e n a t u r a lly o cc u r in g sy stem s has b een r e v iv e d .

F lem ing (1922)

f i r s t ob served and r e p o r te d a b a c t e r i o l y t i c a g en t in th e n a s a l mucus o f a p a tie n t s u f f e r in g from a c o l d .

T h is m a te r ia l

w hich had many o f th e p r o p e r tie s o f an enzyme was c a l l e d " lysozym e" .

The organism u sed t o d em o n stra te l y t i c a c t i v i t y

was M icrococcus l y s o d e i k t i c u s , a sa p ro p h y te i s o l a t e d from la b o r a to r y a i r .

By u s in g th e mucus from t h i s p a t i e n t ,

F lem ing was a b le t o c a u se in h i b i t i o n and l a t e r l y s i s o f th e s u s c e p t ib le organ ism .

A d d itio n a l work by Flem ing showed t h a t X

lysozym e co u ld be found in most body t i s s u e s and s e c r e t io n s * T h is le d t o th e b e l i e f by F lem in g , t h a t lysozym e was an a n t i ­ b a c t e r i a l agen t w hich w a s, " In h eren t in a l l an im al c e l l s " and t h a t th e r e was some r e la t io n s h ip b etw een lysozym e and th e grow th o f b a c t e r ia w it h in th e body. Other a u th o rs have s p e c u la te d on th e p o s s ib le r o le and Im portance o f ly so z y m e .

B radford and R ob erts (1939)

b e lie v e d th a t th e h ig h c o n c e n t r a t io n o f lysozym e i n le u c o c y t e s was a s s o c ia t e d w ith th e b a c t e r i c i d a l power o f b lo o d .

2

B l a t t a n d K e s s le r (1937) a tte m p te d t o c o r r e la t e t h e p r e s e n c e or a b sen ce o f lysozym e i n human b r e a s t m ilk and a lo w er r a t e o f I n fa n t m o r t a lit y ,

M cCulloch ( 1 9 4 5 ), in a d i s c u s s i o n o f

ly so z y m e , a t t r i b u t e s th e rem arkable r e s is t a n c e t o i n f e c t i o n o f th e ey e t o th e h ig h c o n c e n t r a t io n o f lysozym e i n t e a r s . S in c e h ig h lysozym e t i t e r s have been o b ta in e d fo r t e a r s and s a l i v a , many i n v e s t i g a t o r s have c a r r ie d out ex p erim en ts d e a lin g w ith o c u la r and o r a l i n f e c t i o n s and ly s ozyme * C r y s t a llin e lysozym e i n la r g e q u a n t i t ie s was prepared r e c e n t ly by A ld e r to n and F ev o ld (1 9 4 6 ).

P re v io u s

t o t h i s tim e crude p r e p a r a tio n s or u n p u r if ie d m a te r ia ls were u sed i n e x p e r im e n ts .

T h is la c k o f a u n iform enzyme p rep ar­

a t io n has r e s u lt e d i n a c e r t a i n amount o f in c o n s i s t e n t r e ­ s u lts .

Each worker In th e f i e l d d e v is e d h is own method o f

a ssa y .

No r e l i a b l e method fo r th e q u a n t it a t iv e d e te r m in a tio n

o f lysozym e has b een r e p o r te d .

No one h a s d e s c r ib e d a s e r i e s

o f optimum c o n d itio n s w hich would r e s u l t i n maximum l y t i c r e sp o n se o f a t e s t s p e c i e s .

T h is la c k o f d e f i n i t e in fo rm ­

a t i o n , w h ich must o f n e c e s s i t y be known b e fo r e c o n c lu s io n s r e g a rd in g lysozym e can be r e a c h e d , prompted t h i s s t u d y . The f i r s t phase o f t h i s work was co n cern ed w ith th e developm ent o f an a s s a y te c h n iq u e w h ich would be s u f f i ­ c i e n t l y a c c u r a t e , a n d , a t th e same t im e , sim p le and r a p id enough t o be a p p lie d t o a la r g e number o f sa m p le s.

Once a

r e l i a b l e method o f a s s a y was e s t a b lis h e d i t was p o s s ib le t o stu d y some o f th e f a c t o r s w hich a re known t o a f f e c t th e

3

a c t i v i t y o f lysozym e#

The a v a i l a b i l i t y o f h ig h ly p u r if ie d

c r y s t a l l i n e lysozym e added a g r e a t e r s i g n i f i c a n c e t o th e r e s u l t s o b ta in ed #

4

HISTORICAL Lysozyme was f i r s t i d e n t i f i e d and r e p o r te d by F lem ing ( 1 9 2 2 ).

In s tu d y in g th e n a s a l s e c r e t i o n s o f a p a t ie n t

s u f f e r in g from a c o l d , F lem ing ob serv ed t h a t t h e s e s e c r e ­ t i o n s e x e r te d a b a c t e r i o s t a t i c a c t i o n .

By a p p ly in g n a s a l

mucus t o se ed ed agar he was a b le t o show t h e i n h i b i t i o n o f grow th o f a c e r t a i n t e s t s p e c i e s .

L a t e r , F lem ing demon­

s t r a t e d th e l y s i s o f a h eavy b a c t e r i a l s u s p e n s io n by t h i s n a sa l s e c r e tio n .

T h is a c t i v e or l y t i c p r in c ip le he c a l l e d ,

* ly s ozyme", b e c a u se o f i t s en zy m a tic n a tu r e .

The s u s c e p t ib le

organism was a p p r o p r ia te ly named M icrococcu s ly s ode i k t i c u s . P r e p a r a tio n and p u r i f i c a t i o n .

A ft e r F lem ing (1922)

showed t h a t th e r e was a h ig h c o n c e n t r a t io n o f lysozym e in h e n 's egg a lb u m in , eg g w h ite was commonly u s e d as a so u rce o f ly so z y m e .

I n i t i a l i n v e s t i g a t i o n s were c a r r ie d out w ith

u n p u r if ie d raw a lb u m in .

A number o f a tte m p ts were made t o

p repare t h i s enzyme in pure form .

A ll o f th e e a r l i e r , and

l e s s s u c c e s s f u l e f f o r t s a t p u r i f i c a t i o n , a re a d e q u a te ly r e ­ view ed i n th e l i t e r a t u r e .

M eyer, Thompson, Palmer and

Khorazo (1956) r e p o r te d on th e p r e c i p i t a t i o n o f lysozym e by f la v ia n le a c id . and ammonia.

T h is was fo llo w e d by e x t r a c t i o n w ith a lc o h o l

R ob erts (1937) n o ted t h a t t h e method o f M eyer,

e t a l , cau sed a l o s s o f a c t i v i t y and he im proved th e method by u s in g f r a c t i o n a l a c e to n e p r e c i p i t a t i o n .

The lysozym e

prepared i n t h i s manner was an amorphous powder, show ing

5

r e l a t i v e l y h ig h l y t i c a c t i v i t y *

Abraham and R obinson (1937)

c r y s t a l l i z e d s m a ll amounts o f lysozym e u s in g th e R ob erts p r e p a r a tio n a s a s t a r t i n g m a t e r ia l.

A ld e r to n , Ward and F ev old

(1945) r e p o r te d a method o f p u r i f i c a t i o n w h ich was b a sed on th e s e l e c t i v e a d s o r p tio n o f lysozym e on b e n t o n it e . A ld e r to n and F ev o ld (1946) prepared lysozym e d i r e c t ­ l y from egg w h it e .

T h is method made p o s s ib le th e p r o d u c tio n

o f la r g e q u a n t i t ie s o f h ig h l y p u r if i e d and e x tr e m e ly a c t i v e ly so z y m e .

