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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 OF AND  1 4  ISOLATION  C - L A B E L L E D EGG WHITE LYSOZYME  I T S U T I L I Z A T I O N BY I N 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 o l u m b i a , 1974  A THESIS SUBMITTED  I N P A R T I A L FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE.STUDIES i n the Department o f Poultry  Science  We a c c e p t t h i s t h e s i s a s to the r e q u i r e d  conforming  standard  THE' UNIVERSITY OF B R I T I S H COLUMBIA S e p t e m b e r 197 7 (c)  H e n d r i k W i l l e m Bloirimers  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  f u l f i l m e n t o f the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h the L i b r a r y s h a l l I  make i t  freely available  f u r t h e r agree t h a t p e r m i s s i o n  for  Columbia,  I agree  reference and  f o r e x t e n s i v e copying o f  this  that  study. thesis  f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s of  this  representatives. thesis  It  i s understood that copying or p u b l i c a t i o n  f o r f i n a n c i a l gain s h a l l  written permission.  Department of The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Columbia  not be allowed without my  - i i-  ABSTRACT  A preparative  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  to i s o l a t e egg white lysozyme. consisted  The e l e c t r o p h o r e t i c  o f a s l i g h t l y modified glass  funnel  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 s u s p e n d e d . absorbing material  extracted  i n which The  &280  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 polyacrylamide disc gels.  unit  quantitative  Up t o 2 5 mg. o f a p p a r e n t l y p u r e  l y s o z y m e was p r e p a r e d b y t h i s m e t h o d . Radioactive  l y s o z y m e was p r e p a r e d b y i n j e c t i n g 14  young l a y i n g hens w i t h  a  i n j e c t i n g the hydrolysate with  C ammo a c i d h y d r o l y s a t e . 5 hrs.  a specific activity of  prepared. intervals  2 .6  postoviposition, x  10"*"^  Injection of the radioactive l e s s than 5 hrs.  lower a c t i v i t y  (25,  lysozyme  dpm/mole was amino a c i d s a t  postoviposition  resulted i n  lysozyme.  A t h e o r y h a s been s u g g e s t e d w h i c h c o u l d the  By  generation o f antibody d i v e r s i t y .  explain  I n t h i s theory  2 6 ) , t h e RNA p o r t i o n o f t h e R N A — a n t i g e n c o m p l e x f o r m e d  during  a n 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  capacity.  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 radioactive  a n t i g e n was r e q u i r e d .  lysozyme i s o l a t e d as described  The  a b o v e was u s e d .  Nucleic  a c i d e x t r a c t i o n s o f macrophage  w i t h r a d i o a c t i v e lysozyme found l i t t l e nucleic acid fractions.  A thorough  activity  discussion  incubated i n the  in light  o f t h e s e r e s u l t s i s p r e s e n t e d ^ a l o n g w i t h some s u g g e s t i o n s for  further  work.  - iv-  TABLE OF CONTENTS  ABSTRACT  Page  -  i  i  TABLE OF CONTENTS  iv  L I S T OF TABLES  v i  L I S T 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 .  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  3.  6  electrophoresis unit  7  a.  d e s c r i p t i o n of funnel  7  b.  c a s t i n g the acrylamide  c.  s e t t i n g up a r u n  d.  terminating a run  Extraction of purified the polyacrylamide  gel  10 10 .15  lysozyme  from  gel•  4.  D e s a l t i n g o f lysozyme  5.  Quantitative polyacrylamide  6.  . . .  .16 16 disc gel  electrophoresis  17  Scintillation  20  counting  - v -  G.  RESULTS AND DISCUSSION  D.  SUMMARY  22 '  32  PART I I . PREPARATION OF RADIOACTIVE LYSOZYME I N LAYING HENS. . . .  33  A.  INTRODUCTION.  .33  B.  METHODS  35  C.  RESULTS AND DISCUSSION  37  D.  SUMMARY  41 y  PART I I I . I N VITRO CULTURES WITH RADIOACTIVE LYSOZYME  42  A.  INTRODUCTION  42  B.  METHODS  45  1.  I s o l a t i o n o f chicken  lymphocytes  45 14  2.  Iri v i t r o lymphocyte c u l t u r e w i t h labelled  C.  lysozyme  46  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 . 3  4.  I n v i t r o macrophage c u l t u r e w i t h labelled  lysozyme  C.  RESULTS AND DISCUSSION  D.  SUMMARY  . .4 7  H 48 . 