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The preparation and isolation of ¹⁴C-labelled egg white lysozyme and its utilization by in vitro cultured… Blommers, Hendrik Willem 1977

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THE PREPARATION AND ISOLATION OF 1 4C-LABELLED EGG WHITE LYSOZYME AND ITS UTILIZATION BY IN VITRO CULTURED MACROPHAGE by HENDRIK WILLEM BLOMMERS B.Sc. (Hons.) U n i v e r s i t y o f B r i t i s h C olumbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE.STUDIES i n the Department o f P o u l t r y S c i e n c e We a c c e p t t h i s t h e s i s as co n f o r m i n g t o the r e q u i r e d s t a n d a r d THE' UNIVERSITY OF BRITISH COLUMBIA September 197 7 (c) H e n d r i k W i l l e m Bloirimers In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 - i i -ABSTRACT A p r e p a r a t i v e e l e c t r o p h o r e t i c method was devel o p e d t o i s o l a t e egg w h i t e lysozyme. The e l e c t r o p h o r e t i c u n i t c o n s i s t e d o f a s l i g h t l y m o d i f i e d g l a s s f u n n e l i n which a s l a b o f p o l y a c r y l a m i d e g e l was suspended. The &280 a b s o r b i n g m a t e r i a l e x t r a c t e d from the p o l y a c r y l a m i d e g e l t r a v e l l e d as a s i n g l e s t a i n i n g band'on q u a n t i t a t i v e p o l y a c r y l a m i d e d i s c g e l s . Up t o 25 mg. o f a p p a r e n t l y pure lysozyme was p r e p a r e d by t h i s method. R a d i o a c t i v e lysozyme was p r e p a r e d by i n j e c t i n g 14 young l a y i n g hens w i t h a C ammo a c i d h y d r o l y s a t e . By i n j e c t i n g t h e h y d r o l y s a t e 5 h r s . p o s t o v i p o s i t i o n , lysozyme w i t h a s p e c i f i c a c t i v i t y o f 2 .6 x 10"*"^ dpm/mole was p r e p a r e d . I n j e c t i o n o f t h e r a d i o a c t i v e amino a c i d s a t i n t e r v a l s l e s s t h a n 5 h r s . p o s t o v i p o s i t i o n r e s u l t e d i n lower a c t i v i t y lysozyme. A t h e o r y has been s u g g e s t e d which c o u l d e x p l a i n t h e g e n e r a t i o n o f a n t i b o d y d i v e r s i t y . I n t h i s t h e o r y ( 2 5 , 2 6 ) , the RNA p o r t i o n o f t h e RNA—antigen complex formed d u r i n g an immune r e s p o n s e , a c t s i n an i n f o r m a t i o n a l c a p a c i t y . I n o r d e r t o i n v e s t i g a t e one a s p e c t o f t h e f o r m a t i o n o f these complexes, a r a d i o a c t i v e a n t i g e n was r e q u i r e d . The r a d i o a c t i v e lysozyme i s o l a t e d as d e s c r i b e d above was used. N u c l e i c a c i d e x t r a c t i o n s o f macrophage i n c u b a t e d w i t h r a d i o a c t i v e lysozyme found l i t t l e a c t i v i t y i n t h e n u c l e i c a c i d f r a c t i o n s . A thorough d i s c u s s i o n i n l i g h t o f t h e s e r e s u l t s i s presented^ a l o n g w i t h some s u g g e s t i o n s f o r f u r t h e r work. - i v -TABLE OF CONTENTS Page ABSTRACT - i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENT i x GENERAL INTRODUCTION , 1 PART I . ISOLATION OF LYSOZYME 3 A. INTRODUCTION 3 B. METHODS • • • 6 1. I s o l a t i o n o f lysozyme from egg w h i t e . . . . 6 2. P u r i f i c a t i o n o f lysozyme on a p r e p a r a t i v e e l e c t r o p h o r e s i s u n i t 7 a. d e s c r i p t i o n o f f u n n e l 7 b. c a s t i n g t h e a c r y l a m i d e g e l 10 c. s e t t i n g up a r u n 10 d. t e r m i n a t i n g a run .15 3. E x t r a c t i o n o f p u r i f i e d lysozyme from t h e p o l y a c r y l a m i d e g e l • .16 4. D e s a l t i n g o f lysozyme 16 5. Q u a n t i t a t i v e p o l y a c r y l a m i d e d i s c g e l e l e c t r o p h o r e s i s 17 6. S c i n t i l l a t i o n c o u n t i n g 20 - v -G. RESULTS AND DISCUSSION 22 D. SUMMARY ' 32 PART I I . PREPARATION OF RADIOACTIVE LYSOZYME IN LAYING HENS. . . . 33 A. INTRODUCTION. . 3 3 B. METHODS 35 C. RESULTS AND DISCUSSION 37 D. SUMMARY 41 y PART I I I . IN VITRO CULTURES WITH RADIOACTIVE LYSOZYME 42 A. INTRODUCTION 42 B. METHODS 45 1. I s o l a t i o n o f c h i c k e n lymphocytes 45 14 2. I r i v i t r o lymphocyte c u l t u r e w i t h C. l a b e l l e d lysozyme 46 3. I s o l a t i o n of g u i n e a p i g macrophage c e l l s . . .4 7 3 4. In v i t r o macrophage c u l t u r e w i t h H l a b e l l e d lysozyme 48 C. RESULTS AND DISCUSSION . 50 D. SUMMARY 55 GENERAL DISCUSSION 56 BIBLIOGRAPHY 59 APPENDIX 6 2 - v i -LIST OF TABLES Page I A c t i v i t i e s o f lysozyme p r e p a r e d e l e c t r o p h o r e t i c a l l y u s i n g two d i f f e r e n t c athodes 25 I I T y p i c a l y i e l d s o f A „ o n m a t e r i a l from 2. o U egg w h i t e by m o d i f i e d CM-52 method o f Chance and C l a r k (2) 28 I I I T y p i c a l r e c o v e r y d a t a f o r p r e p a r a t i v e e l e c t r o p h o r e s i s r u n (7 h r . d u r a t i o n ) o f CM-52 i s o l a t e d lysozyme p r e p a r a t i o n 29 IV I n v i v o s y n t h e s i s o f lysozyme d u r i n g t h e l a y i n g c y c l e f o l l o w i n g i . v . i n j e c t i o n o f a l 4 C - a m i n o a c i d h y d r o l y s a t e . 3 8 v i i -LIST OF FIGURES Page 1 Dimensions o f f u n n e l used as a p r e p a r a t i v e e l e c t r o p h o r e s i s u n i t 9 2 A p p a r a t u s s e t up f o r p o u r i n g a c r y l a m i d e g e l . 9 3 P r e p a r a t i v e e l e c t r o p h o r e s i s u n i t p r i o r t o a 7 h r . r u n 12 3a The p r e p a r a t i v e e l e c t r o p h o r e s i s u n i t d u r i n g a. r u n • - • 13 4 A p p a r a t u s f o r f i n a l d e s a l t i n g o f lysozyme p r e p a r a t i o n s 19 5 Q u a n t i t a t i v e p o l y a c r y l a m i d e d i s c g e l s o f samples from t h e lysozyme i s o l a t i o n p r o c e d u r e 31 6 S p e c i f i c a c t i v i t i e s o f egg w h i t e lysozyme ("^C l a b e l l e d ) i s o l a t e d from the second 14 egg o f b i r d s i n j e c t e d l . v . w i t h a C amino a c i d h y d r o l y s a t e (75 juCi) a t v a r i o u s t i m e s a f t e r o v i p o s i t i o n 40 7 R a d i o a c t i v i t y p r o f i l e f o r n u c l e i c a c i d e x t r a c t i o n o f lymphocyte c u l t u r e i n c u b a t e d w i t h l 4 C l a b e l l e d lysozyme 51 - v m -R a d i o a c t i v i t y p r o f i l e f o r n u c l e i c ac e x t r a c t i o n o f macrophage c u l t u r e s 3 i n c u b a t e d w i t h H - l a b e l l e d lysozyme i x -ACKNOWLEDGEMEN T The a u t h o r would l i k e t o thank Dr. R.C. Fitzsimmons f o r h i s a d v i c e and a i d d u r i n g t h e d u r a t i o n o f t h i s t h e s i s programme. W i t h o u t t h e many l i v e l y d i s c u s s i o n s , b o t h r e l a t e d and u n r e l a t e d t o t h i s t h e s i s , i t would have been a d u l l M. Sc. programme. v Thanks a l s o t o the Departments o f Food S c i e n c e , P l a n t S c i e n c e , A n i m a l S c i e n c e and P o u l t r y S c i e n c e ( N u t r i t i o n Lab) f o r use o f equipment and r e a g e n t s . S p e c i a l thanks t o Dr. D. C l a r k , U.B.C. C h e m i s t r y Department f o r t h e r a d i o a c t i v e lysozyme. S p e c i a l t h a n k s t o G. G a l z y , Department o f A n i m a l S c i e n c e and Dorothy E m s l i e f o r t h e i r many s p e c i a l e f f o r t s d u r i n g the c o u r s e o f t h e e x p e r i m e n t s . - 1 GENERAL INTRODUCTION A method o f the i s o l a t i o n o f c h i c k e n egg w h i t e lysozyme o f h i g h p u r i t y has been dev e l o p e d as p a r t o f the exp e r i m e n t s f o r t h i s t h e s i s . I n o r d e r f o r the p r o d u c t o f t h i s p r o c e d u r e t o be u s e f u l f o r f u r t h e r e x p e r i m e n t s , p u r i t y was i m p o r t a n t . A l t h o u g h lysozyme i s o l a t i o n t e c h n i q u e s do e x i s t (1, 2) they g e n e r a l l y r e s u l t i n impure p r e p a r a t i o n s and ar e r e l a t i v e l y l e n g t h y . P r e l i m i n a r y work f o r t h i s t h e s i s showed t h a t egg w h i t e e l e c t r o p h o r e s e d on q u a n t i t a t i v e p o l y a c r y l a m i d e g e l s gave r i s e t o a s i n g l e s t a i n i n g band. From t h i s i n i t i a l o b s e r v a t i o n , a p r e p a r a t i v e e l e c t r o p h o r e t i c a p p a r a t u s was dev e l o p e d w h i c h was used t o i s o l a t e m i l l i g r a m q u a n t i t i e s o f p u r i f i e d lysozyme. I n o r d e r t o i n v e s t i g a t e one a s p e c t o f a n t i g e n p r o c e s s i n g lymacrophage c e l l s (PART I I I ) , a r a d i o a c t i v e a n t i g e n was r e q u i r e d . They p r e p a r a t i v e e l e c t r o p h o r e t i c method d e v e l o p e d i n PART I was used t o i s o l a t e r a d i o a c t i v e lysozyme from young l a y i n g hens i n j e c t e d w i t h r a d i o a c t i v e 14 C amino a c i d h y d r o l y s a t e s . Some i n t e r e s t i n g d a t a on the f o r m a t i o n o f lysozyme i n t h e hen o v i d u c t was c o l l e c t e d . The r a d i o a c t i v e lysozyme i s o l a t e d by t h i s method was used i n c e l l c u l t u r e e x p e r i m e n t s (PART I I I ) . - 2 -Macrophage have l o n g been i m p l i c a t e d i n the immune response ( 3 ) . By i n c u b a t i n g macrophage w i t h r a d i o a c t i v e lysozyme, i t was hoped t o d e t e r m i n e the time a t which t h e f o r m a t i o n o f RNA-antigen (RNA-Ag) complexes was maximal. T h i s d e t e r m i n a t i o n would a l l o w i n v e s t i g a t i v e work on t h e n a t u r e o f t h e RNA i n v o l v e d i n t h i s complex. - 3 -PART I . ISOLATION OF LYSOZYME A. INTRODUCTION S i n c e i t s d i s c o v e r y by S i r A l e x a n d e r Fleiraning i n 1922 ( 4 ) , egg w h i t e lysozyme has become a v e r y p o p u l a r p r o t e i n f o r s c i e n t i f i c r e s e a r c h . Lysozyme i s a p r o t e i n composed o f 129 amino a c i d s ( p r i m a r y s t r u c t u r e known (5, 6 ) , the m a j o r i t y o f which are b a s i c a t p h y s i o l o g i c a l pH. Four c y s - c y s bonds m a i n t a i n the t e r t i a r y s t r u c t u r e o f t h e m o l e c u l e . Some q u a t e r n a r y s t r u c t u r e i n t h e form o f pH dependant dimers has been o b s e r v e d a t pH 5-9. H i g h e r polymers a r e formed a t pH v a l u e s above 10 ( 7 ) . Lysozyme i s a l s o known f o r i t s e n z y m a t i c a c t i v i t y (4, 8 ) . I t h y d r o l y s e s the 3 - (1-4) g l y c o s i d i c