P alm er, B a lla n t yne and G a lv in ( 1 9 4 8 ) , u s in g X -ray

d i f f r a c t i o n te c h n iq u e s showed th e m o le c u la r w e ig h t o f th e c r y s t a l l i n e m a t e r ia l t o be 1 3 ,9 0 0 ± 6 0 0 . Go r l ik e

Fromogeot and

(1950) d eterm in ed th e c o n s t it u e n t amino a c id s and

p rep osed t h e f i r s t m o le c u la r form ula of ly so z y m e .

G ly c in e n

a la n in e ^ o serin e^ Q c y s t in e ^ m eth io n in e^ or2 th r e o n in e ^ val i n e g le u c i n e g is o l e u c in e ^ p h o n y la la n in e 2 t y r o s in e ^ t r y p t o phamog a s p a r t ic a c i d ^ g lu ta m ic a e id 4 ( .NHg)^g ly s in e ^ h i s t i ­ d in e ^ a r g in ln e ^ g .

The m o le c u la r w eig h t was c a lc u la t e d t o be

1 4 ,7 0 0 t 2 5 0 . O ccu ren ce.

One o f th e m ost s t r i k i n g o b s e r v a tio n s

made by F lem ing (1922) was th e w ide d i s t r i b u t i o n o f lysozym e or ly s o z y m e -lik e m a t e r ia l.

In a s u r v e y , h e found t h a t th e

f o llo w in g t i s s u e s and s e c r e t i o n s showed b a c t e r i o l y t i c a c t i v i t y : t e a r s , n a s a l m ucus, sputum , s a l i v a , s y n o v ia l f l u i d , b lo o d serum , p le u r a l e f f u s i o n , a s c i t i c f l u i d , o v a r ia n c y s t f l u i d , f l u i d from th e p a r o tid c y s t , sem en, l i v e r , t o n s i l , k id n e y , i n t e s t i n e , sto m a ch , m en in g e s, s k in and c a r t i l a g e .

F lem ing

a l s o r e p o r te d th e p r esen ce o f lysozym e i n th e t i s s u e , c a r -

t l i a g e and eg g s o f p ike*

U sin g p la t e s seed ed w ith M. l y s o -

d e i k t i c u s and th e a g a r cup method o f a s s a y he o b served th e f o llo w in g zon es of i n h i b i t i o n u s in g b ir d egg w h ite s : c h ic k e n 24 mm., moorhen 11 mm., th r u sh 16 mm., and w a g t a il 16 mm. F lo r e y (1930) was a b le t o d em o n stra te lysozym e in s p le e n , k id n e y , lu n g , s a l i v a r y g la n d s , lymph g la n d , stom ach , c o lo n , c a r t i l a g e , l i v e r and t e s t e s .

P r i c k e t t , M ille r and

McDonald (1933) found t h a t m ilk s from humans, d o g s , c a t s , m onkeys, r a t s and lla m a s a l l c o n ta in e d about th e same lysozym e c o n c e n t r a t io n .

M eyer, Thompson, Palmer and Khorazo (1936)

I s o l a t e d an enzyme from a lysozym e s u s c e p t ib le s a r c in a w hich was s i m i l a r t o eg g w h ite ly so z y m e .

B radford and R ob erts (1956)

r e p o r te d t h a t when com p arative t e s t s o f p lasm a, le u c o c y t e s and w h ole b lo o d were c a r r ie d out th e le u c o c y t e s p o s s e s s e d th e h ig h e s t l y t i c a c t i v i t y .

B l a t t and K e s s le r (1937) b e lie v e d

t h a t lysozym e e x i s t s as a b a c t e r i o l y t i c a g e n t i n human m ilk . M eyer, Hahnel and S te in b e r g (1946) I s o la t e d lysozym e from th e l a t e x o f F ic u s t r e e s .

Feldman (1949) d em on strated th e

p r esen ce o f lysozym e i n th e k id n e y , lu n g , s p le e n , l i v e r , m esen tery and b r a in t i s s u e s o f r a t s .

A lthough a la r g e number

o f su r v e y s have b een conducted l i t t l e

d ata i s a v a ila b le r e ­

g a r d in g q u a n t i t a t iv e d e t e r m in a tio n s .

The r e s u l t s in th e

lit e r a tu r e are e s s e n t ia lly q u a lit a t iv e . W eisch (1942) re p o r te d t h a t s t e r i l e f i l t r a t e s o f a c tin o m y c e te s d is s o lv e d h e a t k i l l e d gram p o s i t i v e and gram n e g a t iv e b a c t e r i a .

However, he found no e v id e n c e f o r th e

7

p r o d u c tio n o f lyaoaym ô by a c t in o m y c e t e s . P r o p e r t ie s . b e a b a s ic p o ly p e p tid e ;

Egg w h ite ly so zy m e i s c o n s id e r e d t o M eyer, Thompson, Palmer and Khorazo

( 1 9 5 6 ) , Abraham (1939) and M eyer, Thompson and Palmer ( 1 9 3 6 ) . R oberta (1957) r e p o r te d t h a t lysozym e i s m o d e ra tely s o lu b le i n w ater and s a l i n e , t h a t s o l u b i l i t y i s in c r e a s e d by th e a d d it io n o f a c id s ; t h a t i t i s i n s o lu b l e i n most o r g a n ic s o l ­ v e n ts and t h a t i t g iv e s p o s i t i v e r e s u l t s f o r most o f th e pro­ te in t e s t s .

M eyer, Thompson and Palmer (1936) r e p o r te d t h a t

lysozym e was in a c t iv a t e d by a l k a l i s , p e r o x id e , i o d i n e , and cuprous o x id e .

S in c e M eyer, e t a l ,

(1936) c a r r ie d out r e ­

a c t i v a t i o n w ith h yd rogen s u l f i d e , s u l p h i t e s , and c y a n a te , t h e y co n clu d ed t h a t lysozym e i s a c t i v e o n ly in th e reduced sta te .

Lysozyme a c t i v i t y i s not l o s t a f t e r exp o su re t o t r y p ­

s i n , p e p s in , p a n c r e a tin o r d i a s t a s e . Sm ith and S to k e r (1940) r e p o r te d th a t a l k y l s u l ­ f a t e s , f a t t y a c i d s , sodium s a l t s o f f a t t y a c id s and lo n g c h a in a l i p h a t i c a lc o h o ls in h ib it e d th e a c t i v i t y o f ly so zy m e.

T h is

i n h i b i t i o n was c o r r e la t e d w ith c h a in le n g t h inasm uch as no i n h i b i t i o n co u ld be o b serv ed i f th e c h a in le n g t h was l e s s th an 12 c a r b o n s.

Lawrence and K lin g e l (1943) r e p o r te d t h a t

c e r t a in s u lfo n a m id e -a z o d y e s , e s p e c i a l l y s u l f a t h i a z o l e - d i a z o s y m e t r ic a l carbam ide-2R a c id i n t e r f e r e

w it h th e l y t i c a c t i v ­

i t y o f ly so z y m e . Method o f a s s a y .

A number o f a u th o rs have proposed

d i f f e r e n t methods f o r th e d e te r m in a tio n o f ly so z y m e . a c c u r a te q u a n t it a t iv e method h a s b e e n r e p o r te d .

No

G oldsw orthy

8

and F lo r e y (1930) d e v is e d a scheme o f a s s a y w hich c o n s is t e d o f w ash ing an 18-h au r c u lt u r e o f M, l y s o d e l k t lc u s w ith s a l i n e and a d ju s t in g t h e o p a c it y o f t h a t s u s p e n s io n t o t h a t o f Brow n's barium s u l f a t e sta n d a rd n o . 4 .