50 55  GENERAL DISCUSSION  56  BIBLIOGRAPHY  59  APPENDIX  62  - v i-  L I S T OF TABLES  I  A c t i v i t i e s o f lysozyme  Page  prepared  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 cathodes  II  25  Typical yields of A„  o n  m a t e r i a l from  2. o U  egg  III  white  by m o d i f i e d  Chance and C l a r k  (2)  Typical recovery  data  CM-52 method o f 28  f o r preparative  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 l y s o z y m e p r e p a r a t i o n  IV  I n v i v o s y n t h e s i s o f lysozyme  29  during  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 of a  l 4  C-amino a c i d hydrolysate  .38  vii  -  L I S T OF FIGURES  1  Dimensions o f funnel electrophoresis  2  Page  u s e d as a  preparative  unit  9  A p p a r a t u s s e t up f o r p o u r i n g  acrylamide  gel 3  .9  Preparative  electrophoresis  unit prior to  a 7 hr. run 3a  12  The p r e p a r a t i v e  electrophoresis  d u r i n g a. r u n 4  unit  • - •  Apparatus f o r f i n a l d e s a l t i n g  13  of lysozyme  preparations 5  Quantitative of  19 polyacrylamide disc  samples from the lysozyme  gels  isolation  procedure 6  31  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 l y s o z y m e ("^C  labelled)  egg o f b i r d s  i s o l a t e d from the second 14  injected l . v . with  amino a c i d h y d r o l y s a t e  a  C  (75 juCi) a t v a r i o u s  times a f t e r o v i p o s i t i o n 7  Radioactivity extraction with  l 4  C  profile for nucleic  o f lymphocyte c u l t u r e  l a b e l l e d lysozyme  40 acid incubated 51  -  Radioactivity extraction  vm  profile  -  for nucleic  ac  o f macrophage c u l t u r e s 3 incubated with H - l a b e l l e d lysozyme  ix -  ACKNOWLEDGEMEN T  The a u t h o r w o u l d l i k e  t o t h a n k D r . R.C. F i t z s i m m o n s  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 programme.  W i t h o u t t h e many l i v e l y  discussions,  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 , a d u l l M. S c . programme.  v  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 f o r use o f equipment  both  i t w o u l d have been  Thanks a l s o t o t h e Departments  Lab)  thesis  (Nutrition  and r e a g e n t s .  S p e c i a l t h a n k s t o D r . D. C l a r k , U.B.C. C h e m i s t r y Department  f o rthe radioactive  lysozyme.  S p e c i a l t h a n k s t o G. G a l z y , D e p a r t m e n t S c i e n c e a n d D o r o t h y E m s l i e f o r t h e i r many s p e c i a l during the course o f the experiments.  of Animal efforts  - 1  GENERAL INTRODUCTION  A method o f t h e i s o l a t i o n o f c h i c k e n e g g w h i t e lysozyme o f h i g h p u r i t y has been d e v e l o p e d as p a r t o f t h e experiments f o r t h i s t h e s i s . t h i s procedure t o be u s e f u l  In order f o r the product of f o rfurther  experiments,  p u r i t y was i m p o r t a n t . A l t h o u g h l y s o z y m e 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) t h e y g e n e r a l l y are  relatively  gels  w o r k f o r t h i s t h e s i s showed  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 gave r i s e  observation,  and  lengthy.  Preliminary egg  r e s u l t i n impure p r e p a r a t i o n s  that  polyacrylamide  t o a s i n g l e s t a i n i n g b a n d . From t h i s  a preparative  electrophoretic  initial  a p p a r a t u s was  d e v e l o p e d w h i c h was u s e d t o i s o l a t e m i l l i g r a m  quantities  of 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 processing  lymacrophage c e l l s  a n t i g e n was r e q u i r e d .  (PART I I I ) , a  They p r e p a r a t i v e  radioactive  electrophoretic  method d e v e l o p e d i n PART I was u s e d t o i s o l a t e lysozyme from young l a y i n g hens i n j e c t e d w i t h  radioactive radioactive  14 C amino a c i d h y d r o l y s a t e s . the  Some i n t e r e s t i n g d a t a o n  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  The r a d i o a c t i v e in cell  was c o l l e c t e d .  l y s o z y m e i s o l a t e d b y t h i s method was u s e d  c u l t u r e experiments  (PART I I I ) .  - 2 -  Macrophage  have l o n g been i m p l i c a t e d i n t h e  immune r e s p o n s e ( 3 ) . By i n c u b a t i n g m a c r o p h a g e w i t h r a d i o a c t i v e l y s o z y m e , i t was at which the formation was m a x i m a l .  hoped  t o determine the time  o f RNA-antigen  This determination  work on t h e n a t u r e o f t h e RNA  (RNA-Ag)  would a l l o w  complexes  investigative  involved i n this  complex.  - 3 -  PART I .  ISOLATION OF  A.  INTRODUCTION  LYSOZYME  Since i t s discovery i n 1922  ( 4 ) , egg  by  S i r A l e x a n d e r Fleiraning  w h i t e l y s o z y m e has  protein for scientific  become a v e r y  research.  L y s o z y m e i s a p r o t e i n composed o f 129 (primary basic  s t r u c t u r e known  (5, 6 ) , the m a j o r i t y  a t p h y s i o l o g i c a l pH.  tertiary  structure of  structure  i n the  o b s e r v e d a t pH v a l u e s a b o v e 10  amino  of which  d e p e n d a n t d i m e r s has  Higher polymers are  (NAG)  3 -  muramic a c i d  (1-4)  been  formed a t  (NAM)  glycosidic and  t o g i v e e q u i m o l a r amounts o f NAM  because of  this ability  pH  t h a t i t has  N-acetyl and  NAG.  activity  linkage glucosamine It is  t h e common name  muramidase. C e r t a i n amino a c i d s e q u e n c e s o f l y s o z y m e known t o be residues (one  the  (7).  I t h y d r o l y s e s the  between N - a c e t y l  are  Some q u a t e r n a r y  L y s o z y m e i s a l s o known f o r i t s e n z y m a t i c (4, 8 ) .  acids  Four c y s - c y s bonds m a i n t a i n  the molecule.  f o r m o f pH  5-9.  popular  highly antigenic.  The  amino a c i d  60-8 3, commonly r e f e r r e d t o as  c y s - c y s bond) i s a known a n t i g e n i c  loop  are  sequence peptide  determinant  (9).  _ 4 -  T h i s f e a t u r e , along w i t h the ease of i s o l a t i o n t h i s a convenient  molecule f o r immunological  (10)  research.  Lysozyme w i t h i t s n e t p o s i t i v e c h a r g e p h y s i o l o g i c a l pH  lends  itself  methods o f s e p a r a t i o n . o t h e r egg  white  The  at  to preparative e l e c t r o p h o r e t i c  f a c t t h a t the m a j o r i t y of  p r o t e i n s are apparently  a t p h y s i o l o g i c a l pH  make  the  n e g a t i v e l y charged  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 l y s o z y m e f r o m n o n l y s o z y m e protein. 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 include c r y s t a l l i z a t i o n exchange  (2)  (1), gel f i l t r a t i o n  (11)  Fevold  Their technique  was  b a s e d on  i n s o l u b l e a t pH  t a k e up  t o 12  10.5  the o b s e r v a t i o n  an  acceptable  white.  that  lysozyme  was  the length of  One  time  q u a n t i t y of lysozyme  had  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  could  days.  Molecular  filtration  techniques,  although  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 columns are r e q u i r e d t o prevent o f p r o t e i n p e a k s due -nature  for  i n t h e p r e s e n c e o f 5% N a C l .  f o r t h i s technique  required before precipitated.  ion  (1) r e p o r t e d a t e c h n i q u e  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 l y s o z y m e f r o m egg  disadvantage  and  techniques.  A l t e r t o n and  was  use  o f egg w h i t e  t h e o v e r l a p and  t o an o v e r l o a d e d  column.  faster  (.11) .  Large  tailing The  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  viscous before  - 5 -  l o a d i n g onto t h e 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 a b o v e d i s a d v a n t a g e s , an i o n e x c h a n g e p r o c e d u r e was e m p l o y e d . Chance a n d C l a r k  The i o n e x c h a n g e p r o c e d u r e o f  (2) was u s e d s i n c e i t i s f a s t a n d 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 e m p l o y e d an  initial  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 e x c h a n g e c o l u m n . a c c o m p l i s h e d on a B i o g e l P-2 c o l u m n .  Final desalting The f i n a l  was  product  was j u d g e d t o b e p u r e b y a number o f c r i t e r i a ( 2 ) . In the experiments f o r t h i s t h e s i s , the 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 t h e B i o Rex i o n e x c h a n g e c h r o m a t o g r a p h y .  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 a s a s i n g l e s t a i n i n g b a n 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 d i s c g e l system sodium  dodecyl sulfate  containing  (SDS).  Although other e a s i l y prepared molecules  exist,  l y s o z y m e was u s e d b e c a u s e  i t was r e l a t i v e l y e a s y t o p r e p a r e  i n the radioactive  The f a c t t h a t i t h a s a number o f  form.  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 e v e n more d e s i r a b l e a s t h e p r o t e i n t o u s e f o r t h i s  study.  -  6  -  B.  METHODS  1.  I s o l a t i o n o f lysozyme from egg white  The method f o r i s o l a t i o n o f lysozyme was of Chance and C l a r k  that  (2) w i t h some m o d i f i c a t i o n s .  Seventy-five  mis o f b u f f e r A  added to the albumen o f one f r e s h egg  (appendix 1)  was  (< 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 f o r 1/2  - 3/4 h r .  albumen was  A f t e r t h i s time the m a j o r i t y o f the t h i c k  no longer d i s c e r n a b l e .  The mixture was  through f o u r f o l d s o f c h e e s e c l o t h  and added to 50 ml of  s e t t l e d carboxymethyl c e l l u l o s e *  ( p r e - e q u i l i b r a t e d with  buffer A). stirrer)  The s l u r r y was  moderately s t i r r e d  f o r approximately 1/2  f o r 20 min. a t 4100  x G.  (magnetic  h r . and then c e n t r i f u g e d  The supernatant was  decanted and  the p e l l e t resuspended i n 75 ml o f f r e s h b u f f e r A. wash procedure was (  filtered  o f a l l washings  completed a t o t a l o f 4X.  This  The 280 A  i n t h i s procedure were determined on a  Unicam SP1800 dual beam spectrophotometer. A f t e r the f o u r t h wash w i t h b u f f e r A, the p e l l e t was  resuspended i n 50 ml of b u f f e r B (appendix 2) and  centrifuged. *  This wash was  completed 4X and the r e s u l t a n t  Whatman CM-52, c e l l u l o s e - c a t i o n exchange m a t e r i a l  - 7 -  fractions of  pooled.  lysozyme  followed  was  by  65  ml  was  stirred  a  a  concentrated  preparative  2.  of  electrophoresis  a.  was  Fisher  modified  sides  (vacuum  served  to  filtration) remove  The  ultrafiltration  in  diaflo  (30 p s i  funnel  membrane  by  shown  melting  in Fig.  to  support  the  ( 5 - 1 0 ml)  lysozyme  a  1.  registered  was  a  I,  purified 2).  preparative  (Fig.  a  no-.  glass  equidistant  acrylamide  (PART  on  funnel  cat.  five  The  was  unit  used  Scientific  approximately  as  sample  Description of  The as  UM-2  majority  particles.  electrophoresis unit  Purification  sold  This  cellulose by  #1  a  pressure). The  on  using  containing  Whatman  filtration.*  concentrated  cell  sample  through  carboxymethyl  then  operating  pooled  filtered  Millipore  contaminating sample  The  Pyrex  1)  bottle  10-346E.  The  'fingers' into  around  function of  the  these  .45  pore  funnel the  circumference 'fingers'  gel.  trademark,  funnel  size.  was  -  Key  -8  -  for Fig. 1  €i - g l a s s f i n g e r s - t h e i r  function i s to  the polyacrylamide g e l . A l l dimensions  Key  are i n centimeters  for Fig. 2  C2 - r i n g  stand  b - funnel C - ring d  - #12  © - 250  clamp rubber  stopper  ml e r l e n m e y e r  flask  (cm).  support  F i g . 1.  Dimensions of f u n n e l used as  preparative  electrophoresis unit.  a  c  \ Fig. 2.  Apparatus s e t up f o r pouring  e  acrylamide  gel.  -  10  -  V  b.  Casting  The  the a c r y l a m i d e  was  The  through  t i g h t s e a l between the  complete acrylamide  2)  (Fig.  r i n g clamp e x e r t s p r e s s u r e  f u n n e l t o make a l i q u i d stopper.  gel  funnel  s o l u t i o n (appendix  p o u r e d t h r o u g h t h e stem o f t h e f u n n e l o n t o the  stopper  (#12)  completely.  .  The  s o l u t i o n should  cover  A f t e r p o l y m e r i z a t i o n had  t h e r i n g c l a m p was of the f u n n e l . h e n c e t h e two i n the funnel)  can was  g e l does n o t  e a s i l y be  3)  rubber  ( c a 1/2  occurred  stick  and  the g l a s s f i n g e r s  c a r e f u l l y r e m o v e d f o l l o w e d by  The  the  hr.)  removal  to the stopper  separated.  The  rinsed with buffer E  gel  and  (suspended 4)  (appendix  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  (Fig.  A f t e r t h e g e l had  been c a s t i n t h e  t h e f u n n e l was  s e t up as i n F i g . 3.  t h e f u n n e l and  the  3-fingered  3)  The  funnel,  r i n g clamp  clamp s t a b i l i z e s  the  supports funnel  d u r i n g s u b s e q u e n t movement. A modified i n s e r t e d i n t o the stem. t u b e was  altered until  T h i s t u b e was  The  ( F i g . 3)  p o s i t i o n of the  was  then  subgel-surface  i t s t i p j u s t b a r e l y touched the g e l .  u s e d t o remove t h e a i r w h i c h  by t h e b u f f e r t h a t f i l l e d tube had  rubber stopper  t h i s compartment.  was"displaced Once  this  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  - II  Key f o r F i g . 