l i n k a g e between N - a c e t y l muramic a c i d (NAM) and N - a c e t y l glucosamine (NAG) t o g i v e e q u i m o l a r amounts o f NAM and NAG. I t i s because o f t h i s a b i l i t y t h a t i t has the common name muramidase. C e r t a i n amino a c i d sequences o f lysozyme a r e known t o be h i g h l y a n t i g e n i c . The amino a c i d sequence r e s i d u e s 60-8 3, commonly r e f e r r e d to as l o o p p e p t i d e (one c y s - c y s bond) i s a known a n t i g e n i c d e t e r m i n a n t ( 9 ) . _ 4 -T h i s f e a t u r e , a l o n g w i t h t h e ease o f i s o l a t i o n (10) make t h i s a c o n v e n i e n t m o l e c u l e f o r i m m u n o l o g i c a l r e s e a r c h . Lysozyme w i t h i t s n e t p o s i t i v e charge a t p h y s i o l o g i c a l pH l e n d s i t s e l f t o p r e p a r a t i v e e l e c t r o p h o r e t i c methods o f s e p a r a t i o n . The f a c t t h a t the m a j o r i t y o f the o t h e r egg w h i t e p r o t e i n s a r e a p p a r e n t l y n e g a t i v e l y charged a t p h y s i o l o g i c a l pH makes e l e c t r o p h o r e s i s a d e s i r a b l e method f o r t h e s e p a r a t i o n o f lysozyme from nonlysozyme p r o t e i n . P r o t e i n p u r i f i c a t i o n methods c u r r e n t l y i n use i n c l u d e c r y s t a l l i z a t i o n ( 1 ) , g e l f i l t r a t i o n (11) and i o n exchange (2) t e c h n i q u e s . A l t e r t o n and F e v o l d (1) r e p o r t e d a t e c h n i q u e f o r t h e d i r e c t c r y s t a l l i z a t i o n o f lysozyme from egg w h i t e . T h e i r t e c h n i q u e was based on t h e o b s e r v a t i o n t h a t lysozyme was i n s o l u b l e a t pH 10.5 i n the presence o f 5% N a C l . One d i s a d v a n t a g e f o r t h i s t e c h n i q u e was t h e l e n g t h o f time r e q u i r e d b e f o r e an a c c e p t a b l e q u a n t i t y o f lysozyme had p r e c i p i t a t e d . A 2 o r 3x r e c r y s t a l l i z a t i o n p r o c e d u r e c o u l d take up t o 12 days. M o l e c u l a r f i l t r a t i o n t e c h n i q u e s , a l t h o u g h f a s t e r than d i r e c t c r y s t a l l i z a t i o n have d i s a d v a n t a g e s (.11) . Large columns a r e r e q u i r e d t o p r e v e n t the o v e r l a p and t a i l i n g o f p r o t e i n peaks due t o an o v e r l o a d e d column. The v i s c o u s -nature o f egg w h i t e r e q u i r e s t h a t i t be p r e t r e a t e d b e f o r e - 5 -l o a d i n g onto the column i n o r d e r t o p r e v e n t c l o g g i n g o f the s e p a r a t i o n phase ( g e l f i l t r a t i o n beads) o f t h e column. Due t o t h e above d i s a d v a n t a g e s , an i o n exchange proce d u r e was employed. The i o n exchange proc e d u r e o f Chance and C l a r k (2) was used s i n c e i t i s f a s t and r e l a t i v e l y easy t o a c c o m p l i s h . T h i s method employed an i n i t i a l p u r i f i c a t i o n w i t h C M - c e l l u l o s e f o l l o w e d by p u r i f i c a t i o n on a B i o Rex 70 i o n exchange column. F i n a l d e s a l t i n g was a c c o m p l i s h e d on a B i o g e l P-2 column. The f i n a l p r o d u c t was judged t o be pure by a number o f c r i t e r i a ( 2 ) . In the e x p e r i m e n t s f o r t h i s t h e s i s , t h e p r e p a r a t i v e e l e c t r o p h o r e t i c a p p a r a t u s was s u b s t i t u t e d f o r the B i o Rex i o n exchange chromatography. Lysozyme produced by t h i s p r o c e d u r e t r a v e l l e d as a s i n g l e s t a i n i n g band on q u a n t i t a t i v e p o l y a c r y l a m i d e d i s c g e l system c o n t a i n i n g sodium d o d e c y l s u l f a t e (SDS). A l t h o u g h o t h e r e a s i l y p r e p a r e d m o l e c u l e s e x i s t , lysozyme was used because i t was r e l a t i v e l y easy t o p r e p a r e i n the r a d i o a c t i v e form. The f a c t t h a t i t has a number o f known a n t i g e n i c s i t e s , a l s o e a s i l y p r e p a r e d , made i t even more d e s i r a b l e as t h e p r o t e i n t o use f o r t h i s s t u d y . - 6 -B. METHODS 1. Isolation of lysozyme from egg white The method for i s o l a t i o n of lysozyme was that of Chance and Clark (2) with some modifications. Seventy-five mis of buffer A (appendix 1) was added to the albumen of one fresh egg (< 3 days o l d ) . This mixture was moderately s t i r r e d on a magnetic s t i r p l a t e for 1/2 - 3/4 hr. After t h i s time the majority of the thick albumen was no longer discernable. The mixture was f i l t e r e d through four folds of cheesecloth and added to 50 ml of s e t t l e d carboxymethyl c e l l u l o s e * (pre-equilibrated with buffer A). The s l u r r y was moderately s t i r r e d (magnetic s t i r r e r ) for approximately 1/2 hr. and then centrifuged for 20 min. at 4100 x G. The supernatant was decanted and the p e l l e t resuspended i n 75 ml of fresh buffer A. This wash procedure was completed a t o t a l of 4X. The A280 ( of a l l washings i n t h i s procedure were determined on a Unicam SP1800 dual beam spectrophotometer. After the fourth wash with buffer A, the p e l l e t was resuspended i n 50 ml of buffer B (appendix 2) and centrifuged. This wash was completed 4X and the resultant * Whatman CM-52, ce l l u l o s e - c a t i o n exchange material - 7 -f r a c t i o n s p o o l e d . The p o o l e d sample c o n t a i n i n g a m a j o r i t y o f l y s o z y m e was f i l t e r e d t h r o u g h Whatman #1 (vacuum f i l t r a t i o n ) f o l l o w e d by M i l l i p o r e f i l t r a t i o n . * T h i s s e r v e d t o remove c o n t a m i n a t i n g c a r b o x y m e t h y l c e l l u l o s e p a r t i c l e s . The sample was t h e n c o n c e n t r a t e d by u l t r a f i l t r a t i o n i n a 6 5 ml s t i r r e d c e l l u s i n g a UM-2 d i a f l o membrane ( 3 0 p s i o p e r a t i n g p r e s s u r e ) . The c o n c e n t r a t e d sample ( 5 - 1 0 ml) was p u r i f i e d on a p r e p a r a t i v e e l e c t r o p h o r e s i s u n i t (PART I , 2 ) . 2. P u r i f i c a t i o n o f l y s o z y m e on a p r e p a r a t i v e e l e c t r o p h o r e s i s u n i t a. D e s c r i p t i o n o f f u n n e l ( F i g . 1) The f u n n e l u s e d was a P y r e x b o t t l e f u n n e l s o l d as F i s h e r S c i e n t i f i c c a t . no-. 1 0 - 3 4 6 E . The f u n n e l was m o d i f i e d by m e l t i n g f i v e g l a s s ' f i n g e r s ' i n t o t h e s i d e s a p p r o x i m a t e l y e q u i d i s t a n t a r o u n d t h e c i r c u m f e r e n c e as shown i n F i g . 1. The f u n c t i o n o f t h e s e ' f i n g e r s ' was t o s u p p o r t t h e a c r y l a m i d e g e l . r e g i s t e r e d t r a d e m a r k , .45 p o r e s i z e . - -8 -Key f o r F i g . 1 €i - g l a s s f i n g e r s - t h e i r f u n c t i o n i s t o s u p p o r t t h e p o l y a c r y l a m i d e g e l . A l l d i m e n s i o n s a r e i n c e n t i m e t e r s (cm). Key f o r F i g . 2 C2 - r i n g s t a n d b - f u n n e l C - r i n g clamp d - #12 r u b b e r s t o p p e r © - 250 ml e r l e n m e y e r f l a s k F i g . 1. Dimensions of funnel used as preparative electrophoresis unit. a c \ e F i g . 2 . Apparatus set up for pouring acrylamide g e l . V - 10 -b. C a s t i n g the a c r y l a m i d e g e l ( F i g . 2) The r i n g clamp e x e r t s p r e s s u r e t h rough t h e f u n n e l t o make a l i q u i d t i g h t s e a l between the f u n n e l and s t o p p e r . The complete a c r y l a m i d e s o l u t i o n (appendix 3) was poured t h r o u g h t h e stem o f t h e f u n n e l onto the r u b b e r s t o p p e r (#12) . The s o l u t i o n s h o u l d c o v e r t h e g l a s s f i n g e r s c o m p l e t e l y . A f t e r p o l y m e r i z a t i o n had o c c u r r e d (ca 1/2 hr.) t h e r i n g clamp was c a r e f u l l y removed f o l l o w e d by removal o f t h e f u n n e l . The g e l does n o t s t i c k t o the s t o p p e r and hence the two can e a s i l y be s e p a r a t e d . The g e l (suspended i n t h e f u n n e l ) was r i n s e d w i t h b u f f e r E (appendix 4) p r e p a r a t i o n f o r sample a p p l i c a t i o n . c. S e t t i n g up a r u n ( F i g . 3) A f t e r t h e g e l had been c a s t i n t h e f u n n e l , t h e f u n n e l was s e t up as i n F i g . 3. The r i n g clamp s u p p o r t s t h e f u n n e l and the 3 - f i n g e r e d clamp s t a b i l i z e s t h e f u n n e l d u r i n g subsequent movement. A m o d i f i e d rubber s t o p p e r ( F i g . 3) was then i n s e r t e d i n t o t h e stem. The p o s i t i o n o f t h e s u b g e l - s u r f a c e tube was a l t e r e d u n t i l i t s t i p j u s t b a r e l y touched t h e g e l . T h i s tube was used t o remove t h e a i r w h i c h w a s " d i s p l a c e d by t h e b u f f e r t h a t f i l l e d t h i s compartment. Once t h i s tube had been p o s i t i o n e d p r o p e r l y , the r u b b e r s t o p p e r was - I I Key for F i g . 3 a - anode b -i buffer E, upper reservoir c - sample to be electrophoresed; 10% sucrose added d -5.6% polyacrylamide gel e - supporting r i n g clamp f - supporting 3 fingered clamp g -modified #6 rubber stopper h - subsurface gel tube • i - buffer E "out 1 tube © 1 " buffer E ' i n ' tube k - cathode •i - buffer E charge tube for lower reservoir. \ - .12 -F i g . 3. P r e p a r a t i v e e l e c t r o p h o r e s i s u n i t p r i o r t o a 7 - h r r u n . - 13 -- 14 -t a p e d (3M masking tape) to the stem. The lower compartment was charged w i t h b u f f e r E v i a t h e b u f f e r charge tube. As t h e b u f f e r f i l l e d t h i s compartment, e q u i v a l e n t volumes o f a i r were removed i n o r d e r t o p r e v e n t r u p t u r e o f the g e l . By t i l t i n g t h e f u n n e l , a l l the a i r from t h e lower compartment was removed. Once t h i s s t a g e had been completed the a p p a r a t u s was t r a n s f e r r e d t o a r e f r i g e r a t o r . B u f f e r E was added t o t h e upper r e s e r v o i r t o a h e i g h t 2 cm from t h e top o f the f u n n e l . The r e q u i r e d c o n n e c t i o n s on the g l a s s "T" were made and t h e e l e c t r o d e s c o n n e c t e d t o t h e power s u p p l y . An optimum f l o w r a t e o f c a . 500 m l / h r . was e s t a b l i s h e d by a d j u s t i n g the clamps on t h e b u f f e r E " i n " and "out" tubes ( F i g . 