Lysozyme was s e r i a l l y

d i l u t e d s o t h a t each su c c e e d in g d i l u t i o n c o n ta in e d o n ly h a l f as much o f t h e enzyme as th e one p r e v io u s .

Then 0 .5 m l. o f

each d i l u t i o n was m ixed w ith an eq u a l q u a n tity o f c e l l s u s ­ p e n s io n . one h o u r .

The m ix tu re s were a llo w e d t o in c u b a te a t 38°G. f o r A u n i t was d e fin e d as th e l e a s t amount o f l y s o ­

zyme n e c e s s a r y t o produce co m p lete l y s i s . R o se n th a l and Lieberm an (1931) i n d eterm in in g th e lysozym e c o n te n t o f in fa n t s t o o l s m ixed a s u s c e p t ib le s a r c in a w ith s t o o l e x t r a c t s .

V is u a l o b s e r v a tio n s o f th e m ix­

tu r e were made under t h e m ic r o s c o p e . s a r c in a c e l l s

A d isa p p e a ra n ce o f th e

in d ic a t e d lysozym e a c t i v i t y .

B oasson (1938) d e v e lo p e d a te c h n iq u e u s in g o p t i c a l m easurem ents o f t u r b i d i t y .

A p h e n o l- k ille d s u sp e n s io n o f th e

t e s t organism was m ixed w ith v a r io u s d i l u t i o n s o f ly so z y m e . The amount and r a t e o f c l e a r i n g was c a r e f u l l y m easured in a M oll e x t in c t o m e te r and c o r r e la t e d w ith th e c o n c e n t r a t io n o f ly so z y m e .

S in c e t h e a c t i v i t y f o r th e known c o n c e n tr a tio n

co u ld be o b serv ed a c c u r a t e ly , i t was p o s s ib le t o compare th e e x t e n t o f a c t i v i t y o f an unknown and i n t h i s manner t o d e ­ term in e t h e amount o f lysozym e p r e s e n t. Meyer and Hahnel (1946) d e v e lo p e d a v is c o s im e t r ie method f o r m easuring t h e m u c o ly tic a c t i v i t y of ly so zy m e.

9

A m u co p o ly sa cch a rid e was u se d as a s u b s t r a t e f o r th e enzyme • The t e s t was b ased on th e d e p o ly m e r iz a tio n o f t h i s s u b s t r a t e . The change i n v i s c o s i t y c o u ld be m easured and c o r r e la t e d w ith th e lysozym e c o n c e n tr a tio n #

The p r e p a r a tio n o f t h i s m ucopoly­

s a c c h a r id e f r a c t i o n i s a v e r y in v o lv e d and la b o r io u s p ro ­ ced u re , w hich does not le n d i t s e l f t o a ra p id method o f a s s a y . W ilson (1950) found t h a t t h e v is c o s im e t r ie method c o u ld n ot be u sed t o d eterm in e lysozym e i n u r i n e .

The a c t io n o f o th e r

enzymes p r e s e n t i n th e u r in e cau sed a change i n th e v i s c o s i t y o f th e s u b s t r a t e prepared i n th e manner d e s c r ib e d by Meyer and Hahnel (1 9 4 6 ). \

M eyer, H ahnel and S te in b e r g ( 1 9 4 6 ) , r e p o r tin g on

lysozym e o f p la n t o r i g in u sed th e v i s c o s im e t r ie method t o m easure th e m u c o ly tic a c t i v i t y and an o p t i c a l method t o mea­ su re th e b a c t e r i o l y t i c a c t i v i t y .

The o p t i c a l method was not

v ery d i f f e r e n t from th o s e u sed by o th e r in v e s t ig a t o r s #

M.

ly s o d e i k t ic u s c e l l s w ere suspended in a M/15 phosphate s o lu ­ t i o n and m ixed w ith lysozym e d i l u t i o n s .

A fte r 1 hour in c u ­

b a tio n a t 3 7 ° C ., 2 drops o f normàl sodium h y d ro x id e were added and th e c l e a r i n g was read v i s u a l l y .

A u n it was e x ­

p r e sse d as th e h ig h e s t d i l u t i o n g iv in g com p lete v i s i b l e c l e a r ­ in g under th e c o n d it io n s s p e c i f i e d . I t i s i n t e r e s t i n g t o n o te h e r e t h a t th e Nakamura r e a c t io n was em ployed by Meyer e t a l in th e d e te r m in a tio n o f ly s is #

Nakamura (1923) ob served t h a t t h e a d d it io n o f a l k a l i

t o a ly s o z y m e -b a e te r ia m ix tu re f o llo w in g in c u b a t io n , w i l l r e s u l t i n a v e r y r a p id l y s i s o f th e c e l l s .

T his has b een e x -

10

p la in e d b y B easaon (1958) on t h e b a s i s o f s o l u b i l i t y o f th e c e l l u l a r m a t e r ia ls . H arts e 11 (1948) u sed a s u s p e n s io n o f M. l y s o d e i k t ic u s i n p h osp h ate b u f f e r , pH 6 , 2 , mixed w ith lysozym e c o n ta in e d in r e h y d r a te d , sp ra y d r ie d , w hole egg powder.

An in c u b a tio n

tem p era tu re o f 5 2 ° C, was u s e d , and t h e c l e a r i n g o f th e su sp en ­ s i o n was o b ser v ed v i s u a l l y .

The lysozym e c o n c e n t r a t io n i n a

sam ple was e x p r e ss e d as th e r e c ip r o c a l o f th e h ig h e s t d i l u t i o n c a u sin g c l e a r i n g , W ilson (1950) d e s c r ib e d a t u r b id im e t r ic method o f lysozym e d e te r m in a tio n w hich he a p p lie d t o u r in e sa m p le s,

A

s ta n d a r d iz e d s u s p e n s io n o f 40 t o 48 hour M, ly s ode i k t ic u s grow th was m ixed w ith d i l u t i o n s o f u r in e i n M/30 phosphate b u ffe r .

In c u b a tio n was c a r r ie d out a t 4 5 ° C, f o r 2 hours a t

a pH o f 6 , 5 ,

F o llo w in g in c u b a tio n th e m ix tu res were ob served

v i s u a l l y and th e c o n c e n t r a t io n was e x p r e sse d as th e r e c ip r o ­ c a l o f th e h ig h e s t d i l u t i o n w hich ca u sed c l e a r i n g .

An a lt e r n ­

a t i v e method f o r end p o in t d e te r m in a tio n was th e a d d it io n o f m eth lyen e b lu e ,

Lysed M, ly s o d e i k t lc u s c e l l s were u nab le t o

ca u se a r e d u c tio n of the d y e . F a c to r s a f f e c t i n g lysozym e a c t i v i t y , (1929) r e p o r te d th a t th e r a t e o f l y s i s

F lem ing ( 1 9 2 2 ) ,

o f a b a c t e r i a l su sp en ­

s io n was a f u n c t io n o f th e tem p era tu re o f in c u b a tio n . imum l y s i s t a k in g p la c e a t ab out 6 0 ° C,

Max­

Much o f th e same

ty p e o f d a ta has b een p r e s e n te d b y o th e r i n v e s t i g a t o r s .

S in c e

lysozym e i s en zy m a tic in n a tu re and i s h e a t l a b i l e a t extrem e te m p e r a tu r e s , th e tem p era tu re f o r maximum l y s i s i s dependent

11

upon th e r a t e o f d é n a tu r a t io n .