3  a - buffer anode  b -  i  E, upper r e s e r v o i r  c - sample t o be e l e c t r o p h o r e s e d ; 1 0 % sucrose added  d -5.6%  polyacrylamide g e l  e - s u p p o r t i n g r i n g clamp f - s u p p o r t i n g 3 f i n g e r e d clamp m o d i f i e d #6 rubber  g h  -  subsurface  •  g e l tube  i - b u f f e r E "out ©  1  "  stopper  1  tube  b u f f e r E ' i n ' tube  k - cathode •i  -  b u f f e r E charge tube f o r lower r e s e r v o i r .  \  - .12 -  Fig.  3.  Preparative electrophoresis unit p r i o r to a 7-hr  run.  - 13  -  - 14 -  taped  (3M m a s k i n g The  tape) to the  stem.  l o w e r c o m p a r t m e n t was  b u f f e r E v i a the b u f f e r charge tube. t h i s compartment, e q u i v a l e n t volumes in  charged w i t h  As t h e b u f f e r  o f a i r were removed  order to prevent rupture of the g e l .  By t i l t i n g  f u n n e l , a l l t h e a i r f r o m t h e l o w e r c o m p a r t m e n t was Once t h i s  s t a g e had been c o m p l e t e d t h e a p p a r a t u s  transferred to a  added t o t h e upper  a h e i g h t 2 cm f r o m t h e t o p o f t h e f u n n e l .  c o n n e c t i o n s on t h e g l a s s  500 m l / h r . was  removed. was  reservoir The  required  "T" w e r e 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 . ca.  the  refrigerator.  B u f f e r E was to  filled  An o p t i m u m f l o w r a t e o f  e s t a b l i s h e d by a d j u s t i n g t h e  on t h e b u f f e r E " i n " a n d " o u t " t u b e s  (Fig. 3).  clamps This  b u f f e r f l o w s e r v e s t o remove h y d r o g e n b u b b l e s g e n e r a t e d by t h e c a t h o d e f r o m t h e s y s t e m . was  The b u f f e r  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  reservoir  (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 " t u b e v i a a l e n g t h o f 6 mm  i . d . surgical tubing.  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 , v o l u m e c o u l d be r e a l i z e d  sufficient  to l a s t over n i g h t  ( i f required).  The b u f f e r E " o u t " t u b e 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  e m p t i e d when r e q u i r e d .  - 15  The  sample  -  (5-10  ml,  10%  added  sucrose)  was  l a y e r e d under the upper b u f f e r r e s e r v o i r w i t h a  and  short length of tubing or a pasteur  s u p p l y * was  turned  on  and  adjusted  pipette.  t o 45 ma.  syringe  The  The  power  system  r u n s a t 10-12°.  d.  Terminating  The supply  and  r e m o v e d by  terminated  of the run.  s u c t i o n and  The  and  t h e power  upper b u f f e r  the surface of  sides of the funnel r i n s e d very  b u f f e r E. m u s t be  run  b u f f e r f l o w was  s h u t o f f a t t h e end  r e s e r v o i r was gel  a  thoroughly  A l l t r a c e s o f s p e n t b u f f e r E and  b i t s of  the with carbon  removed. The  g e l was  b e t w e e n t h e g e l and  r e m o v e d by  funnel w a l l .  inserting a spatula  Enough a i r was  l e t into  t h e space b e l o w t h e g e l t o n e u t r a l i z e t h e convex shape assumed b y  the g e l during e l e c t r o p h o r e s i s .  c a r e f u l l y removed w i t h water. no  t h e f i n g e r s and  gel  was  rinsed in cold  tap  Should the g e l f l i p over during removal, there i s  cause f o r alarm The  with a pasteur  i f i t s s u r f a c e was lower  pipette.  the i n i t i a l  cleaned  r e s e r v o i r b u f f e r was The  f u n n e l and  a s s e m b l y w e r e r i n s e d w i t h b u f f e r E and  *  The  volume o f lower  properly. collected  lower  electrode  t h i s wash a d d e d  reservoir buffer.  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  to  - 16 -  3.  Extraction of 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  removal from the f u n n e l . macerated i n a c e l l is  5.4  mm  of  b u f f e r E was  stirrer)  The p l a s t i c  f o r c a . 1/2  pestle  to allow ready maceration of the g e l .  p r e p a r e d i n t h i s way.  added  following  cubes were t h o r o u g h l y  homogenizer.  under s i z e  The w h o l e g e l was  These  cubes  A s m a l l volume  t o t h i s m i x t u r e and s t i r r e d hr.  The m i x t u r e was  (magnetic  filtered  through  Whatman #1 f i l t e r p a p e r on a b u c h n e r 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 h o u r s a t 4°, respectively.  