3 ) . T h i s b u f f e r f l o w s e r v e s t o remove hydrogen b u b b l e s g e n e r a t e d by t h e cathode from t h e system. The b u f f e r r e s e r v o i r was p l a c e d on t o p o f t h e r e f r i g e r a t o r (130 cm p r e s s u r e head) and c o n n e c t e d t o t h e b u f f e r E " i n " tube v i a a l e n g t h o f 6 mm i . d . s u r g i c a l t u b i n g . By c o n n e c t i n g a number o f 4L e r l e n m e y e r f l a s k s i n s e r i e s , s u f f i c i e n t volume c o u l d be r e a l i z e d to l a s t o v e r n i g h t ( i f r e q u i r e d ) . The b u f f e r E "out" tube d r a i n s i n t o t h e c r i s p e r o f t h e r e f r i g e r a t o r , w h i c h was emptied when r e q u i r e d . - 15 -The sample (5-10 m l , 10% added sucrose) was l a y e r e d under t h e upper b u f f e r r e s e r v o i r w i t h a s y r i n g e and s h o r t l e n g t h o f t u b i n g o r a p a s t e u r p i p e t t e . The power s u p p l y * was t u r n e d on and a d j u s t e d t o 45 ma. The system runs a t 10-12°. d. T e r m i n a t i n g a r u n The b u f f e r f l o w was t e r m i n a t e d and t h e power s u p p l y s h u t o f f a t t h e end o f t h e r u n . The upper b u f f e r r e s e r v o i r was removed by s u c t i o n and t h e s u r f a c e o f t h e g e l and s i d e s o f t h e f u n n e l r i n s e d v e r y t h o r o u g h l y w i t h b u f f e r E. A l l t r a c e s o f spent b u f f e r E and b i t s o f carbon must be removed. The g e l was removed by i n s e r t i n g a s p a t u l a between the g e l and f u n n e l w a l l . Enough a i r was l e t i n t o t h e space below t h e g e l t o n e u t r a l i z e t h e convex shape assumed by t h e g e l d u r i n g e l e c t r o p h o r e s i s . The g e l was c a r e f u l l y removed w i t h the f i n g e r s and r i n s e d i n c o l d t a p w a t e r . S h o u l d t h e g e l f l i p o v e r d u r i n g r e m o v a l , t h e r e i s no cause f o r a l a r m i f i t s s u r f a c e was c l e a n e d p r o p e r l y . The lower r e s e r v o i r b u f f e r was c o l l e c t e d w i t h a p a s t e u r p i p e t t e . The f u n n e l and lower e l e c t r o d e assembly were r i n s e d w i t h b u f f e r E and t h i s wash added t o t h e i n i t i a l volume o f lower r e s e r v o i r b u f f e r . * VOKAM C o n s t a n t C u r r e n t 0-50 ma; C o n s t a n t DC V o l t a g e 0-400 VDC - 16 -3. E x t r a c t i o n o f p u r i f i e d lysozyme from g e l 3 The g e l was c u t up i n t o c a . 2 cm cubes f o l l o w i n g removal from t h e f u n n e l . These cubes were t h o r o u g h l y macerated i n a c e l l homogenizer. The p l a s t i c p e s t l e i s 5.4 mm under s i z e t o a l l o w ready m a c e r a t i o n o f t h e g e l . The whole g e l was p r e p a r e d i n t h i s way. A s m a l l volume of b u f f e r E was added t o t h i s m i x t u r e and s t i r r e d (magnetic s t i r r e r ) f o r c a . 1/2 h r . The m i x t u r e was f i l t e r e d t h r o u g h Whatman #1 f i l t e r paper on a buchner f u n n e l a p p a r a t u s . The g e l was e x t r a c t e d a t o t a l o f 3X w i t h t h e l a s t two e x t r a c t i o n s b e i n g o v e r n i g h t a t 4° and t h r e e hours a t 4°, r e s p e c t i v e l y . The m a j o r i t y o f t h e A 2 g Q a b s o r b i n g m a t e r i a l was e x t r a c t e d from t h e g e l i n t h e f i r s t two e x t r a c t i o n s . The f i r s t and second e x t r a c t i o n s were p o o l e d and l y o p h i l i z e d i n p r e p a r a t i o n f o r d e s a l t i n g . 4. D e s a l t i n g o f p u r i f i e d lysozyme The l y o p h i l i z e d samples were r e c o n s t i t u t e d i n a s m a l l volume o f d e i o n i z e d w a t e r ( d i r ^ O ) and a p p l i e d t o a Sephadex* G-25 (2.4 cm x 39 cm) column (90 cm p r e s s u r e head, ca 70 ml/hr f l o w r a t e ) . The i n i t i a l major peak was l y o p h i . l i . z e d . * trademark - Pharmacia f i n e c h e m i c a l s . - 17 -The l y o p h i l i z e d peak was r e c o n s t i t u t e d i n 2.5 ml d i H 2 0 and p l a c e d i n d i a l y s i s t u b i n g (1 cm f l a t w i d t h ) . T h i s t u b i n g was p l a c e d i n t h e a p p a r a t u s shown i n F i g . 4. The samples were d i a l y s e d a g a i n s t ca. 30 1 o f d i H 2 0 i n t h i s way (ca. 700 m l / h r . f l o w r a t e ) . A f t e r c o m p l e t i o n o f d i a l y s i s the samples were removed q u a n t i t a t i v e l y from t h e d i a l y s i s bags. These washings were p o o l e d and l y o p h i l i z e d . The s p e c i f i c r a d i o a c t i v i t y d e t e r m i n e d from t h e s e f r e e z e d r i e d p r o d u c t s were used i n a l l subsequent c a l c u l a t i o n s where r a d i o a c t i v e lysozyme was p r e p a r e d and used. 5. Q u a n t i t a t i v e p o l y a c r y l a m i d e d i s c g e l e l e c t r o p h o r e s i s The method o f P e t r i (12) w i t h some m o d i f i c a t i o n s was used. Samples were added t o sample b u f f e r ,(appendix 5) which c o n t a i n e d sodium d o d e c y l s u l f a t e (SDS) and 2-m e r c a p t o e t h a n o l and p l a c e d i n a b o i l i n g w a t e r b a t h f o r 2 min. Up t o 50 u l o f t h e s e samples were used p e r g e l (appendix 6 ) . The sample was c a r e f u l l y o v e r l a y e d w i t h b u f f e r E (and 1% SDS) and t h e power t u r n e d on. A c o n s t a n t c u r r e n t o f 2 ma/tube was a p p l i e d u n t i l t h e t r a c k i n g dye (bromophenol b l u e ) had e n t e r e d the g e l ( c o n f i r m e d v i s u a l l y ) , the c u r r e n t was then i n c r e a s e d t o 5 ma/tube. - 18 -Key t o F i g . 4. Q - d e i o n i z e d w a t e r r e s e r v o i r (ca 30L) b - d i r e c t i o n o f water f l o w C - p u r i f i e d lysozyme samples i n d i a l y s i s membranes d - g l a s s column (20 x 400 mm). - 19..: -a F i g . 4. A p p a r a t u s f o r f i n a l d e s a l t i n g o f lysozyme p r e p a r a t i o n s . - 20 -When the dye was 1-2 cm from t h e bottom o f t h e g e l , t h e power was s h u t o f f . The g e l s were removed from t h e g l a s s tubes (6 mm i d x 75 mm 1) by r i n g i n g w i t h a s y r i n g e and 30 gauge n e e d l e ( d i H 2 0 as l u b r i c a n t ) . The g e l s were s t a i n e d f o r a t l e a s t 2 h r s . i n 0.25% Coomassic b l u e s o l u t i o n (appendix 7) f o l l o w e d by e l e c t r i c d e s t a i n i n g (Pharmacia e l e c t r i c g e l d e s t a i n e r ) i n 5% a c e t i c a c i d s o l u t i o n . ' G e l s were s t o r e d i n f r e s h d e s t a i n i n g s o l u t i o n f i n s t o p p e r e d t e s t t u b e s . 6. L i q u i d S c i n t i l l a t i o n c o u n t i n g - I s o c a p 300 s c i n t i l l a t i o n c o u n t e r Dry samples (e.g. f r e e z e d r i e d lysozyme) were weighed o u t i n t o g l a s s s c i n t i l l a t i o n v i a l s ( p o l y e t h y l e n e ) l i n e d c a p s ) . The sample was s o l u b i l i z e d i n 0.25 ml d i H 2 0 and d i g e s t e d by a d d i t i o n o f 1 ml o f P r o t o s o l . * D i g e s t i o n o f t h e sample was c o n s i d e r e d complete when t h e s o l u t i o n was c o m p l e t e d l y c l e a r (2-5 h r . ) . E c o n o f l u o r * (10 ml) was used as t h e s c i n t i l l a t i o n f l u i d . Wet samples (e.g. albumen) were t r e a t e d i d e n t i c a l l y e x c e p t t h a t t h e sample volume was 0.25 ml and d i r ^ O was t h e r e f o r e o m i t t e d . In t h e d e t e r m i n a t i o n o f s p e c i f i c a c t i v i t y , <• samples were counted f o r a p e r i o d o f 10 min. * trademark NEN Canada L t d . - 21 -Counts per minute (CPM) were c o r r e c t e d t o d i s i n t e g r a t i o n s per minute (DPM) by a p p l y i n g CPM d a t a 14 t o a quench c u r v e d e r i v e d from C quenched s t a n d a r d s s u p p l i e d by t h e m a n u f a c t u r e r . E f f i c i e n c i e s r a n g i n g from 50-85% were r e a l i z e d i n , t h i s t h e s i s . . - 22 -C. RESULTS & DISCUSSION For t h i s s t u d y , an a n t i g e n was r e q u i r e d t h a t was i n e x p e n s i v e and easy t o i s o l a t e . Lysozyme was s u g g e s t e d by Dr. D. C l a r k o f t h e U.B.C. C h e m i s t r y Dept. The complete p r i m a r y s t r u c t u r e o f lysozyme i s known (13) i n c l u d i n g t h e sequence o f some s p e c i f i c a n t i g e n i c d e t e r m i n a n t s c o n t a i n e d i n t h i s m o l e c u l e (14, 15). T h i s f a c t was a n e c e s s i t y f o r f u r t h e r s t u d i e s as a p a r t o f t h i s t h e s i s . The i n i t i a l o b s e r v a t i o n t h a t d i l u t e d albumen gave r i s e t o a s i n g l e s t a i n i n g band when e l e c t r o p h o r e s e d on q u a n t i t a t i v e d i s c g e l s s u g g e s t e d t h a t lysozyme c o u l d be p r e p a r e d i n pure form by s i m p l e p r e p a r a t i v e e l e c t r o p h o r e s i s o f egg w h i t e . A l t h o u g h t h i s i d e a was c o n c e p t u a l l y sound, t e c h n i c a l problems w i t h t h e e l e c t r o p h o r e t i c u n i t p r e v e n t e d complete s u c c e s s . I n o r d e r t o o b t a i n p r e p a r a t i v e amounts o f lysozyme ru n t i m e s o f 20-33 h r s . d u r a t i o n were r e q u i r e d . In runs o f t h i s l e n g t h , one o f t h e f i r s t problems e n c o u n t e r e d was t h e r a t h e r dynamic p r o p e r t y o f t h e g e l i t s e l f . D u r i n g v a r i o u s runs t h e g e l was o b s e r v e d t o assume convex and/or concave c o n f i g u r a t i o n s (16) . A b r i e f l i t e r a t u r e s u r v e y r e v e a l e d t h a t t h i s was a common phenomena and t h a t i n t h e - 23 -p a r t i c u l a r papers examined, t h e r e was no s o l u t i o n ( 1 6 ) . Sim p l e removal o r a d d i t i o n o f b u f f e r d i d n o t r e s t o r e the g e l t o i t s o r i g i n a l h o r i z o n t a l c o n f i g u r a t i o n ( 1 6 ) . I t was f i n a l l y d e t e r m i n e d f o r the u n i t d e s c r i b e d here t h a t t h e d i a m e t e r o f t h e t u b i n g and hence f l o w r a t e t h r ough t h e cathode was r e s p o n s i b l e . I n i t i a l l y , an u n m o d i f i e d p a s t e u r p i p e t t e was immersed i n t o t h e b u f f e r r e s e r v o i r and a c t e d as t h e i n l e t . When t h i s p i p e t t e was r e p l a c e d w i t h l a r g e r d i a m e t e r g l a s s t u b i n g ( 5 mm i d . i