U sin g egg w h it e , sputum ,

s a l i v a or t i s s u e e x t r a c t s , F lem ing found t h a t i n a c t i v a t i o n to o k p la c e a t 7 5 ° C ., a f t e r a 30 m inute e x p o su r e .

Sandow

(1926) r e p o r te d t h a t h e a t l a b i l i t y was dependent upon th e pH o f t h e medium.

A 1 :2 0 d i l u t i o n o f egg w h ite f o r ex a m p le, was

i n a c t iv a t e d by ex p o su re t o 6 5 ° C. fo r 15 m in u tes a t pH 8 . 0 , a t pH 6 . 8 30 m in u tes w ere r e q u ir e d fo r i n a c t i v a t i o n , w h ile a t pH 5 .0 h e a t in g f o r ov er 60 m in u tes d id n o t r e s u l t in a lo s s of a c t iv ity .

I t must be remembered t h a t most o f th e e x p e r i­

m ents i n th e l i t e r a t u r e d e a l t w ith impure p r e p a r a tio n s o f th e enzym e, w h ich may have in flu e n c e d th e r e s u l t s somewhat. A lth ou gh t h e in d ic a t io n s are t h a t a t a c id pH v a lu e s lysozym e i s not p a r t ic u la r l y h e a t l a b i l e . The optimum pH f o r lysozym e a c t i v i t y has b een d e ­ s c r ib e d a s b e in g somewhere a t n e u t r a l i t y or b e lo w .

Flem ing

(1922) found t h a t th e a d d it io n o f a l k a l i or a c id t o a neu­ t r a l lysozym e m ixtu re ca u sed an i n h i b i t i o n or a d e s t r u c t io n o f ly tic

p r o p e r t ie s .

Sandow (1926) r e p o r te d t h a t th e optimum

pH was 6 .0 when a s a r c in a s t r a i n was th e t e s t o rg a n ism , but a g a in s t S tr e p to c o c c u s h e m o ly tic u s i t was 9 . 2 .

B oasson (1938)

c o n sid e r e d th a t l y s i s by lysozym e was a two f o l d r e a c t io n ; an a c id pH fa v o r s t h e a c t i v i t y o f ly so z y m e , b u t an a lk a lin e pH r e s u l t s i n an in c r e a s e in v i s i b l e l y s i s .

On th e b a s is o f h is

work h e d eterm in ed th e optimum t o be pH 6 . 2 .

F e in e r , Meyer

and S te in b e r g (1946) a l s o found t h a t an a c id pH was optimum f o r lysozym e a c t i v i t y .

Webb (1948) showed th a t th e pH most

s u i t e d f o r th e a c t i v i t y o f lysozym e upon C lo str id iu m w e lc h ii

12

and S tap h ylocoecm s alb ua ( h e a t - k i l l e d ) was b etw een 6 .0 and 7 .0 .

H ow ever, Webb d eterm in ed th e a c t i v i t y on th e b a s is o f

c o n v e r s io n o f t h e Oram s t a i n r e a c t io n o f t h e s e two organism s r a t h e r th a n l y s i s , as I s u s u a l ly d o n e. The e f f e c t o f s a l t s on lysozym e a c t i v i t y h a s been g iv e n o n ly p r e fu n e to r y c o n s id e r a t io n by p rev io u s i n v e s t i g a t o r s . Flem ing (1922) r e p o r te d t h a t l y s i s was most r a p id when a 0 .5 per c e n t s a l t c o n c e n t r a t io n was u s e d , 2 per c e n t I n h ib it e d and 5 per c e n t I n h ib it e d c o m p le te ly th e l y t i c a c t i v i t y . Sandow (1926) found t h a t optimum sodium c h lo r id e c o n c e n tr a tio n was 0 .1 4 5 N. B oasson (1938) r e p o r te d t h a t h ig h c o n c e n tr a tio n s o f e l e c t r o l y t e s ca u sed i n h i b i t i o n .

In a d d it io n , he found

t h a t t h e * valency'* o f t h e io n s i n th e medium co u ld be c o r r e ­ la t e d w ith th e e x t e n t o f I n h i b i t i o n , in t h a t th e h ig h e r th e v a le n c y th e g r e a t e r th e i n h i b i t i o n .

S in c e th e e a r ly ex p er­

im en ts w ere n o t b a sed on pure ly so z y m e , no d e f i n i t e c o n c lu ­ s io n s can be drawn. F e in e r , Meyer and S te in b e r g (1946) a ttem p ted t o i n ­ c r e a s e c e l l s u s c e p t i b i l i t y t o lysozym e b y th e a d d it io n o f v a r io u s m a t e r ia ls . any e f f e c t .

Not many o f th e su b s ta n c e s t e s t e d showed

F erro u s s u l f a t e in h ib it e d l y s i s ; sodium pyro­

phosphate c o u n te r a c te d t h i s i n h i b i t i o n .

Treatm ent o f th e

c e l l s w it h t y r o t h r i e i n , try p to p h a n e and th e su p ern a ta n t wash­ in g s o f M. l y s ode i k t ic u s showed no c o n s is t e n t e f f e c t .

Sodium

a r s e n i t e a t a c o n c e n t r a t io n o f M/200 ca u sed th e t i t e r o f a s u s p e n s io n t o In c r e a s e fo u r t im e s .

13

A c t i v i t y o f lyaozym e a g a in s t m icro o rg a n ism s»

S in c e

lysozym e was o r i g i n a l l y c o n s id e r e d t o have a n t i b i o t i c p o s s i ­ b i l i t i e s , a g r e a t many ex p erim en ts have been c a r r ie d o a t t o d eterm in e what m icroorgan ism s a re s u s c e p t i b l e .

A resume o f

th e d a ta i n t h e l i t e r a t u r e i s p r e se n te d by Thompson ( 1 9 4 0 ). T here i s g e n e r a l agreem ent a t p r e s e n t , t h a t l y s o ­ zyme a c t s on th e b a c t e r i a l membrane.

Meyer (1946) c o n s id e r s

th e a c t i v i t y o f lysozym e in term s o f “h y d r o ly s is o f a su b ­ s t a n c e o f mucoid n a tu re w hich i s c o n ta in e d i n th e b a c t e r i a l membrane” .

I t Is n o t , h o w ev er, c o n sid e r e d t h a t lysozym e i s

s o l e l y r e s p o n s ib le f o r a l l o f th e changes w hich occu r w ith ­ in a b a c t e r i a l c e l l d u rin g l y s i s .

Meyer (1946) c o n s id e r s

th e m u co p o ly sa cch a rid e t o be an i n t e g r a l p art o f th e c e l l membranee

11The prim ary mechanism o f lysozym e a c t io n appar­

e n t l y c o n s i s t s o f a d e p o ly m e r iz a tio n o f t h i s m ucopoly­ s a c c h a r id e , le a d in g t o w a ter im b ib it io n by th e organism and t o d is o r g a n iz a t io n o f th e m ic r o b ia l c e l l . ”

The su b seq u en t

breakdown o f t h e c e l l u l a r s t r u c t u r e he c o n s id e r s t o be due p r im a r ily t o th e a u t o l y t i c enzymes p r e s e n t In th e organ ism . M eyer, Thompson, Palmer and Khorazo (1936) found t h a t r e d u c in g su g a rs w ere lib e r a t e d when s u s c e p t ib le c e l l s were l y s e d ,

E p s t e in and Chain (1940) i s o l a t e d a h ig h m olec­

u la r w e ig h t p o ly s a c c h a r id e w hich was a c te d upon by ly so zy m e. They found t h a t t h i s p o ly s a c c h a r id e was h y d r o ly ze d in t o w ater s o lu b le m a t e r i a l s ,

Meyer and H ahnel (1946) d e s c r ib e d th e

i s o l a t i o n and c h a r a c t e r i s t i c s o f th e lysozym e s u b s t r a t e . They d e s c r ib e d a h ig h ly p o ly m erized m u co p o ly sa cch a rid e which

14

s e r v e d a s a s p e c i f i c s u b s tr a te f o r ly so z y m e .