The m a j o r i t y o f t h e A g 2  Q  was  e x t r a c t e d from the g e l i n the f i r s t  The  first  in 4.  absorbing material two  and s e c o n d e x t r a c t i o n s w e r e p o o l e d and  Desalting of p u r i f i e d  lysozyme  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  (dir^O)  a Sephadex*  column  G-25  (2.4 cm x 39 cm)  h e a d , c a 70 m l / h r f l o w r a t e ) .  *  lyophilized  preparation for desalting.  The  was  extractions.  The  and a p p l i e d  to  (90 cm p r e s s u r e  i n i t i a l major  lyophi.li.zed. trademark - Pharmacia f i n e c h e m i c a l s .  peak  - 17  The 2.5  ml  -  l y o p h i l i z e d p e a k was  d i H 0 and 2  T h i s t u b i n g was  placed placed  in dialysis  reconstituted in tubing  (1 cm  width).  i n t h e a p p a r a t u s shown i n F i g .  The  s a m p l e s w e r e d i a l y s e d a g a i n s t ca. 30  way  (ca. 700  ml/hr. flow  1 of d i H 0 i n 2  4. this  rate).  A f t e r completion  of 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. washings were p o o l e d  flat  and  lyophilized.  r a d i o a c t i v i t y determined from these  The  These  specific  freeze dried  products  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 l y s o z y m e was  5.  and  used.  Quantitative polyacrylamide  The was  prepared  method o f P e t r i  (12)  disc gel electrophoresis  w i t h some m o d i f i c a t i o n s  used. S a m p l e s w e r e added t o s a m p l e b u f f e r ,(appendix  which contained  sodium dodecyl  m e r c a p t o e t h a n o l and 2 min.  Up  t o 50  (appendix 6 ) . buffer E  (and  and  c u r r e n t o f 2 ma/tube was (bromophenol blue) t h e c u r r e n t was  had  2for  samples were used p e r  s a m p l e was  SDS)  and  i n a b o i l i n g water bath  u l of these  The 1%  placed  s u l f a t e (SDS)  carefully overlayed  t h e power t u r n e d o n . applied until  entered  the g e l  A  gel with constant  the t r a c k i n g (confirmed  t h e n i n c r e a s e d t o 5 ma/tube.  5)  dye  visually),  -  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 ( c a 30L)  b  - d i r e c t i o n of water  C  - purified  d  - g l a s s column  flow  lysozyme samples (20 x 400  i n d i a l y s i s membranes  mm).  - 19..  :  -  a  Fig.  4.  Apparatus f o r f i n a l d e s a l t i n g lysozyme  preparations.  of  When t h e dye gel,  t h e power was  the glass tubes syringe  and  30  - 20  -  1-2  cm  was  shut o f f .  (6 mm  The  1) by  ( d i H 0 as 2  s o l u t i o n ( a p p e n d i x 7)  (Pharmacia e l e c t r i c  a  lubricant).  f o l l o w e d by  gel destainer)  from  ringing with  g e l s w e r e 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% blue  the  g e l s were removed  i d x 75 mm  gauge n e e d l e  from the bottom of  Coomassic  electric  i n 5%  The  destaining  acetic acid  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 i n stoppered  6.  test  L i q u i d S c i n t i l l a t i o n counting scintillation  Dry weighed out  samples  The  d i g e s t e d by  of the  (e.g.  f r e e z e d r i e d lysozyme) were  ml  a d d i t i o n o f 1 ml o f P r o t o s o l . * considered  completedly  clear  was  u s e d as t h e  scintillation  samples  (2-5  diH 0 2  Digestion  c o m p l e t e when t h e s o l u t i o n  hr.).  Econofluor*  (10  ml)  fluid.  ( e . g . albumen) w e r e t r e a t e d  e x c e p t t h a t t h e s a m p l e v o l u m e was  0.25  ml  and  identically  dir^O  was  omitted. In the d e t e r m i n a t i o n  of s p e c i f i c a c t i v i t y ,  s a m p l e s w e r e c o u n t e d f o r a p e r i o d o f 10 *  (polyethylene)  s o l u b i l i z e d i n 0.25  was  Wet  300  counter  s a m p l e was  s a m p l e was  therefore  - Isocap  into glass s c i n t i l l a t i o n v i a l s  lined caps). and  f  tubes.  t r a d e m a r k NEN  Canada L t d .  min.  <•  -  Counts  per minute  d i s i n t e g r a t i o n s per minute  -  21  (CPM) (DPM)  were c o r r e c t e d by a p p l y i n g CPM  to data  14  to a quench curve d e r i v e d from C s u p p l i e d by t h e m a n u f a c t u r e r . 50-85% w e r e r e a l i z e d  i n ,this  quenched  Efficiencies thesis..  standards r a n g i n g from  - 22  C.  RESULTS & DISCUSSION  For was  -  t h i s study,  inexpensive  s u g g e s t e d by The known  (13)  and  Dr.  D.  easy to i s o l a t e .  required  Lysozyme  that  was  C l a r k o f t h e U.B.C. C h e m i s t r y D e p t .  