n c r e a s e ) t h i s problem was g r e a t l y reduced. The narrow o p e n i n g o f t h e p i p e t t e had r e s t r i c t e d t o some e x t e n t , t h e maximum f l o w r a t e t h rough t h e lo w e r e l e c t r o d e assembly. Hence t h e lower e l e c t r o d e a c t e d as a vacuum d i a l y s i s mechanism r e s u l t i n g i n removal o f b u f f e r from t h e lo w e r compartment. T h i s r e s u l t e d i n d e f l e c t i o n o f t h e g e l towards t h e lower e l e c t r o d e . When t h e l a r g e r d i a m e t e r tube was r e p l a c e d f o r t h e p a s t e u r p i p e t t e , g r e a t e r f l o w r a t e s were p o s s i b l e . The problem here was t h a t t h e i n c r e a s e d f l o w r a t e caused c o l l a p s e o f the d i a l y s i s membrane around t h e e l e c t r o d e . T h i s p r e v e n t e d t h e hydrogen b u b b l e s from b e i n g c a r r i e d o u t o f t h e system and subsequent c e s s a t i o n o f b u f f e r f l o w . I n o r d e r t o p r e v e n t membrane c o l l a p s e t h e f l o w r a t e was reduced by p l a c i n g clamps on the b u f f e r E "out" and " i n " t u b e s . By a d j u s t i n g t h e two - 24 -clamps t h e p r o p e r membrane d i s t e n t i o n and f l o w r a t e c o u l d be a c h i e v e d . O c c a s i o n a l l y , i f t h e s e s e t t i n g s were n o t c o r r e c t , t h e l o w e r e l e c t r o d e assembly a c t e d as a r e v e r s e vacuum d i a l y s i s mechanism. T h i s caused d e f l e c t i o n o f t h e g e l i n t h e o p p o s i t e d i r e c t i o n as p r e v i o u s l y d e s c r i b e d . The amount o f d e f l e c t i o n n o r m a l l y p r e s e n t a f t e r two hours was g r e a t l y reduced w i t h t h e s e m o d i f i c a t i o n s . A l t h o u g h s i g n i f i c a n t l y r educed, th e d e f l e c t i o n problem c o u l d n o t be n u l l i f i e d c o m p l e t e l y . D u r i n g t h e c o u r s e o f t h e 20-30 h r . r u n s , t h e o v e r n i g h t p e r i o d became most c r i t i c a l . R a r e l y c o u l d t h e f l o w r a t e o f b u f f e r t h r o u g h t h e l ower e l e c t r o d e be s e t so t h a t z e r o d e f l e c t i o n c o u l d be m a i n t a i n e d o v e r n i g h t . When t h e d e f l e c t i o n became l a r g e enough, the g e l would l o o s e n from th e g l a s s s i d e s o f t h e f u n n e l ; t h e r e f o r e , a l l o w i n g m i x i n g o f the upper and l o w e r b u f f e r r e s e r v o i r s . Any m i x i n g would t h e r e f o r e contaminate the l o wer r e s e r v o i r w i t h non-lysozyme p r o t e i n ; an u n d e s i r a b l e s i t u a t i o n . The second problem e n c o u n t e r e d was the low e n z y m a t i c a c t i v i t y o f t h e lysozyme p r e p a r e d by t h i s method. Ta b l e I summarizes t h e a c t i v i t i e s o f lysozyme b e f o r e and a f t e r an e l e c t r o p h o r e t i c s e p a r a t i o n u s i n g copper o r p l a t i n u m (Pt) as t h e cathode. Copper i s known t o have a d e l e t e r i o u s e f f e c t on t h e s t a b i l i t y o f lysozyme (13) and i t was thought t h a t by s u b s t i t u t i n g a P t e l e c t r o d e , Table I . Enzymatic a c t i v i t i e s o f lysozyme p r e p a r e d e l e c t r o p h o r e t i c a l l y u s i n g two d i f f e r e n t c athodes. D e s c r i p t i o n o f lysozyme sample Lysozyme a c t i v i t y (% - o f c o n t r o l ) Copper cathode P l a t i n u m cathode C o n t r o l , commercial lysozyme^ 100% = 21,000 u n i t s / A . U . 2 8 Q . 3 Egg w h i t e i s o l a t e d lysozyme pre 33 h r . e l e c t r o p h o r e s i s . As 2 b u t f o l l o w e d by d i a l y s i s as i n 5. 3 Egg w h i t e i s o l a t e d lysozyme p o s t 33 h r . e l e c t r o p h o r e s i s . As 4 f o l l o w e d by d i a l y s i s a g a i n s t pH 6.24 0.066M KH 2P0 4 b u f f e r f o r 48 h r s . 2 C o n t r o l , " c o m m e r c i a l lysozyme p o s t 33 h r . e l e c t r o p h o r e s i s . As 6 f o l l o w e d by d i a l y s i s as i n 5. 100 87 70 25 62 67 71 36 26 1 Assay as d e s c r i b e d i n Sigma t e c h n i c a l b u l l e t i n L6876. (Sigma C h e m i c a l Co., S t . L o u i s , Mo., U.S.A.) 2 C o m m e r c i a l l y p r e p a r e d by Sigma Ch e m i c a l Co. 3X r e c r y s t a l l i z e d from egg w h i t e , 3 R e f e r s t o lysozyme i s o l a t e d from egg w h i t e as d e s c r i b e d i n PART I , A ( l ) . - 26 -the a c t i v i t y o f t h e f i n a l p r o d u c t c o u l d be i n c r e a s e d . The a c t i v i t y o f t h e f i n a l p r o d u c t was i n c r e a s e d by a p p r o x i m a t e l y 10% when a p l a t i n u m cathode was used (Table I ) . U n f o r t u n a t e l y , t h i s 10% i n c r e a s e was l o s t when t h e sample was d i a l y s e d a g a i n s t b u f f e r K (appendix 8 ) . When a copper cathode was used, the a c t i v i t y o f lysozyme was a p p r o x i m a t e l y 2-1/2 times g r e a t e r than p r i o r t o d i a l y s i s . These r e s u l t s s u g g e sted t h a t copper w i r e be used i n a l l f u t u r e s e p a r a t i o n s . When one compares t h e a c t i v i t i e s o f lysozyme p u r i f i e d from egg w h i t e by t h e method d e s c r i b e d i n PART I v s . commercial grade lysozyme* p u r i f i e d on t h e e l e c t r o p h o r e t i c a p p a r a t u s , d i f f e r e n c e s i n a c t i v i t y a r e ob s e r v e d . Commercial lysozyme p u r i f i e d on t h i s a p p a r a t u s was a p p r o x i m a t e l y 3 ti m e s more a c t i v e than egg w h i t e lysozyme p r e p a r e d s i m i l a r i l y . W h i l e t h e a c t i v i t y o f egg w h i t e lysozyme i n c r e a s e d 2-1/2 f o l d a f t e r d i a l y s i s , t h e a c t i v i t y o f lysozyme p u r i f i e d from commercial s o u r c e s i n c r e a s e d a few p e r c e n t . S i n c e PART I I I o f t h i s t h e s i s r e q u i r e d p r e p a r a t i v e amounts o f r a d i o a c t i v e lysozyme, which had t o be p r e p a r e d i n t h i s l a b o r a t o r y , the b e h a v i o u r o f commercial lysozyme on t h e p r e p a r a t i v e e l e c t r o p h o r e s i s u n i t d e s c r i b e d was no t c r i t i c a l . S h o u l d t h i s a p p a r a t u s be used t o p u r i f y c o mmercial lysozyme, i t would c e r t a i n l y be i m p o r t a n t t o r m a i n t a i n e n z y m a t i c a c t i v i t y . * Sigma Ch e m i c a l Co. - 27 -A n o t h e r problem e n c o u n t e r e d d u r i n g a p r e p a r a t i v e e l e c t r o p h o r e t i c r u n was d i s i n t e g r a t i o n o f t h e anode (a carbon r o d s a l v a g e d from a "D" s i z e b a t t e r y ) . D u r i n g a 33 h r . r u n , t h e r o d c o n t i n u a l l y r e l e a s e d t i n y carbon p a r t i c l e s w h i c h s l o w l y sank t o the s u r f a c e o f t h e g e l . T h i s n o t o n l y c o n t a m i n a t e d the upper b u f f e r r e s e r v o i r b u t may have i n t e r f e r r e d w i t h t h e homogeneity o f t h e e l e c t r i c f i e l d a c r o s s t h e g e l . Due t o the t e c h n i c a l d i f f i c u l t i e s d e s c r i b e d , t h e p r e p a r a t i v e e l e c t r o p h o r e t i c method was a l t e r e d . I n s t e a d o f a t t e m p t i n g t o e l e c t r o p h o r e s e t h e lysozyme i n t o the lo w e r r e s e r v o i r b u f f e r , i t was d e c i d e d t o reduce t h e r u n time c o n s i d e r a b l y and e x t r a c t t h i s enzyme from t h e g e l . I t was a l s o d e c i d e d t o use an i n i t i a l p u r i f i c a t i o n s t e p from raw egg w h i t e , u s i n g t h e CM-52 (Table I I ) method o f Chance and C l a r k ( 2 ) . T h i s i n i t i a l s t e p would s e r v e t o remove t h e m a j o r i t y o f c o n t a m i n a t i n g p r o t e i n and hence make th e e l e c t r o p h o r e s i s more e f f i c i e n t i n terms o f t h e y i e l d o f lysozyme. The y i e l d o f lysozyme was s u b s t a n t i a l a t t h i s s t a g e . T a b l e I I I shows t y p i c a l r e s u l t s o f t h e &28O u n i t s a p p l i e d and s u b s e q u e n t l y e x t r a c t e d from t h e p r e p a r a t i v e 1 & p o l y a c r y l a m i d e g e l . Assuming E ^ m . 2 g o nm = ^6.3 ( 2 ) , up t o 25 mg. p e r egg were p u r i f i e d by t h i s method. Table I I . T y p i c a l y i e l d s o f A 2 g 0 m a t e r i a l from egg w h i t e by m o d i f i e d CM-52 method o f Chance and C l a r k (2) . PH Wash number 280/ml Volume (ml) T o t a l A 280 ph 9.0' 1 2 3 4 13.64 3.2 0.62 0.18 81 72 74 75 1105 230 46 13 ph 9.2' 1 2 3 4 2.0 0.9 0 . 38 0.23 49 54 51 52 98 49 19 12 2 3 Carboxymethyl c e l l u l o s e i s c e n t r i f u g e d (4080 x G, 20 min) and resuspended i n -75 ml b u f f e r A. pH 9.0 washings c o n t a i n t h e m a j o r i t y , o f non lysozyme egg w h i t e p r o t e i n s . The ph 9.2 washings a r e p o o l e d and c o n c e n t r a t e d i n an u l t r a f i l t r a t i o n d e v i c e i n p r e p a r a t i o n f o r p r e p a r a t i v e e l e c t r o p h o r e s i s . T h i s i s t h e lysozyme e n r i c h e d f r a c t i o n . T a b l e I I I . T y p i c a l r e c o v e r y d a t a f o r p r e p a r a t i v e e l e c t r o p h o r e s i s r u n (7 h r . d u r a t i o n ) o f CM-52 i s o l a t e d lysozyme p r e p a r a t i o n . Absorbance* 2 80 nm Nature o f Sample A p p l i e d A.U^gQ** m a t e r i a l e x t r a c t e d from p o l y a c r y l a m i d e g e l Upper Lower f i r s t second t h i r d R e s e r v o i r R e s e r v o i r e x t r a c t , e x t r a c t , e x t r a c t . CM-52 lysozyme 228 193 60 28 10 Background ( s u c r o s e i n B u f f e r E) 0.25 84 3.5 3.4 2.5 1 mg lysozyme has A 2 80 ~ 2.0. A.U. A b s o r p t i o n U n i t . The amount o f m a t e r i a l w h i c h has an absorbance o f 1.0 i n a 1 cm p a t h l e n g t h c e l l a t a wavelength o f 280 - 30 -A l t h o u g h up t o 5 0 mg were e x t r a c t e d from the g e l , l a r g e l o s s e s w h i c h o c c u r r e d d u r i n g t h e d e s a l t i n g s t e p s p r e v e n t e d q u a n t i t a t i v e r e c o v e r y . Y i e l d s o f 5-25 mg. o f p u r i f i e d m a t e r i a l were t y p i c a l . Lysozyme p r e p a r e d by t h i s method t r a v e l l e d as a s i n g l e s t a i n i n g band on SDS d i s c g e l s ( F i g . 