A fte r In c u b a tio n

w ith ly so z y m e , th e p o ly s a c c h a r id e was found t o be depolym er­ iz e d ; th e g l u e o s i d l e lin k a g e s w ere h y d ro ly zed and th e r e d u c in g su g a rs and a c e ty lh e x os amine were l ib e r a t e d . B o a sso n (1938) em ploying dark f i e l d illu m in a t io n r e p o r te d on th e m orphology o f ly s l n g M. ly s ode i k t ic u s c e l l s . He n o te d t h a t th e c e l l s d id n o t change in form or m agnitude and t h a t th e c e l l w a ll was n o t d e str o y e d by ly so zy m e.

The

p r in c ip le change was c o n s id e r e d t o be one o f p e r m e a b ility o f th e c e l l membrane.

S u b se q u e n tly , o n ly d if f e r e n c e s in d en s­

i t y c o u ld be o b se r v e d .

S w e llin g o f th e c e l l s , when i t was

o b se r v e d , was a t t r ib u t e d t o a m ech a n ica l f l a t t e n i n g o f th e organism s a f t e r l y s i s . B a b u d ie r i and B l e t t i (1945) s t u d ie d th e l y t i c a c t io n o f lysozym e w ith th e e l e c t r o n m ic r o sc o p e .

U sin g a

”s a r c i n a - l i k e organ ism ” and t e a r s t h r e e s t a g e s o f l y s i s were o b ser v ed ; 2.

1 . ”R a r e fa c tio n ” o f th e p ro to p la sm ic m ass.

P ro to p la sm ic fr a g m e n ta tio n .

3.

Complete fr a g m e n ta tio n .

B ab u d iere and B l e t t i co n clu d ed t h a t th e l y t i c p r o c e ss i s r e ­ la t e d t o membrane p e r m e a b ility ; and th a t th e i n t e g r i t y o f th e membrane was m a in ta in ed th ro u g h o u t l y s i s .

T hese i n v e s t ig a t o r s

d id n ot u se t h e shadow c a s t te c h n iq u e in p rep a rin g e l e c t r o n m icr o sco p e s p e c im e n s . W elshim er and Robinow (1949) d e sc r ib e d th e l y s i s o f fo r m a lin k i l l e d B a c ill u s m egatherium .

These a u th o rs

f o llo w e d th e c o u r s e o f th e m o r p h o lo g ic a l ch an ges w ith a l i g h t m ic r o sc o p e ; th e JB. m egatherium c e l l s underwent l y s i s in an

o r d e r ly manner.

F i r s t , c e l l p e r m e a b ility was a l t e r e d , w hich

was fo llo w e d by a d e c r e a s e in s t a i n a b i l i t y .

A fte r p ro lo n g ed

ex p o su re t o t h e enzym e, th e c e l l w a ll was d e str o y e d ; f i n a l l y o n ly c e l l u l a r d e b r is rem ain ed . A n t ig e n ic it y o f ly so z y m e.

Smolens and Charney

(1947) r e p o r te d t h a t i t was p o s s ib le t o c a u se an a n t ig e n ic r e sp o n se i n r a b b its by i n j e c t i n g c r y s t a l l i n e ly so z y m e .

How­

e v e r , th e y o b serv ed th a t t h e a n t i g e n i c i t y o f lysozym e i s not as marked a s t h a t o f a t y p i c a l p r o t e in .

A s im ila r o b serv a ­

t i o n was r e p o r te d by W o lff ( 1 9 2 7 ), he found th a t lysozym e i s r a p id ly e lim in a t e d from th e b lo o d when in j e c t e d in t o e x p e r i­ m en ta l a n im a ls . C l i n i c a l s i g n i f i c a n c e o f ly so z y m e.

B ecause th e

a c t io n o f ly so zy m e a s d em on strated by th e l y s i s o f a s u s c e p ­ t i b l e s p e c i e s i s v er y s t r i k i n g and th e d i s t r i b u t i o n i s v e r y w id e , a tte m p ts h ave been made t o c o r r e la t e lysozym e w ith some p a t h o lo g ic a l c o n d it io n w ith man.

Corper (1932) r e p o r te d th a t

lysozym e does not p la y a s i g n i f i c a n t part in th e h i s t o r y o f t u b e r c u lo s is i n man.

K o p e lo f f , H a rris and McGinn (1932) ob­

s e r v e d th a t no d if f e r e n c e s co u ld be found betw een th e l y s o ­ zyme c o n te n t of p o st e n c e p fo a litic and normal p a t i e n t s , when a c c u r a te sta n d a r d s were em ployed.

Ju n gb lu t (1935) r e p o r te d

t h a t u n d ilu te d human t e a r s showed p o l l o c l d a l p r o p e r t ie s . P r i c k e t t , M ille r and McDonald (1937) in s tu d y in g th e t i s s u e s o f v ita m in A d e f i c i e n t an im als found t h a t c o n s i s t e n t l y h ig h ­ e r lysozym e t i t e r s were a s s o c ia t e d w ith th e s e a n im a ls. S u ll iv a n and Manv i l l e

(1937) found t h a t r a b b it s , w hich were

16

d e f i c i e n t i n v ita m in A and u r o n ic a c i d , showed a lysozym e t i t e r 2 t o 4 tim e s h ig h e r th a n th o s e on co m p lete d i e t s .

They

b e lie v e d t h a t v ita m in A and u r o n lc a c id a c t a s c o n t r o l l i n g m echanisms i n t h e s e c r e t io n o f ly so zy m e.

Gunderson (1958)

found t h a t when 0 .5 m l. o f egg album in was mixed w ith 5 ml# o f C lo str id iu m b o tu lln u m (ty p e C) c u lt u r e and f e d t o p ig e o n s , s u r v i v a l was p ro lon ged t o 8 and as many as 15 d a y s .

When th e

c u lt u r e a lo n e was f e d , d ea th u s u a lly to o k p la c e w it h in 24 h ou rs.

Voronova (1941) r e p o r te d on th e trea tm en t o f d ia r r h e a

in c h ild r e n u s in g a s a l i n e d i l u t i o n o f a s o p t i c a l l y drawn egg alb u m in .

The egg w h ite was a d m in ister ed o r a l ly and in enem as.

Voronova con clu d ed t h a t th e tim e ly u se o f lysozym e i s i n s t r u ­ m en tal in p r e v e n tin g common d y sp e p sia from d e v e lo p in g i n t o a t o x i c c o n d it io n ,

Ponomereva (1946) u sed f r e s h h en s' egg w h ite

in th e trea tm en t o f wounds. The w r it e r w ish es t o r e f e r th e re a d e r t o th e e x ­ c e l l e n t r e v ie w s i n th e l i t e r a t u r e which co v e r a d e q u a te ly th e e a r ly c l i n i c a l s t u d ie s o f ly so zy m e, Mesrobeanu and N oeppel ( 1 9 3 8 ) , D obraja ( 1 9 3 9 ), Thompson (1940) and Thompson (1 9 4 1 ). Much o f th e work t o d a te has b een in co m p lete and i n many e a s e s s e r v e s o n ly as an in tr o d u c t io n t o t h e c l i n i c a l s i g n i f ­ ic a n c e o f ly so z y m e .