complete primary s t r u c t u r e of lysozyme i s  i n c l u d i n g t h e s e q u e n c e o f some s p e c i f i c  determinants contained f a c t was  an a n t i g e n was  i n t h i s molecule  (14,  antigenic  15).  This  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  this  thesis. The gave r i s e  i n i t i a l observation  that diluted  t o a s i n g l e s t a i n i n g b a n d when  albumen  electrophoresed  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 l y s o z y m e be  p r e p a r e d i n p u r e f o r m by  o f egg  white.  lysozyme run  conceptually  sound,  the e l e c t r o p h o r e t i c u n i t prevented  to o b t a i n p r e p a r a t i v e  times of  amounts o f  20-33 h r s . d u r a t i o n w e r e  I n r u n s o f t h i s l e n g t h , one  of the  the r a t h e r dynamic p r o p e r t y  various  electrophoresis  success. In order  was  preparative  A l t h o u g h t h i s i d e a was  t e c h n i c a l problems w i t h complete  simple  could  r u n s t h e g e l was  f i r s t problems  encountered  of the g e l i t s e l f .  During  o b s e r v e d t o assume c o n v e x a n d / o r  concave c o n f i g u r a t i o n s  (16) .  revealed  a common phenomena and  t h a t t h i s was  required.  A brief literature  survey  that i n  the  -  -  23  p a r t i c u l a r p a p e r s e x a m i n e d , t h e r e was no s o l u t i o n ( 1 6 ) . Simple removal o r a d d i t i o n o f b u f f e r d i d not 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 t h e u n i t d e s c r i b e d that the diameter o f the tubing t h r o u g h t h e c a t h o d e was Initially,  and hence f l o w  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  When t h i s p i p e t t e was r e p l a c e d (  reduced.  5 mm i d . i n c r e a s e )  with  as t h e i n l e t .  l a r g e r diameter  glass  t h i s p r o b l e m was g r e a t l y  The n a r r o w o p e n i n g o f t h e p i p e t t e h a d r e s t r i c t e d  t o some e x t e n t , electrode  rate  responsible.  i m m e r s e d 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  tubing  here  t h e maximum f l o w r a t e t h r o u g h t h e l o w e r  assembly.  Hence t h e l o w e r e l e c t r o d e  acted  as  a vacuum d i a l y s i s m e c h a n i s m r e s u l t i n g i n r e m o v a l o f b u f f e r from t h e l o w e r compartment.  This  resulted i n deflection  of 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 t u b e was r e p l a c e d  f o r the pasteur pipette,  f l o w r a t e s were p o s s i b l e .  The p r o b l e m h e r e was t h a t t h e  increased  flow  r a t e caused c o l l a p s e o f the d i a l y s i s  around t h e e l e c t r o d e . from being  greater  membrane  This prevented t h e hydrogen bubbles  c a r r i e d o u t o f t h e system and subsequent  cessation of buffer flow.  In order  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 r e d u c e d b y p l a c i n g c l a m p s o n the b u f f e r E " o u t " and " i n " tubes.  By a d j u s t i n g t h e two  - 24  -  c l a m p s t h e p r o p e r membrane d i s t e n t i o n and be  achieved.  Occasionally,  c o r r e c t , the  g e l i n the opposite The two  i f t h e s e s e t t i n g s were  lower electrode  vacuum d i a l y s i s m e c h a n i s m .  h o u r s was  flow rate  assembly acted This  as a  could not  reverse  caused d e f l e c t i o n of  d i r e c t i o n as p r e v i o u s l y  amount o f d e f l e c t i o n n o r m a l l y g r e a t l y reduced w i t h  these  the  described. present a f t e r  modifications.  Although s i g n i f i c a n t l y reduced, the d e f l e c t i o n problem c o u l d n o t be the  nullified  20-30 h r .  Rarely  could  the  e l e c t r o d e be overnight.  runs,  completely.  During the course  t h e o v e r n i g h t p e r i o d became m o s t  flow r a t e of b u f f e r through the  s e t so  t h a t zero  d e f l e c t i o n c o u l d be  therefore,  from the g l a s s  sides of the  a l l o w i n g m i x i n g o f t h e u p p e r and  reservoirs.  Any  mixing would t h e r e f o r e  critical.  lower maintained  When t h e d e f l e c t i o n became l a r g e e n o u g h ,  gel would loosen  of  the  funnel; lower  buffer  contaminate  l o w e r r e s e r v o i r w i t h n o n - l y s o z y m e p r o t e i n ; an  the  undesirable  situation. The  s e c o n d p r o b l e m e n c o u n t e r e d was  the  e n z y m a t i c a c t i v i t y o f t h e l y s o z y m e p r e p a r e d by T a b l e I summarizes the a c t i v i t i e s of before  and  a f t e r an  copper or platinum  electrophoretic separation ( P t ) as t h e  cathode.  