5 ) . T h i s lysozyme was found t o c o n t a i n c o n t a m i n a n t s a f t e r h a v i n g been s t o r e d a t 4° f o r p e r i o d s e x c e e d i n g one month. The n a t u r e o f th e s e c o n t a m i n a n t s were not c h a r a c t e r i z e d . I t was n o t e d however, t h a t t h e major c o n t a m i n a n t t r a v e l l e d a t t h e same p o s i t i o n on 7.5% a c r y l a m i d e g e l s as d i d t h e major contaminant o f a sample of commercial lysozyme. When thes e bands were c u t o u t and counted ( i n P r o t o s o l & E c o n o f l o u r ) i n a l i q u i d s c i n t i l l a t i o n c o u n t e r , a l l d e t e c t a b l e r a d i o a c t i v i t y was found i n the lysozyme band. r HIS l l l l I I / 2 3 ? PH?.O WASH 5 6 ? 8 / > / / * 2 to II 0 0 1—' 1 / a p#?2 *£S. AES. STMT). F i g . 5 . Q u a n t i t a t i v e p o l y a c r y l a m i d e d i s c g e l s o f samples from t h e lysozyme i s o l a t i o n p r o c e d u r e . See a l s o T a b l e I I . Lower r e s . and upper r e s . r e f e r t o t h e upper b u f f e r r e s e r v o i r and l o w e r b u f f e r r e s e r v o i r ( i n c l u d i n g m a t e r i a l e x t r a c t e d from t h e g e l ) o f t h e p r e p a r a t i v e e l e c t r o p h o r e s i s u n i t . - 32 -D. SUMMARY An i n e x p e n s i v e , s i m p l e p r e p a r a t i v e p o l y a c r y l a m i d e e l e c t r o p h o r e t i c a p p a r a t u s was d e v e l o p e d f o r t h e i s o l a t i o n o f egg w h i t e lysozyme. T h i s method i s based on t h e o b s e r v a t i o n t h a t lysozyme appears t o be t h e o n l y p o s i t i v e l y c h a r g e d p r o t e i n i n egg w h i t e a t pH 7.6. Up t o 25 mg. o f a p p a r e n t l y pure lysozyme was i s o l a t e d w i t h t h i s t e c h n i q u e . Some c o n t a m i n a t i o n was found i n samples s t o r e d f o r p e r i o d s e x c e e d i n g one month. However, t h i s c o n t a m i n a t i o n i s a l s o o b s e r v e d i n commercial p r e p a r a t i o n s . - 33 -PART I I . PREPARATION OF RADIOACTIVE LYSOZYME IN LAYING HENS A. INTRODUCTION. A b r i e f survey of the l i t e r a t u r e did not suggest an optimum time at which to i n j e c t a "^C-amino acid hydrolysate into laying hens i n order to get maximum incorporation of label into egg white lysozyme. Oades and Brown (17) found that the oviduct of a mature laying hen contains s u f f i c i e n t water soluble oviduct proteins (WSOP) for about two eggs. Most of i t appears to be formed i n the magnum during the inter v a l s between the passage of successive eggs down the oviduct (18, 19-21). Secretory goblet c e l l s of the magnum discharge t h e i r contents computely during the passage of an egg and are replenished a few hours l a t e r (22, 23); a similar discharge from the tubular gland c e l l s has also been reported (21, 24). The rate at which these c e l l s are recharged with d i f f e r e n t proteins varies. Mandel and Ducay (18) found that 14 birds injected (i.v.) with C l a b e l l e d ammo acids did not lay a radioactive egg u n t i l the second day a f t e r i n j e c t i o n . They also found that ovalbumin and conalbumin were 50% more radioactive than lysozyme. These workers did not 14 state the exact time of i n j e c t i o n of the C l a b e l l e d - 34 -amino a c i d s t hey used. P r e l i m i n a r y work f o r t h i s t h e s i s showed t h a t 14 l . v . i n j e c t i o n w i t h 5 jaCi o f a C amino a c i d h y d r o l y s a t e d i r e c t l y a f t e r o v i p o s i t i o n r e s u l t e d i n lysozyme w i t h z e r o d e t e c t a b l e r a d i o a c t i v i t y . These r e s u l t s s u g g e sted t h e p o s s i b i l i t y t h a t a l a r g e r dose o f r a d i o a c t i v i t y i n j e c t e d a f t e r o v i p o s i t i o n would produce r a d i o a c t i v e lysozyme. - 35 -B. METHODS White l e g h o r n hens k e p t a t t h e U.B.C. Pou l t r y -Farm were used. A commercial l a y e r r a t i o n and wa t e r were s u p p l i e d ad l i b i t u m - . The c l u t c h l e n g t h o f 6 b i r d s was r e c o r d e d and o n l y b i r d s i n the b e g i n n i n g o r m i d d l e o f t h e i r c l u t c h were used. On the day o f i n j e c t i o n t h e e x a c t time o f o v i -p o s i t i o n was d e t e r m i n e d and t h e b i r d was i n j e c t e d i . v . w i t h 14 50 u C i o f a C amino a c i d h y d r o l y s a t e * 0.75 h r . a f t e r o v i p o s i t i o n . The b i r d was s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n a f t e r f o u r eggs had been c o l l e c t e d . A l i q u o t s o f albumen (0.25 ml) were counted f o r r a d i o a c t i v i t y (PART I , A, v i ) and t h e " h o t t e s t " eggs were used f o r subsequent lysozyme i s o l a t i o n . The method o f i s o l a t i o n was t h a t as d e s c r i b e d i n PART I , A. In a l l c a s e s , the second egg l a i d f o l l o w i n g i n j e c t i o n had t h e " h o t t e s t " albumen f o l l o w e d by t h e second and t h i r d eggs. I n t h e i n i t i a l e x p e r i m e n t d e s c r i b e d above, the hen was i n j e c t e d a t 0.75 h r . a f t e r o v i p o s i t i o n . The lysozyme i s o l a t e d from t h e second and t h i r d eggs was not c o n s i d e r e d "hot" enough f o r t h e c u l t u r e e x p e r i m e n t s t o be * NEN Canada L t d . see appendix 10. - 36 -completed i n PART I I I . I n e f f o r t s t o a c h i e v e "hot" lysozyme, t h e dosage o f r a d i o a c t i v i t y was i n c r e a s e d t o 75 u C i and t h e b i r d was i n j e c t e d 3 h r s . p o s t o v i p o s i t i o n . A t h i r d a t t e m p t was a l s o made. The dosage remained c o n s t a n t bu the b i r d was i n j e c t e d 5 h r s . p o s t o v i p o s i t i o n . \ - 37 -C. RESULTS AND DISCUSSION A supply of radioactive lysozyme was required to complete the experiments described i n PART I I I . I n i t i a l l y i t was thought that i . v . i n j e c t i o n of 5 jaCi of an amino acid hydrolysate into a laying hen 45 min. after o v i p o s i t i o n would r e s u l t i n high s p e c i f i c a c t i v i t y lysozyme i n the albumen of the egg l a i d the following day. This was based on the assumption that the majority of the egg white lysozyme was synthesized and l a i d down i n the albumen as the yolk passed through the magnum. I t was found that a dosage of 75 uCi injected at 3 or 5 hours postoviposition -was required to produce lysozyme with a s p e c i f i c a c t i v i t y greater than 2 x l O 1 ^ dpm/mole i n the second egg l a i d following ovposition. This data suggests that the bulk of the lysozyme for an egg i s synthesized up to 44 hrs. pr i o r to ovipo s i t i o n , and not as the yolk passes through the magnum. Table IV summarizes the results obtained i n attempts to prepare h i s p e c i f i c r a d i o a c t i v i t y lysozyme. 14 The i n j e c t i o n of a C-amino acid hydrolysate 3 hrs. postoviposition results i n lysozyme ca. 6 times "hotter" than when a 0.75 hr. i n j e c t i o n postoviposition was made. Increasing the i n t e r v a l to 5 hrs. results i n an increase / - 38 -Ta b l e IV. I n v i v o s y n t h e s i s o f lysozyme d u r i n g the l a y i n g c y c l e f o l l o w i n g I.V. i n j e c t i o n o f 14 a C-ammo a c i d h y d r o l y s a t e . Time p o s t -o v i p o s i t i o n i n j e c t i o n H r s . Lysozyme s p e c i f i c a c t i v i t y Egg Number  2 3 2-5' 1 0 .75 3.0 5.0 0.36" 2.22 2.60 0.45 1.10 1.18 0.76 S p e c i f i c a c t i v i t y , DPM/mole x 10"^; assume m o l e c u l a r w e i g h t o f 14,300 gm/mole o f lysozyme. Egg l a i d a f t e r f i r s t day p o s t i n j e c t i o n , b u t n o t a f t e r f i f t h day. Id . . Only 50 j i C i "C-ammo a c i d h y d r o l y s a t e i n j e c t e d . i - 39 -i n a c t i v i t y o f c a . 1.2 o v e r t h a t o f t h e 3 h r . i n j e c t i o n . F i g . 6 d e p i c t s the s p e c i f i c a c t i v i t i e s o f egg w h i t e 14 lysozyme ( *C l a b e l l e d ) i s o l a t e d from t h e second egg 14 o f b i r d s i n j e c t e d w i t h a C-amino a c i d h y d r o l y s a t e (75 jiCi) a t v a r i o u s t i m e s a f t e r o v i p o s i t i o n . The a c t i v i t y seems t o have p l a t e a u e d a t about 5 h r s . p o s t -14 o v i p o s i t i o n , w h i c h s u g g e s t s t h a t i n j e c t i n g t h e C-amino a c i d h y d r o l y s a t e l a t e r than 5 h r s . p o s t o v i p o s i t i o n would n o t r e s u l t i n a s i g n i f i c a n t i n c r e a s e i n t h e s p e c i f i c r a d i o a c t i v i t y o f "^C l a b e l l e d lysozyme. T h i s s u g g e s t i o n s h o u l d be v e r i f i e d e x p e r i m e n t a l l y . T h i s would suggest t h a t i n c r e a s i n g t h e dose o f r a d i o a c t i v i t y b u t k e e p i n g the i n j e c t i o n time c o n s t a n t c o u l d r e s u l t i n h i g h e r s p e c i f i c a c t i v i t y lysozyme. S i n c e the n a t u r e o f t h e i n c o r p o r a t i o n 14 o f the C-amino a c i d s i n t o lysozyme was not d e t e r m i n e d , t h i s s u g g e s t i o n s h o u l d a l s o be v e r i f i e d e x p e r i m e n t a l l y . V I * - * A • " * n T -0 ] 2 3 4 5 6 7 8 hrs 14 F i g . 6. S p e c i f i c a c t i v i t i e s o f egg w h i t e lysozyme ( C l a b e l l e d ) i s o l a t e d from the second egg of b i r d s i n j e c t e d i . v . w i t h a 1 4 C - a m i n o a c i d h y d r o l y s a t e (7 5 u C i ) a t v a r i o u s t i m e s a f t e r o v i p o s i t i o n . (Note: the f i r s t p o i n t i s t h e r e s u l t of a 50 u C i i n j e c t i o n , n o t 75 jiCi as f o r t h e r e m a i n i n g p o i n t s ) . - 41 -D. SUMMARY R a d i o a c t i v e lysozyme was i s o l a t e d from eggs o f l a y i n g hens (PART I) i n j e c t e d w i t h a m i c r o c u r i e amounts 14 o f C-amino a c i d h y d r o l y s a t e s . The second egg l a i d f o l l o w i n g an i . v . i n j e c t i o n p o s t o v i p o s i t i o n c o n t a i n e d t h e h i g h e s t s p e c i f i c a c t i v i t y lysozyme. The optimum time f o r i n j e c t i o n was 5 h r s . p o s t o v i p o s i t i o n w h i c h r e s u l t e d i n lysozyme w i t h an a c t i v i t y o f 2.6 x lO^dpm/mole. 14 I n j e c t i n g t h e C-amino a c i d h y d r o l y s a t e l e s s than 5 h r s . p o s t o v i p o s i t i o n r e s u l t e d i n lower s p e c i f i c a c t i v i t y lysozyme. 