For t h i s r e a so n a l l of th e o r i g i n a l r e ­

p o r ts are n o t in c lu d e d h e r e . R e c e n t ly , Meyer (1947) induced n o n - s p e c i f ic u le r a t i v e c o l i t i s i n la b o r a to r y anim als by th e a d m in is tr a tio n o f ly so z y m e .

M eyer, Pruden, Lehman and S te in b e r g (1947) found

t h a t th e lysozym e c o n te n t o f human g a s t r i c j u ic e s i s in c r e a s e d

17

when pQ ptic u l c e r s a re p r e s e n t #

A number o f t i s s u e s o b ta in e d

d u rin g r e s e c t i o n f o r p e p tic u l c e r a l s o showed in c r e a s e d l y s o ­ zyme c o n t e n t .

M eyer, G e llh o r n , Prudden, Lehman and S te in b e r g

(1947) were a b le t o show t h a t i n c a s e s o f c h r o n ic u l c e r a t i v e c o l i t i s th e lysozym e c o n te n t o f th e c o lo n was in c r e a s e d many t im e s .

F u r th e r work by Meyer (1 9 4 8 ) d em o n stra ted t h a t p a t ie n t s

s u f f e r i n g from u l c e r a t i v e c o l i t i s showed in c r e a s e d lysozym e c o n c e n tr a tio n s i n t h e i r s t o o l s .

G race, S e t o n , W olf and W o lff

( 1 9 4 9 ) , who a l s o s t u d ie d t h i s p rob lem , found t h a t lysozym e c o n c e n tr a tio n s v a r ie d w ith e m o tio n a l c o n d itio n s o f p a t i e n t s . The im p lic a t io n s o f t h e s e s t u d ie s are c l e a r — th e r e i s a v er y good p o s s i b i l i t y t h a t th e h y p e r s e c r e tio n o f lysozym e ca n be c o r r e la t e d w ith th e p resen ce o f u l c e r a t i v e c o l i t i s . W ilso n and H adley (1950) d e te n n in e d th e lysozym e c o n te n t i n th e u r in e o f 595 h e a lt h y in f a n t s and c h ild r e n . T h is was p r e lim in a r y t o c e r t a in c l i n i c a l s t u d i e s .

They found

th a t 96 p e r c e n t o f t h e s e c h ild r e n e x c r e te d lysozym e i n th e u r in e .

F u rth er i t was r e p o r te d t h a t fe m a le s e x c r e te d l y s o ­

zyme 5 tim e s more o f t e n th an m a le s . r a b b its .

T h is was a l s o tr u e f o r

In an exp erim en t d e a lin g w ith th e i n g e s t i o n o f a

la r g e amount of raw eg g album in i t was o b serv ed t h a t one p in t o f egg w h ite d id n ot c a u se th e e x c r e t io n o f lysozym e i n t h e u r in e o f a normal m a le .

A d d it io n a l s t u d ie s on th e e x c r e t io n

o f u r in a r y lysozym e by c h ild r e n s u f f e r i n g from a v a r i e t y o f d is e a s e s showed t h a t th e r e was no s i g n i f i c a n t d if f e r e n c e from th e e x c r e t io n s o f h e a lt h y c h ild r e n .

In a l a t e r p u b lic a t io n ,

W ilson and H adley (1950) re p o r te d on th e e x c r e t io n o f l y s o -

18

zyme by c h ild r e n w ith a n e p h r o tic syndrom e.

A lth ou gh th e r e

w ere no ob v io u s c o r r e la t i o n s b etw een th e e x c r e t io n o f l y s o ­ zyme and

th e d i s e a s e , 10 o f 14 c h ild r e n t e s t e d e x c r e te d i n ­

c r e a s e d q u a n t i t ie s o f u r in a r y ly so z y m e . Regan (1950) s tu d ie d th e lysozym e c o n te n t o f t e a r s o f p a t ie n t s s u f f e r i n g from v a r io u s o c u la r i n f e c t i o n s .

U sin g

t h e v is c o s im e t r ie method f o r lysozym e d e t e r m in a tio n , Regan showed t h a t , i n t h e p a t h o lo g ic a l c o n d it io n s s t u d ie d , th e l y s o ­ zyme c o n c e n t r a t io n i n t e a r s i s low er th a n norm al. E xcep t f o r the s t u d ie s c o n c e r n in g p e p tic u l c e r s , no a b s o lu te c o r r e la t i o n s e x i s t b etw een lysozym e and i n f e c t i o n s or p a t h o lo g ic a l c o n d it i o n s .

D e s p ite t h e a b sen ce o f o b viou s

im p lic a t io n s i t i s s t i l l p o s s ib le t h a t lysozym e has some f u n c t io n in th e m ainten an ce o f good h e a lt h .

Dub os (1947)

had t h i s t o sa y : “Even in h ig h d i l u t i o n s , t h i s enzyme c a u se s th e d e a th o f b a c t e r ia o f s e v e r a l s a p r o p h y tic s p e c i e s •

S in c e

i t i s in a c t iv e a g a in s t m ost p a th o g e n s, or a t b e s t o n ly s l i g h t ­ l y a c t iv e in h ig h c o n c e n t r a t io n s , i t has b een c o n s id e r e d t o be o f no s i g n i f i c a n c e in a n t i b a c t e r i a l r e s i s t a n c e .

The p o s s i ­

b i l i t y r e m a in s, h ow ever, th a t t h e r e e x i s t a number o f m icro ­ organism s w hich a re p o t e n t i a l l y p a th o g en ic and w hich are p re­ v en ted from m a n if e s tin g t h e i r p a th o g en ic p r o p e r t ie s s o l e l y b eca u se th e y are s u s c e p t ib le t o lysozym e and t h e r e f o r e cannot e s t a b l i s h th e m se lv e s in th e anim al body."

EXPERIMENTAL M ic r o b io lo g ic a l A ssay o f Lysozyme None o f th e lysozym e a s s a y m ethods d e s c r ib e d i n th e l i t e r a t u r e appeared t o be ca p a b le o f p r o v id in g a c c u r a te r e s u l t s w ith a minimum e x p e n d itu r e o f tim e*

The i s o l a t i o n

o f c r y s t a l l i n e lysozym e by A ld e r to n and F ev o ld (1946) su g ­ g e s te d th e p o s s i b i l i t y o f a method f o r d o in g s o .

S in c e th e

most s t r i k i n g e v id e n c e o f lysozym e a c t i v i t y i s th e l y s i s o f s u s c e p t ib le b a c t e r i a l c e l l s , an a s s a y m ethod, b a sed on t h e b a c t e r i o l y s i s o f M lcrococcu s l y s ode i k i t i c u s c e l l s , was e v o lv e d To adapt t h i s te c h n iq u e t o la r g e s c a l e a s s a y s , a s ta n d a r d iz e d c e l l p r e p a r a tio n was d e v e lo p e d .

Some o f th e f a c t o r s a f f e c t ­

in g l y t i c a c t i v i t y were s t u d ie d and an a ttem p t was made t o s ta n d a r d iz e them th rou gh ou t th e a s s a y p ro ced u re. P r e p a r a tio n o f C e lls f o r Use in th e A ssa y .

M.

l y s ode i k t i c u s ATCC 4 6 9 8 , w hich was th e o r i g i n a l Flem ing s t r a i n was u sed as a t e s t organ ism th ro u g h o u t a l l o f t h e s e e x p e r i­ ments .