42 -PART I I I . IN VITRO CULTURES WITH RADIOACTIVE LYSOZYME A, INTRODUCTION These e x p e r i m e n t s (PART I I I ) w i l l d e a l w i t h some a s p e c t s o f a new t h e o r y c o n c e i v e d and d e v e l o p e d i n t h i s l a b o r a t o r y ( P o u l t r y Embryology L a b o r a t o r y , U.B.C.) over t h e l a s t s i x y e a r s (25) . T h i s t h e o r y a t t e m p t s t o d e s c r i b e a mechanism f o r t h e g e n e r a t i o n o f a n t i b o d y d i v e r s i t y d u r i n g an immune r e s p o n s e . B r i e f l y s t a t e d , t h e t h e o r y i s as f o l l o w s . When a f o r e i g n p r o t e i n e n t e r s t h e h o s t i t i s i m m e d i a t e l y e n g u l f e d by p h a g o c y t i c c e l l s (most l i k e l y K u p f e r c e l l s , t i s s u e h i s t i o c y t e s and p e r i t o n e a l macrophage). Some o f t h i s a n t i g e n r e a c t s w i t h RNA m o l e c u l e s t o g i v e an RNA a n t i g e n complex h e r e a f t e r r e f e r r e d t o as RNA-Ag. T h i s complex i s p r o c e s s e d by v a r i o u s p r o t e o l y t i c and RNase enzymes so t h a t the s u r v i v i n g e n t i t y i s o f t h e s i z e as d e s c r i b e d by many r e s e a r c h e r s (elsewhere t h i s r e v i e w ) . A c c o r d i n g t o Fit z s i m m o n s (25, 2 6 ) , t h e RNA-Ag complex a c t s t o p r e v e n t e x c e s s i v e h y d r o l y s i s o f t h e a n t i g e n i c d e t e r m i n a n t and a s s o c i a t e d RNA m o l e c u l e , i . e . mutual p r o t e c t i o n o c c u r s through b i n d i n g f o r t h e sequence o f n u c l e o t i d e s and t h e a n t i g e n i c fragment. Each p r o t e c t s the o t h e r from exc e s s p r o c e s s i n g . We now have a - 43 -p r o c e s s e d RNA-Ag complex. T h i s complex i s t r a n s f e r r e d t o lymphocytes e i t h e r v i a c y t o p l a s m i c i n f o r m a t i o n exchange b r i d g e s o r r e l e a s e d from the macrophage t o be p i c k e d up by r e c e p t i v e lymphocytes. One i n s i d e t h e lymphocyte the RNA may e i t h e r be ( i ) r e p l i c a t e d and th e n r e v e r s e t r a n s c r i b e d ( v i a Reverse T r a n s c r i p t a s e ) t o a c o r r e s p o n d i n g DNA sequences, w h i c h may then be i n t e g r a t e d i n t o t h e lymphocyte genome a t t h e h y p e r - v a r i a b l e s i t e s ; o r ( i i ) r e v e r s e t r a n s c r i b e d d i r e c t l y as DNA fragments and i n t e g r a t e d i n t o the genome. I n b o t h c a s e s , the RNA codes (through DNA) f o r an amino a c i d sequence which would be i d e n t i c a l t o t h a t o f one o r more o f the hyp e r -v a r i a b l e r e g i o n s i n an immunoglobulin m o l e c u l e . The immunoglobulins produced would be s p e c i f i c f o r the a n t i g e n w h i c h i n i t i a t e d t h i s sequence o f e v e n t s . RNA has been i m p l i c a t e d t o have the r o l e d i s c u s s e d above ( C l t s i n c e Garvey and Campbell (27) f i r s t o b s e r v e d RNA complexed w i t h fragments o f r a d i o a c t i v e BSA i n t h e u r i n e o f BSA c h a l l e n g e d r a b b i t s . S i n c e t h a t time the l i t e r a t u r e p u b l i s h e d on the r o l e o f RNA i n the immune response has been voluminous (28, 2 9 ) . Most exponents o f t h e C l o n a l (6) t h e o r y suggest t h a t t h e RNA a c t s s i m p l y as an a d j u v a n t f o r the Ag, hence the word " s u p e r a n t i g e n " (28, 30-33). R o e l a n t s and Goodman - 44 -(341 c l a i m t h a t t h e s e complexes a r e a r t i f a c t u a l . In a l a t e r paper (.30) t h e s e same a u t h o r s i n v e s t i g a t e d t h e b i n d i n g o f s e v e r a l m o l e c u l e s o f v a r y i n g charge and s i z e t o t o t a l c e l l u l a r RNA, d e r i v e d from macrophages. From t h e i r r e s u l t s t hey c o n c l u d e d t h a t a l l macrophage RNA-Ag complexes appear t o be magnesium i o n complexes, j o i n i n g n e g a t i v e groups on t h e p o l y p e p t i d e c h a i n w i t h n e g a t i v e l y c harged phosphate groups on the RNA. G o t t l i e b and Schwartz C29) q u e s t i o n e d t h e s e c o n c l u s i o n s and s u b s e q u e n t l y >{ demonstrated t h a t s p e c i f i c i t y may o r may not be shown depending on v a r i o u s f a c t o r s o n e o f w h i c h was i n c u b a t i o n c o n d i t i o n s . L i t t l e i s known o f t h e mechanics o f RNA-Ag f o r m a t i o n . By i n c u b a t i n g macrophage c u l t u r e s w i t h r a d i o -a c t i v e lysozyme ( a n t i g e n ) and e x t r a c t i n g RNA a t v a r i o u s t imes a f t e r i n i t i a l e x p o s u r e , i t was hoped t h a t the time a t which RNA-Ag f o r m a t i o n was maximum c o u l d be d e termined. Once t h i s was d e t e r m i n e d , a method c o u l d then be e s t a b l i s h e d f o r t h e r o u t i n e i s o l a t i o n o f RNA-Ag complexes. T h i s would a l l o w more i n t e n s i v e s t u d y o f t h e RNA i n v o l v e d i n the RNA-Ag c omplex. - 45 -B. METHODS 1. I s o l a t i o n o f lymphocytes Whole b l o o d (1 ml) was c o l l e c t e d from t h e wing v e i n o f mature w h i t e l e g h o r n hens. H e p a r i n (15 ug per ml. blood) was used as t h e a n t i c o a g u l a n t . The b l o o d was mixed w i t h 0.25 ml 0.9% s a l i n e and l a y e r e d o v er 3 ml o f f i c o l l -hypaque s o l u t i o n (appendix 9) i n a 11 x 100 mm t e s t tube. A f t e r c e n t r i f u g a t i o n f o r 20 min (400 x G) the lymphocytes w h i c h had c o l l e c t e d a t the f i c o l l - h y p a q u e plasma i n t e r f a c e were a s p i r a t e d i n t o a s t e r i l e t e s t t ube. The lymphocytes i n s u s p e n s i o n were s e t t l e d by c e n t r i f u g a t i o n (10 min 150 x G). The plasma was d e c a n t e d and t h e p e l l e t washed t w i c e w i t h RPM1 1640 (appendix 1 1 ) . The f i n a l p e l l e t was suspended i n 10 ml o f RPMl 164 0 and c o u n t e d i n a hemocytometer. The s u s p e n s i o n was a d j u s t e d t o 2 x 1 0 6 c e l l s / m l w h i c h was t h e . c e l l c o n c e n t r a t i o n used i n a l l i n v i t r o c u l t u r e s . C e l l v i a b i l i t y was d e t e r m i n e d by the t r y p a n b l u e e x c l u s i o n method (appendix 12) and a d i f f e r e n t i a l w i t h Leishmans s t a i n completed a t t h i s p o i n t . I n a l l c a s e s , v i a b i l i t y was > 97% and n o n - l y m p h o c y t i c c e l l s were n o t d e t e c t e d . - 46 -2. I n v i t r o lymphocyte c u l t u r e w i t h C l a b e l l e d lysozyme S t e r i l e d i s p o s a b l e p l a s t i c t e s t tubes (16 x 95 mm) w i t h snap f i t t i n g caps were used t h r o u g h o u t th e c u l t u r e e x p e r i m e n t s . Two ml o f c e l l s u s p e n s i o n was used per tube. Lysozyme ( i n 0.9% s a l i n e ) was added a t the r a t e o f 0.4 mg per c u l t u r e tube. The tube was i n v e r t e d and s l o w l y r o t a t e d t o make sure t h a t a l l t h e lysozyme mixed w i t h t h e c e l l s u s p e n s i o n . The completed c u l t u r e was i n c u b a t e d a t 37° w i t h the cap snapped i n p l a c e . Gas exchange w i t h the i n c u b a t o r environment was t h e r e f o r e i m p o s s i b l e . D u p l i c a t e samples were removed from the i n c u b a t o r and c e n t r i f u g e d f o r 10 min. (150 x G) a t 0, 2, 5, 8, 16 and 24 h r s . A 0.25 ml sample was removed f o r s c i n t i l l a t i o n c o u n t i n g . The r e m a i n i n g s u p e r n a t a n t was decanted and 2 ml PBS (appendix 13) added t o the p e l l e t . T h i s a d d i t i o n was f o l l o w e d by 2 ml p h e n o l . * T h i s b i p h a s i c system was shaken f o r c a . 1 min and then t r a n s f e r r e d t o p o l y p r o p y l e n e c e n t r i f u g e tubes (13 x 104 mm). A f t e r c e n t r i f u g a t i o n f o r 10 min (3090 x G) a 0.25 ml a l i q u o t was removed from the aqueous phase f o r c o u n t i n g . A l l counts were c o r r e c t e d t o DPM by t h e c h a n n e l s r a t i o method. * ' M a l l i n c k r o d t - no p r e s e r v a t i v e s . - 47 -c 3. I s o l a t i o n o f g u i n e a p i g macrophage c e l l s The m o d i f i e d method o f Sabet e t a l (35) was used t o i s o l a t e macrophage c e l l s . Mature male g u i n e a p i g s were i n j e c t e d IP w i t h a s t e r i l e , 3% s a l i n e s u s p e n s i o n o f Sephadex G-50 f i n e . * A p p r o x i m a t e l y 72 h r s . p o s t i n j e c t i o n the g u i n e a p i g was s a c r i f i c e d by c h l o r o f o r m a n e s t h e s i a . The s k i n was c u t from t h e r e c t a l a r e a t o a p o i n t between t h e f r o n t l e g s and r e f l e c t e d p e r p e n d i c u l a r to t h e l o n g a x i s o f t h e c u t . The u n d e r l y i n g muscle t i s s u e was c u t s i m i l a r l y and t h e i n t e s t i n e s p a r t i a l l y e x t r a c t e d . T w e n t y - f i v e t o 30 ml o f s t e r i l e s a l i n e was dec a n t e d i n t o t h e p e r i t o n e u m and a s p i r a t e d a g a i n s t t h e p e r i t o n e a l l i n i n g . The r e s u l t i n g c e l l s u s p e n s i o n was a s p i r a t e d i n t o s t e r i l e p l a s t i c c u l t u r e tubes and c e n t r i f u g e d f o r 10-15 min (150 x G). The s u p e r n a t a n t was dec a n t e d and t h e p e l l e t washed w i t h 5 ml o f RPM1 1640. The r e s u l t i n g p e l l e t s were resuspended i n RPMl 1640 and p o o l e d t o a t o t a l volume o f 20 m l . A b s o l u t e and d i f f e r e n t i a l c o u n t s were made i n a hemocytometer. The s u s p e n s i o n was a d j u s t e d t o 2 x 10 macrophage/ml which was the f i n a l c o n c e n t r a t i o n used i n subsequent c u l t u r e e x p e r i m e n t s . * trademark - Pharmacia F i n e C h e m i c a l s . - 48 -C e l l v i a b i l i t y was d e t e r m i n e d by t h e t r y p a n b l u e e x c l u s i o n method (appendix 12). C e l l v i a b i l i t y was > 97% i n a l l c a s e s . C e l l s h a r v e s t e d by t h i s t e c h n i q u e were 40-50% macrophage. The r e m a i n i n g c e l l s were r e d b l o o d c e l l s . 3 4. I n v i t r o macrophage c u l t u r e d w i t h H l a b e l l e d lysozyme S t e r i l e d i s p o s a b l e p l a s t i c t e s t tubes (16 x 95 mm) w i t h snap f i t t i n g caps were used t h r o u g h o u t t h e c u l t u r e e x p e r i m e n t s . Two ml o f c e l l s u s p e n s i o n was used per tube. Lysozyme was used a t t h e r a t e o f 0.4 mg per c u l t u r e tube. The tube was i n v e r t e d and s l o w l y r o t a t e d to make s u r e t h a t a l l the lysozyme mixed w i t h t h e c e l l s u s p e n s i o n . The completed c u l t u r e was i n c u b a t e d a t 37° w i t h t h e cap snapped i n p l a c e . Gas exchange w i t h t h e i n c u b a t o r environment was t h e r e f o r e i m p o s s i b l e . D u p l i c a t e samples were removed from t h e i n c u b a t o r and c e n t r i f u g e d (150 x G, 10 min) a t 0, 2, 4, 6, 8, 12 & 24 h r s . A.0.25 ml a l i q u o t was removed f o r s c i n t i l l a t i o n c o u n t i n g (0.25 ml sample t o 15 ml A q u a s o l * ) . The r e m a i n i n g s u p e r n a t a n t was decanted and 2 ml PBS added to t h e p e l l e t . T h i s a d d i t i o n was f o l l o w e d by 2 ml p h e n o l . T h i s b i p h a s i c system was shaken * A q u a s o l - NEN trademark - 49 -f o r c a . 1 min and then t r a n s f e r r e d t o p o l y p r o p y l e n e c e n t r i f u g e t u b e s . A f t e r c e n t r i f u g a t i o n (12,350 x G, 10 min) a 0.25 ml a l i q u o t was removed from t h e aqueous phase f o r c o u n t i n g . A l l c o u n t s were c o r r e c t e d t o DPM by the c h a n n e l s r a t i o method. - 50 -C. RESULTS & DISCUSSION Lysozyme t r i t i a t e d a t t h e two m e t h i o n i n e p o s i t i o n s 14 was used i n t h e macrophage c u l t u r e e x p e r i m e n t s . C l a b e l l e d lysozyme was used i n lymphocyte c u l t u r e e x p e r i m e n t s . The 3 t r i t i a t e d lysozyme (2.1 x 10 DPM/mg) was t h e g i f t o f Dr. D. C l a r k (U.B.C. C h e m i s t r y D e p t . ) . A c c o r d i n g t o t h e t h e o r y d i s c u s s e d i n t h e I n t r o d u c t i o n , c u l t u r e o f macrophage w i t h a n t i g e n s h o u l d l e a d t o RNA-Ag complexes soon a f t e r i n i t i a l e xposure. S i n c e t h e a n t i g e n used i n t h e s e e x p e r i m e n t s was r a d i o a c t i v e , t h i s a s s o c i a t i o n between RNA and lysozyme s h o u l d be d e t e c t a b l e i n the aqueous ( n e c l e i c a c i d f r a c t i o n ) phase o f a p h e n o l i c e x t r a c t i o n o f macrophage c e l l s . F i g u r e s 7 and 8 a r e t h e r e s u l t s o f i n v i t r o c u l t u r e e x p e r i m e n t s w i t h r a d i o a c t i v e lysozyme. T h e o r e t i c a l l y one s h o u l d e x p e c t a r a t h e r l e v e l c u r v e f o r t h e lymphocyte c u l t u r e s ( F i g . 7 ) . S i n c e the lymphocyte i s n o t e x p e c t e d t o a c t i v e l y i n g e s t the a n t i g e n , th e f o r m a t i o n o f RNA-Ag complexes would n o t o c c u r and hence r a d i o a c t i v i t y would n o t appear i n t h e aqueous phase o f a ph e n o l e x t r a c t i o n . The r e s u l t s o f t h e macrophage c u l t u r e s ( F i g . 8) s h o u l d have r e f l e c t e d p r o t e i n Ag upta k e . The macrophage s h o u l d have i n g e s t e d l a r g e amounts o f lysozyme r e s u l t i n g i n r a d i o a c t i v i t y i n t h e n u c l e i c a c i d p o p u l a t i o n . T h i s - 51, -1 dpm 110 30 f " ' ' ' culture medium f r a c t i o n 60 nucleic acid f r a c t i o n 20 F i g . 7, 8 12 16 20 24 irs Radioactivity p r o f i l e for nucleic acid extraction of lymphocyte cultures incubated with l^C l a b e l l e d lysozyme. Results of one experiment. F i g . 8. Radioactivity p r o f i l e for nucleic acid extraction of macrophage cultures incubated with l a b e l l e d lysozyme. Results of one experiment. - 52 - \ w o u l d have r e s u l t e d i n an i n c r e a s e i n r a d i o a c t i v i t y o v e r b a c k g r o u n d . Whether o r n o t t h i s i n c r e a s e w o u l d have t h e n f a l l e n a g a i n t o b a c k g r o u n d l e v e l s o r r e m a i n e d a t a p l a t e a u i s q u e s t i o n a b l e . Had t h e a c t i v i t y d r o p p e d t o b a c k g r o u n d f o l l o w i n g t h i s r i s e , t h e n one c o u l d s u g g e s t a breakdown o f RNA-Ag c o m p l e x e s . I f a p l a t e a u h a d f o r m e d one c o u l d s u g g e s t a c o n s t a n t t u r n o v e r o f c o m p l e x e s a c c o r d i n g t o t h e f o l l o w i n g f o r m u l a ; RNA + Ag > RNA-Ag ^ RNA + Ag. The above s t a t e m e n t s a r e c o n j e c t u r e . An i n v i v o s y s t e m m i g h t have g i v e n c o m p l e t e l y d i f f e r e n t r e s u l t s . F i g u r e s 7 and 8 a r e t h e r e s u l t s o f t h e i n v i t r o e x p e r i m e n t s . The c o u n t s f r o m t h e aqueous p h a s e o f t h e p h e n o l e x t r a c t i o n h o v e r a b o u t t h e . b a c k g r o u n d l e v e l and hence a r e p r o b a b l y i n s i g n i f i c a n t . One c o u l d l o o k a t t h e amount o f r a d i o a c t i v e l y s o z y m e r e m a i n i n g i n t h e c u l t u r e m e d i a o f e a c h sample p e r i o d b u t t h i s w o u l d n o t d i f f e r e n t i a t e between i n t e r i o r i z e d l y s o z y m e a n d / o r n u c l e i c a c i d a s s o c i a t e d l y s o z y m e ( o r l y s o z y m e a s s o c i a t e d w i t h t h e e x t e r i o r c e l l s u r f a c e membrane). V a r i o u s f a c t o r s c o u l d have been r e s p o n s i b l e f o r t h e s e r e s u l t s . The most l i k e l y one i s t h a t t h e l y s o z y m e u s e d was n o t " h o t " enough and h e n c e c o u l d n o t be d e t e c t e d i n t h e n u c l e i c a c i d , f r a c t i o n s o f t h e s e v a r i o u s c e l l p o p u l a t i o n s . The s o l u t i o n h e r e i s o b v i o u s ; a h i g h e r s p e c i f i c a c t i v i t y l y s o z y m e i s r e q u i r e d . - 53 -S i n c e lysozyme i s a s o l u b l e a n t i g e n , t h e r a t e o f i n g e s t i o n by macrophage might not be as g r e a t as f o r m a t e r i a l s such as r e d b l o o d c e l l s , b a c t e r i a and macro-m o l e c u l e s (e.g. hemoglobin, h o r s e r a d i s h p e r o x i d a s e ) . I f t h e r a t e i s d e p r e s s e d , low a c t i v i t y i n t h e n u c l e i c a c i d p o p u l a t i o n c o u l d be e x p e c t e d . T h i s r e a s o n i n g c o u l d e x p l a i n the r e s u l t s o b t a i n e d from t h e macrophage c u l t u r e e x p e r i m e n t s . The s o l u t i o n h e r e , a l t h o u g h q u i t e s t r a i g h t f o r w a r d i s l e s s d e s i r a b l e i n terms o f i n t e r p r e t a t i o n o f e x p e r i m e n t a l r e s u l t s . By a t t a c h i n g t h e lysozyme t o some l a r g e r c a r r i e r , such as -a CM sephadex o r CM c e l l u l o s e p a r t i c l e , t h e lysozyme would be p h a g o c y t i z e d a l o n g w i t h the p a r t i c l e . T h i s s h o u l d r e s u l t i n a l a r g e amount o f lysozyme i n t e r i o r i z e d by t h e macrophage; e s p e c i a l l y i f t h e CM p a r t i c l e s were s a t u r a t e d v w i t h t h e a n t i g e n . T h i s t e c h n i q u e s h o u l d i n c r e a s e t h e amount o f r a d i o a c t i v i t y a s s o c i a t e d w i t h t h e n u c l e i c a c i d f r a c t i o n . One d i s a d v a n t a g e o f t h i s method c o u l d be t h a t t h e c a r r i e r p a r t i c l e might i n t e r f e r e w i t h t h e normal p r o c e s s i n g o f a n t i g e n ( i . e . lysozyme) w i t h i n t h e macrophage. Ano t h e r s o l u t i o n would be t o use a l a r g e r r a d i o -a c t i v e macromolecule. E x p e r i m e n t a l d e s i g n would d i c t a t e however, t h a t t h i s m o l e c u l e have a known p r i m a r y s t r u c t u r e and a number of known a n t i g e n i c d e t e r m i n a n t s . - 54 -Although the results i n these two experiments.1 were inconclusive, a number of constructive suggestions have been made. Using these technique i n conjunction with very high s p e c i f i c a c t i v i t y lysozyme, some l i g h t should be shed on the manner of lysozyme processing and nucleic acid i n t e r a c t i o n . - 55 -D. SUMMARY RNA e x t r a c t s o f c h i c k e n lymphocytes and g u i n e a p i g p e r i t o n e a l macrophage i n c u b a t e d w i t h r a d i o a c t i v e lysozyme (PART I I ) c o n t a i n e d l i t t l e r a d i o a c t i v i t y . The optimum time o f RNA-Ag complex f o r m a t i o n was t h e r e f o r e n o t e s t a b l i s h e d . Reasons f o r t h e s e i n c o n c l u s i v e r e s u l t s a r e s u g g e s t e d and f o l l o w e d by a d i s c u s s i o n o f e x p e r i m e n t s w h i c h c o u l d g i v e more p o s i t i v e r e s u l t s . - 56 GENERAL DISCUSSION P a r t s I and I I o f t h i s t h e s i s were concerned m a i n l y w i t h the p r e p a r a t i o n o f m i l l i g r a m q u a n t i t i e s o f h i g h s p e c i f i c r a d i o a c t i v i t y lysozyme. Both p a r t s were s u c c e s s f u l i n t h a t a s i m p l e p r e p a r a t i v e e l e c t r o p h o r e t i c method was d e v e l o p e d t o i s o l a t e up t o 25 mg o f 2.6 x lO^dpm/mole lysozyme. Some s u g g e s t i o n s have been made (PART I I ) which c o u l d p o s s i b l y i n c r e a s e t h e s p e c i f i c r a d i o a c t i v i t y o f lysozyme p r e p a r e d from egg w h i t e . These s u g g e s t i o n s , when a p p l i e d t o the e x p e r i m e n t s o f PART I I I s h o u l d shed some l i g h t on t h e mechanics o f RNA-Ag f o r m a t i o n . F u r t h e r e x p e r i m e n t s i n the a r e a o f RNA-Ag i n t e r a c t i o n s s h o u l d be d i r e c t e d s p e c i f i c a l l y a t the n a t u r e o f t h e i n t e r a c t i o n s between the n u c l e o t i d e sequences o f the RNA and t h e p r i m a r y s t r u c t u r e o f t h e a n t i g e n . One s e r i e s o f e x p e r i m e n t s s h o u l d i n v o l v e c o n t i n u i n g t h e work o f S a x i n g e r and Ponnamperuma ( 3 6 ) . These workers have p u b l i s h e d a t a b l e o f i n t e r a c t i o n s o f two amino a c i d s ( t r p and g l y ) w i t h a number o f d i - and t r i n u c l e o t i d e s . One e n t r y from t h i s t a b l e shows t h a t t h e t r y p t o p h a n s e l e c t i v i t y c o e f f i c i e n t f o r ApApUp was t e n times g r e a t e r t h a n t h a t f o r ApGpUp. A l t h o u g h o n l y a few d i f f e r e n t c o m b i n a t i o n s were t e s t e d , a r u d i m e n t a r y p r e f e r e n t i a l scheme o f i n t e r a c t i o n s was shown. - 57 -S i n c e the p r i m a r y s t r u c t u r e o f t h e a n t i g e n i c d e t e r m i n a n t s o f lysozyme are known (e.g. l o o p p e p t i d e ) , work i n these i n t e r a c t i o n e x p