A grow th curve ex p erim en t showed t h a t t h i s c u lt u r e

w ould re a ch th e peak grow th p erio d a t 18 hours on y e a s t w a ter v e a l in f u s io n a g a r ’*' a t 3 7 ° G.

The s u s c e p t i b i l i t y o f an organ -

■*■ Ten p e r c e n t v e a l i n f u s i o n , 10 p erc en t y e a s t w a te r , 0 .5 p e r c e n t NaC l, 0 .5 p erc en t p ep ton e and 2 p e r c e n t a g a r .

20

ism t o e x t e r n a l s t i m u l i has b een shown by E l l i k e r and F r a z ie r (1938) and H egarty and W ells (1940) t o be g r e a t e s t a t th e tim e o f maximum re p r o d u c tio n #

On t h i s b a s i s y ou n g, a c t i v e l y

grow ing c e l l s w hich w ere u n d er g o in g r a p id d i v i s i o n were ch o se n f o r ex p o su re t o lysozym e#

The medium f i n a l l y u sed f o r

th e grow th o f c e l l s was YWVI ag a r p lu s 0 .2 p erc en t g lu c o s e , pH 7 . 0 .

A s t o c k c u lt u r e o f th e organ ism was k ep t on th e

same medium a t 4 ° C.

P r io r t o u se th e s to r e d organism s were

su b c u ltu r e d e v e r y 24 hours f o r 3 days#

The c e l l s were con ­

d it io n e d in t h i s manner t o p ro v id e a s u n iform and s t a b l e a c e l l p r e p a r a tio n a s p o s s i b l e . Lysozyme a c t i v i t y was m ost e a s i l y d em on strated by m ixin g s u s c e p t i b l e c e l l s w ith a d i l u t i o n o f th e enzym e. Under proper c o n d ito n s and w it h in a v e r y s h o r t tim e th e c e l l s u s p e n s io n was c le a r e d and th e in d iv id u a l c e l l s no lo n g e r m ain ta in ed t h e i r i n t e g r i t y .

When a s e r i e s o f tw o -fo ld d i l u ­

t io n s were mixed and in c u b a te d w ith a c e l l s u s p e n s io n , th e r e s u l t i n g t u r b i d i t i e s c o u ld be e x p r e s se d g r a p h ic a ll y .

It

was p o s s ib le t o p lo t th e l i g h t tr a n s m is s io n v a lu e s o f th e m ix tu res a g a in s t lysozym e c o n c e n t r a t io n s .

One p o r tio n o f

t h i s cu rv e becom es a s t r a i g h t l i n e . T ab le 1 , F ig u re 1 .

In

d e v e lo p in g t h i s a s s a y method an a ttem p t was made t o p ro v id e th o s e c o n d itio n s w hich would p r e s e n t a s t r a i g h t l i n e r e l a t i o n ­ s h ip when th e t u r b i d i t i e s and lysozym e c o n c e n tr a tio n s were e x p r e sse d g r a p h ic a ll y .

21

TABLE 1 l ys o

T u r b id im e tr ic d e ik tlc u s and

R e sp o n se o f M. t lysozy m e d i l u t i o n s v'

Lysozyme D ilu t io n

A ve, %T

Lysozyme D ilu t i o n

A ve. % T

1 :8 0 ,0 0 0 1 :1 6 0 ,0 0 0 1 : 3 2 0 ,0 0 0 1 : 6 4 0 ,0 0 0 1 : 1 ,2 8 0 ,0 0 0 1 : 2 ,5 6 0 ,0 0 0 1 : 5 ,1 2 0 ,0 0 0

6 4 .1 6 6 .7 6 8 .8 67 67 6 5 .5 6 1 .2

1 : 1 0 ,2 4 0 ,0 0 0 1 : 2 0 ,4 8 0 ,0 0 0 1 : 4 0 ,9 6 0 ,0 0 0 1 : 8 1 ,9 2 0 ,0 0 0 1 : 1 6 3 ,8 4 0 ,0 0 0 1 : 3 2 7 ,6 8 0 ,0 0 0 0

4 7 .4 5 8 .5 3 2 .2 2 9 .7 28 2 7 .1 2 6 .7

'x' In p h osp h ate b u f f e r pH 6 .2 ; 20 m in. in cu b a ­ t i o n a t room te m p e r a tu r e . B a c t e r i o l y s i s was d eterm in ed in a Coleman s p e c t r o ­ p h o to m eter.

The p rocedure c o n s is t e d o f m ix in g a c e l l s u s ­

p e n sio n w it h a d i l u t i o n o f ly so z y m e ; a f t e r an a p p r o p r ia te in c u b a tio n t im e , t h e e x t e n t o f l y s i s was o b serv ed by a d e ­ te r m in a tio n o f th e t u r b i d i t y o f th e m ix tu r e .

In a l l o f th e s e

ex p erim en ts th e sp e c tr o p h o to m e te r was u sed a t a wave le n g th o f 540 mu w ith a g a lv a n o m eter s e t t i n g w hich showed 100 p er­ c e n t l i g h t tr a n s m is s io n fo r d i s t i l l e d w a te r .

The s o f t g la s s

15 x 150 mm. tu b e s u sed in th e sp e c tr o p h o to m e te r were c a l ­ ib r a te d b e fo r e u s e .

C a lib r a t io n c o n s is t e d o f o r ie n t in g each

tu b e in th e c a r r ie r so t h a t i t showed th e same tr a n s m is s io n f o r d i s t i l l e d w a ter a s t h e r e fe r e n c e tu b e .

The t e s t tu b es

were marked s o t h a t i t was p o s s ib le t o lo c a t e th e same p o in t each t im e .

P r io r t o u s e ea ch tu b e and a l l o th e r g la ssw a r e

was c le a n e d w ith a c id c le a n in g s o l u t i o n and r in s e d w ith ta p and d i s t i l l e d w a te r .

T h is was e s p e c i a l l y im portan t b eca u se

22

FIGURE 1 T u r b id im e tr ic R esponse o f M. ly s ode i k t ic u s and Lysozyme D ilu t i o n s

0.300

8* 't

«

0 .4 0 0

0.500

0.600

0.0186

0.0051

0.0007

0.000042

Lysozyme C o n c e n tr a tio n mg. p er m l.

0.0000026

23

m inute c o n c e n tr a tio n s o f c o n ta m in a tin g m a te r ia ls can e x e r t an in f lu e n c e on th e r e s u l t s . The i n i t i a l d e n s it y o f th e c e l l s u s p e n s io n w hich i s ex p o sed t o lysozym e in f lu e n c e s the t u r b i d i t y a f t e r in c u ­ b a tio n ,

T h is i s due t o th e enzyme s u b s tr a te r e l a t i o n s h i p o f

th e m ix tu r e ,

I t was n e c e s s a r y t o prepare a c e l l su s p e n s io n

w hich would f u r n is h m easu reab le t u r b i d i t y d if f e r e n c e s when a w ide range o f lysozym e d i l u t i o n s were u s e d .

C e ll s u sp e n sio n s

o f t h r e e d e n s i t i e s were u se d ; 3 , 1 0 , and 25 p e r c e n t.

The p er­

c e n t v a lu e was th e p e r c e n ta g e o f l i g h t tr a n s m itte d by th e s u sp e n sio n s in com parison w ith a d i s t i l l e d w a ter r e fe r e n c e whose tr a n s m itta n c e v a lu e was 100 p e r c e n t.