e r i m e n t s s h o u l d i n c l u d e those amino a c i d s . * T h i s would r e s u l t i n t h e g e n e r a t i o n o f a s e r i e s o f t r i - n u c l e o t i d e sequences f o r t h e amino a c i d s w h i c h make up l o o p p e p t i d e . By t r a c i n g t h i s code through the r e v e r s e t r a n s c r i p t i o n and i n t e g r a t i o n s t a g e s , f o l l o w e d by t r a n s c r i p t i o n and t r a n s l a t i o n o f t h e r e s u l t a n t DNA, t h e p r o t e i n sequence f o r t h e a n t i l o o p p e p t i d e a n t i b o d y c o u l d be a s c e r t a i n e d ( t h i s p o r t i o n c o u l d be done t h e o r e t i c a l l y ) . The f i n a l s t e p o f t h i s e x p e r i m e n t would be t o sequence th e h y p e r v a r i a b l e r e g i o n s o f a n t i b o d y c h a i n s d i r e c t e d a g a i n s t t h e a n t i g e n i c d e t e r m i n a n t i n q u e s t i o n . I f the amino a c i d sequence was i d e n t i c a l o r v e r y s i m i l a r t o t h e sequence coded f o r by the o r i g i n a l RNA which i n t e r a c t e d w i t h t h e d e t e r m i n a n t ( i . e . RNA-Ag com p l e x ) , t h i s would be s t r o n g s u p p o r t i n g e v i d e n c e f o r t h e t h e o r y (PART I I I ) . These e x p e r i m e n t s a r e e n t i r e l y p l a u s i b l e . Methods a l r e a d y e x i s t w hich d e s c r i b e the methodology f o r i s o l a t i o n o f l o o p p e p t i d e d i r e c t e d a n t i b o d i e s (9, 1 0 ) . P r o t e i n s e q u e n c i n g methods a r e r a t h e r l e n g t h y and e x p e n s i v e b u t have been s t a n d a r d i z e d . The methodology f o r amino a c i d , l o o p p e p t i d e i s a 23 amino a c i d l o n g sequence ( r e s i d u e s 60-83) t h a t i s e a s i l y i s o l a t e d from egg w h i t e lysozyme ( 1 7 ) . - 58 -t r i n u c l e o t i d e b i n d i n g s t u d i e s a r e numerous and i n c l u d e column chromatography, e q u i l i b r i u m d i a l y s i s and n i t r o c e l l u l o s e b i n d i n g s t u d i e s . One f o r e s e e a b l e p r o blem i s t h e l a c k o f c o m m e r c i a l l y a v a i l a b l e t r i n u c l e o t i d e sequences. To-date t h e r e a r e a p p r o x i m a t e l y t e n o f t h e s e sequences a v a i l a b l e . T h i s r e s t r a i n t c o u l d p r e v e n t the d e t e c t i o n o f c e r t a i n i m p o r t a n t i n t e r a c t i o n s s i m p l y because t h e t r i n u c l e o t i d e sequences were n o t a v a i l a b l e . A n o t h e r problem, p o s s i b l y more s e r i o u s , i s t h e l a r g e number o f i n t e r a c t i o n s p o s s i b l e . Four b a s e s , i . e . A, U, G, C r e s u l t i n 64 c o m b i n a t i o n s o f t r i p l e t sequences. T h i s does n o t i n c l u d e t h e v a r i a t i o n s o f c e r t a i n bases t h a t e x i s t . There a r e 2 0 odd amino a c i d s , a g a i n n o t i n c l u d i n g v a r i a t i o n s . T h i s p e r m i t s a l a r g e number o f i n t e r a c t i o n s . S a x i n g e r and Ponnamperuma (36) have a l r e a d y shown a r u d i m e n t a r y p r e f e r e n t i a l scheme o f i n t e r a c t i o n s . I f t h i s scheme c o u l d be extended w i t h t h e m a t e r i a l s c u r r e n t l y a v a i l a b l e , a type o f code f o r RNA n u c l e o t i d e s - amino a c i d s c o u l d be f o r m u l a t e d . To be c o m p l e t e , a l l i n t e r a c t i o n s s h o u l d be t e s t e d . C e r t a i n p a t t e r n s might d e v e l o p which would negate t e s t i n g o f c e r t a i n c o m b i n a t i o n s . T h i s would be good. As mentioned p r e v i o u s l y , o n l y c e r t a i n a n t i g e n i c d e t e r m i n a n t s need be t e s t e d . Once t h e s e i n t e r a c t i o n s were c o n f i r m e d , the r e m a i n i n g work be u s e f u l i n e s t a b l i s h i n g f u r t h e r the v a l i d i t y o f t h e s e i d e a s . - 59 -BIBLIOGRAPHY 1. A l d e r t o n , G. and H.L. T e v o l d . 1946. 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Immunochemical s t u d i e s on t h e p o l y - - D - g l u t a m y l c a p s u l e o f b a c i l l u s a n t h r a c i s IV. The A s s o c i a t i o n w i t h p e r i t o n e a l exudate c e l l r i b o n u c l e i c a c i d o f t h e p o l y p e p t i d e i n immunogenic and non immunogenic forms. Biochem. 7:1432. Sabet, T., W.C. H s i a , M. S t a n i s z , A. E l - D o m e i r i and P. V a n A l t e n . 1977. A s i m p l e method f o r o b t a i n i n g p e r i t o n e a l macrophages from c h i c k e n s . J . Immunolog. Meth. 14:103. S a x i n g e r , C. and C. Ponnamperuma. 1974. I n t e r a c t i o n s between amino a c i d s and n u c l e o t i d e s i n t h e p r e b i o t i c m i l i e u . O r i g i n s o f L i f e 5:189. - 62 -APPENDIX Formulae f o r S o l u t i o n s and C u l t u r e Media B u f f e r A: 0.1M Ammonium A c e t a t e pH 9.0 15.42 gm Ammonium A c e t a t e t o 2000 ml d i H 2 0 a d j u s t t o pH 9.0 w i t h c o n c e n t r a t e d NH^OH B u f f e r B: 0.4M Ammonium Carbonate pH 9.2 76.8 gm Ammonium Carbonate t o 2000 d i H 2 0 a d j u s t t o pH 9.2 w i t h c o n c e n t r a t e d NH^OH 5.6% Complete A c r y l a m i d e S o l u t i o n f o r P r e p a r a t i v e E l e c t r o p h o r e s i s 12 ml s t o c k a c r y l a m i d e s o l u t i o n ( 3 i ) 7.5 ml 5xconc. b u f f e r E ( 3 i i ) 12 ml d i H 2 0 0.025 ml Temed (N,N,N',N' t e t r a m e t h y l e t h y l e n e diamine 0.375 ml Ammonium P e r s u l f a t e S o l u t i o n ( 3 i i i ) i . S t o c k A c r y l a m i d e S o l u t i o n A c r y l a m i d e & B i s - a c r y l a m i d e a r e BIORAD* E l e c t r o p h o r e s i s P u r i t y Reagents 15 gm a c r y l a m i d e 0.75 m b i s a c r y l a m i d e d i H 2 0 t o 100 ml f i l t e r t hrough Whatman #1 and s t o r e a t 4° i n brown b o t t l e . B i o r a d L a b o r a t o r i e s , M i s s i s s a u g a , O n t a r i o . - 63 -i i . 5X c o n c e n t r a t e d b u f f e r E 21.8 gm TRIS (hydroxymethylamino methane) 20.7 gm NaH2PC>4 1.85 gm EDTA ( e t h y l e n e d i a m i n e t e t r a c e t a t e - N a 2 ) d i H 2 0 t o 1000 m l . i i i . Ammonium p e r s u l f a t e s o l u t i o n 0.5 gm Ammonium p e r s u l f a t e add 5 ml d i H 2 0 p r e p a r e d f r e s h . d a i l y . 4. B u f f e r E: 17.44 gm TRIS o r 69.8 gm TRIS 16.56 gm NaH 2P0 4 66.3 gm NaH 2P0 4 1.4 8 gm EDTA 5.9 gm EDTA d i H 2 0 t o 4 , l i t e r s d i H 2 0 t o 16 l i t e r s pH 7 . 6 - 7 . 8 5. Sample b u f f e r f o r Q u a n t i t a t i v e P o l y a c r y l a m i d e D i s c G e l E l e c t r o p h o r e s i s . 0.2 gm Sodium Dodecyl S u l f a t e (SDS) 9.8 ml B u f f e r E 0.2 ml 2-mercaptoethanol (BME) 1 gm Sucrose S p a t u l a t i p f u l l o f bromophenol b l u e t r a c k i n g dye u n t i l s o l u t i o n i s deep b l u e . 6. Complete S o l u t i o n f o r Q u a n t i t a t i v e P o l y a c r y l a m i d e D i s c G e l s . - 64 -Volume Q. "O 5.6 a c r y l a m i d e 7.5 9.5 Stock a c r y l a m i d e s o l u t i o n 3 ( i ) 8 12.6 16.0 5X cone b u f f e r E 5 5.0 5.0 d i H 2 0 8 7.4 4.0 Temed .01 .01 .01 Ammonium P e r s u l f a t e .25 .25 .25 7. .25% Coomassie B l u e S t a i n i n g S o l u t i o n d i s s o l v e 1.25 gm Coomassie B r i l l i a n t B l u e i n 227 ml Methanol add 45 ml A c e t i c A c i d Make up t o 5 00 ml w i t h d i H 2 0 f i l t e r Whatman #1 S t o r e a t 4° i n brown b o t t l e a l l o w t o come t o room temperature b e f o r e use. 8. B u f f e r K: 0.066 M K H 2 P 0 4 9 gm KH 2P0 4. H 20 t o 1000 ml a d j u s t t o pH 6.24 w i t h NaOH* 9. F i c o l l - HYPAQUE:* F i c o l l 9 gms d i H 2 0 t o 100 ml. 30 ml Hypaque s o l u t i o n add 14.25 ml d i H 2 0 w o r k i n g s o l u t i o n 10 p a r t s hypaque 24 p a r t s f i c o l l 3 ml w o r k i n g s o l u t i o n can s u p p o r t 3-5 ml d i l u t e d b l o o d . Pharmacia F i n e C h e m i c a l s , U p s a l a , Sweden. - 65 " 10. Amino A c i d H y d r o l y s a t e Used t o P r e p a r e R a d i o a c t i v e Lysozyme (Amersham S e a r l e ) : Ammo a c i d % L - a l a n i n e 9.3% L - a r g i n i n e 6.3% L - a s p a r t i c a c i d 9.0% L - g l u t a m i c a c i d 11. 8% g l y c i n e 4.6% L - h i s t i d i n e 4.0% L - l e u c i n e 11.8% L - i s o l e u c i n e 4.8% L - l y s i n e 5.1% L - p h e n y l a l a n i n e 6.7% L - p r o l i n e 5.6% L - s e r i n e 4.8% L - t y r o s i n e 3.6% L - v a l i n e 6.8% S p e c i f i c a c t i v i t y 56 mCi/mAtom c a r b o n r a d i o a c t i v e c o n c e n t r a t i o n 50 u C i / m l . 11. RPMl 1640 T i s s u e C u l t u r e Medium ( G i b c o ) : mg/L 100. 0 2000.0 100.0 400.0 Component C a ( N 0 3 ) 2 . 4 H 2 0 G l u c o s e MgSO 4.7H 20 KC1 - 66 -Na^HPO, . 7H„0 1512.0 2 4 2 N c C l 6000.0 L-'arginine ( f r e e base) 200.0 L - a s p a r a g i n e 50.0 L - a s p a r t i c a c i d 20.0 L - c y s t i n e 50.0 L - g l u t a m i c a c i d 2 0.0 L - g l u t a m i n e 300.0 G l u t a t h i o n e (reduced) 1.0 G l y c i n e 1 . 1 0 . 0 L - h i s t i d i n e ( f r e e base) 15.0 L - h y d r o x y p r o l i n e 20.0 L - i s o l e u c i n e ( a l i o f r e e ) 50.0 L - l e u c i n e ( m e thionine f r e e ) 50.0 L - l y s i n e HCl 40.0 L-meth i o n i n e 15.0 L - p h e n y l a l a n i n e 15.0 L - p r o l i n e (hydroxy f r e e ) 20.0 L - s e r i n e 30.0 L - t h r e o n i n e ( a l l o w f r e e ) 20.0 L - t r y p t o p h a n 5.0 L - t y r o s i n e 20.0 L - v a l i n e 20.0 B i o t i n 0.2 V i t a m i n B 1 2 0.005 D-Ca p a n t o t h e n a t e 0.25 C h o l i n e CI 3.0 F o l i c a c i d 1.0 i - I n o s i t o l 35.0 N i c o t i n a m i d e 1.0 Para - a m i n o b e h z o i c a c i d 1.0 R i b o f l a v i n 0.2 Thiamine HCl 1.0 P h e n o l r e d -5.0 NaHCO-, 2000.0 - 6 7 -12. Trypan B l u e E x c l u s i o n Method f o r D e t e r m i n a t i o n o f C e l l V i a b i l i t y 5 5 i . t o 0.5 ml o f c e l l s u s p e n s i o n (1 x 10 t o 20 x 10 ) add 0.1 ml 0.4% t r y p a n b l u e ( i n phosphate b u f f e r e d s a l i n e ) and mix t h o r o u g h l y , i i . a l l o w t o s t a n d f o r 5-10 min. i i i . c o u n t v i a b l e c e l l s i n a hemocytometer and e x p r e s s as % v i a b l e c e l l s (dead c e l l s a r e b l u e ) . 13. Phosphate B u f f e r e d S a l i n e (PBS): Na 2HP0 4.7H 20 NaH 2P0 4.H 20 NaCl 17.0 4.44 gm 0.45 gm gm Add diH„0 to'2 l i t e r s pH 7.4 

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