Equal p o r tio n s

o f t h e s e s u s p e n s io n s were mixed w ith s e r i a l lysozym e d i l u ­ t io n s #

A fte r a s u i t a b l e in c u b a tio n p e r io d , th e t u r b i d i t i e s

w ere d e te r m in e d . T ab le 2 , p e n sio n s a re sh ow n ),

(o n ly th e 10 and 25 p ercen t s u s ­

The 10 p e r c e n t tr a n s m is s io n p rep ara­

t i o n was c h o se n f o r a s s a y u s e ,

At t h i s c e l l t u r b i d i t y , the

maximum d if f e r e n c e b etw een th e ly s o z y m e -fr e e c o n t r o l and th e e n z y m e -c e ll m ix tu re was o b se r v e d . F igu re 2 .

24

TABLE 2 E f f e c t of C e ll S u sp e n sio n D e n s ity on B a c t e r io ly s is * 5, 10 P ercen t C e ll S u sp e n sio n A ve. Lys ozyme D ilu t io n % T 1 : 1 0 0 ,0 0 0 1 :2 0 0 ,0 0 0 1 : 4 0 0 ,0 0 0 1 :8 0 0 ,0 0 0 1 : 1 ,6 0 0 ,0 0 0 1 : 3 ,2 0 0 ,0 0 0 1 : 6 ,4 0 0 ,0 0 0 1 : 1 2 ,8 0 0 ,0 0 0 1 : 2 5 ,6 0 0 ,0 0 0 1 : 5 1 ,2 0 0 ,0 0 0 0

6 5 .4 6 6 .1 65 65 6 3 .4 6 0 .4 5 0 .4 41 3 2 .6 2 9 .6 2 7 .6

D 3 7 .8 3 9 .5 3 7 .4 3 7 .4 3 5 .8 3 2 .8 2 2 .8 1 3 .4 5 2

25 P ercen t C e ll S u sp e n sio n Lys ozyme A ve. D ilu t io n %T 1 :1 0 0 ,0 0 0 1 :2 0 0 ,0 0 0 1 :4 0 0 ,0 0 0 1 :8 0 0 ,0 0 0 1 : 1 ,6 0 0 ,0 0 0 1 : 3 ,2 0 0 ,0 0 0 1 : 6 ,4 0 0 ,0 0 0 1 : 1 2 ,8 0 0 ,0 0 0 1 : 2 5 ,6 0 0 ,0 0 0 1 : 5 1 ,2 0 0 ,0 0 0 0

D

8 0 .9 8 2 .5 82 82 8 1 .7 8 2 .5 7 6 .4 69 5 9 .9 5 6 .5 50

3 0 .9 3 2 .5 32 32 3 1 .7 3 0 .5 2 6 .4 19 9 .9 6 .5 ——

* pH 6 . 2 , 20 m in. in c u b a tio n a t room te m p e r a tu r e . To e x p e d it e th e t e s t i n g procedure and t o redu ce th e p o s s i b i l i t y o f d a i l y v a r i a t io n s a s s o c ia t e d w ith th e u se o f l i v e M. l y s ode i k t i c u s c e l l s , Meyer and H ahnel ( 1 9 4 6 ), an e f f o r t was made t o u se k i l l e d c e l l s in th e a s s a y s . zyme i s c a p a b le o f c a u s in g th e l y s i s c e lls .

L yso­

o f b o th l i v e and dead

E ig h te e n hour c e l l s were h a r v e s te d from agar s l a n t s

and exp osed t o 1 p erc en t phenol f o r v a r io u s tim e i n t e r v a l s . A fte r exp osu re t o p h e n o l, t h e c e l l s were washed and r e s u s ­ pended in s a l i n e . was n o t s u c c e s s f u l .

S to r a g e a t 4 ° C. o f su ch a p r e p a r a tio n D a ily d e te r m in a tio n s showed a c o n s id e r ­

a b le v a r i a t i o n in r e s p o n s e . P h e n o l- k ille d c e l l s were th e n t r e a t e d w ith ic e c o ld a c e to n e t o f a c i l i t a t e s t o r a g e .

By d eh y d r a tin g th e c e l l u l a r

m a t e r ia l in t h i s f a s h io n , i t was p o s s ib le t o s t o r e la r g e

25

FIGURE 2 E f f e c t o f C e ll S u sp e n sio n D e n s ity On B a c t e r i o l y s i s

0.100

50

2 - l o g %T

0 .2 0 0

-4 0

10

25# 2 5 # tr

El

0 .3 0 0

30

0 .4 0 0

20

0 .5 0 0

0 .6 0 0

10#

0 .0 1

0 .0 0 0 0 1 7 0 .0 0 0 3 0 .0 0 2 5 Lysozyme Coonncceenntr traa t1 tioon n mg mg** per m mll.

10

D iffe r e n c e

2 - log

%T

2 -lo g

26

q u a n t i t ie s o f c e l l s a t i c e box te m p e r a tu r e s•

S in c e th e e f f e c t

o f a c e to n e on k i l l e d M. ly s o d e ik tI c u s i s n ot known, an a l t e r n ­ a t i v e method o f d r y in g was s o u g h t.

A fte r w a sh in g , th e c e l l s

were resu sp en d ed in d i s t i l l e d w a te r , s h e l l fr o z e n and d r ie d from th e f r o z e n s t a t e . t a b l e fo r u s e in a s s a y s ,

These c e l l p r e p a r a tio n s were a c c e p ­ L y o p h iliz a t io n has b een recommend­

ed as a method o f p r e s e r v in g b i o l o g i c a l m a te r ia ls w ith a m in­ imum o f a l t e r a t i o n , P lo s d o r f and Mudd (1 9 3 8 ).

D eh yd ration

w ith a c e to n e or e t h e r ca u sed th e rem oval o f c e r t a in f a t t y or l i p o i d m a te r ia ls w hich was n o t c o n sid e r e d d e s i r a b l e .

Any

d e h y d r a tio n p r o c e ss w i l l a f f e c t a change in th e arrangem ent o f c e llu la r m a te r ia ls ,

L y o p h iliz a t io n , w hich cau sed th e

l e a s t a l t e r a t i o n , a l s o p r e se n te d an added advantage in th a t c e l l s s o t r e a t e d underwent ra p id r e h y d r a tio n . In an a ttem p t t o p repare a k i l l e d - c e l l p r e p a r a tio n , w hich was o f th e same ord er o f s e n s i t i v i t y t o lysozym e a c t i v ­ i t y as l i v i n g c e l l s , a number o f d i f f e r e n t k i l l i n g methods were t r i e d .

The r e s u l t s o f u s in g t h e s e a g e n t s , as d em o n strat­

ed by t h e l y t i c r e s p o n s e , are p r e se n te d in T able 3 and F ig ­ u re 3 . An e x a m in a tio n o f th e d a ta shows t h a t trea tm en t o f c e l l s by any method p r io r t o ex p o su re t o ly so z y m e , in v a r ­ i a b ly cau sed a d e c r e a se d l y t i c r e s p o n s e ,

T his was c o n tr a r y

t o t h e f in d in g s o f F e in e r , Meyer and S te in b e r g (1 9 4 6 ), r e p o r te d t h a t l i t t l e

They

or no change in c e l l s u s c e p t i b i l i t y was

ob served a f t e r th e organism was s u b je c te d t o r e p e a te d wash­ in g s w ith s a l i n e , l y o p h i l i z a t i o n , p r e c i p i t a t i o n w ith i c e - c o l d

27

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t3 M

03 tO E> Tf* tO # # # • • to 03 xp i> - 1> oo œ to to to 03 03 03

Pu,

I> I> I> I> 03 • • • • • 0> 0- O) a> to 0» 'sh IO IO rh 60 to 03 03

pi OS

to to to H to # * » * * rH CO 0> Oî t0 CJ> tO tO tO xH 60 tO 03 03

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