t h i s method. lysozyme using  C o p p e r i s known  t o h a v e a d e l e t e r i o u s e f f e c t on  the  (13)  s u b s t i t u t i n g a Pt  and  i t was  t h o u g h t t h a t by  low  s t a b i l i t y of  lysozyme electrode,  Enzymatic a c t i v i t i e s o f lysozyme prepared 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 a t h o d e s .  Table I .  D e s c r i p t i o n o f lysozyme  Lysozyme a c t i v i t y  sample  Copper cathode C o n t r o l , commercial lysozyme^ 100% = 21,000 u n i t s / A . U . . 3 Egg w h i t e i s o l a t e d l y s o z y m e p r e 33 h r . e l e c t r o p h o r e s i s .  70  3 Egg w h i t e i s o l a t e d l y s o z y m e p o s t 33 h r . e l e c t r o p h o r e s i s . 2  P l a t i n u m cathode  87  by d i a l y s i s  As 4 f o l l o w e d b y d i a l y s i s a g a i n s t pH 6.24 0.066M K H P 0 b u f f e r f o r 48 h r s .  control)  100  2 8 Q  As 2 b u t f o l l o w e d a s i n 5.  (% - o f  4  25  36  62  26  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 .  67  As 6 f o l l o w e d as i n 5.  71  by d i a l y s i s  1  A s s a y a s 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 L 6 8 7 6 . 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 b y S i g m a C h e m i c a l Co.  3  R e f e r s t o lysozyme  (Sigma C h e m i c a l C o . ,  3X r e c r y s t a l l i z e d  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  from egg white,  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 c a t h o d e was u s e d (Table I ) .  Unfortunately, this  10% i n c r e a s e was l o s t  when t h e s a m p l e was d i a l y s e d a g a i n s t b u f f e r K ( a p p e n d i x 8 ) . When a c o p p e r c a t h o d e was u s e d , t h e a c t i v i t y o f l y s o z y m e was a p p r o x i m a t e l y 2-1/2 t i m e s g r e a t e r t h a n p r i o r t o dialysis.  These r e s u l t s  used i n a l l f u t u r e  s u g g e s t e d t h a t copper w i r e be  separations.  When one c o m p a r e s t h e a c t i v i t i e s o f l y s o z y m e purified vs.  f r o m e g g w h i t e b y t h e m e t h o d d e s c r i b e d i n PART I  commercial grade lysozyme* p u r i f i e d  apparatus, differences i n a c t i v i t y 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  are observed.  o n 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 t i m e s more a c t i v e t h a n e g g w h i t e l y s o z y m e similarily.  W h i l e t h e a c t i v i t y o f egg w h i t e  i n c r e a s e d 2-1/2 f o l d lysozyme p u r i f i e d  Commercial  after  dialysis,  prepared lysozyme  the activity of  from commercial sources 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  thesis required  preparative  amounts o f r a d i o a c t i v e l y s o z y m e , w h i c h h a d t o be p r e p a r e d in this  l a b o r a t o r y , the behaviour 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 not c r i t i c a l .  Should t h i s  a p p a r a t u s be used t o p u r i f y  commercial lysozyme, i t would c e r t a i n l y r  maintain enzymatic *  activity.  S i g m a C h e m i c a l Co.  be i m p o r t a n t t o  - 27 -  Another problem encountered during 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 salvaged  f r o m a "D"  size battery).  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  During carbon  p a r t i c l e s w h i c h s l o w l y sank t o t h e s u r f a c e o f t h e g e l . This not only contaminated  the upper b u f f e r r e s e r v o i r b u t  may h a v e i n t e r f e r r e d w i t h t h e h o m o g e n e i t y o f t h e e l e c t r i c f i e l d across  the g e l .  Due t o t h e t e c h n i c a l d i f f i c u l t i e s  described, the  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 . of attempting  to electrophorese  Instead  the lysozyme i n t o the lower  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 a n d e x t r a c t t h i s enzyme f r o m t h e g e l . I t was a l s o d e c i d e d  t o u s e an i n i t i a l  purification  s t e p f r o m raw e g g w h i t e , u s i n g t h e CM-52 ( T a b l e I I ) method o f Chance and C l a r k  (2). This i n i t i a l  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  step would serve to  p r o t e i n and h e n c e  make t h 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 t e r m s o f t h e y i e l d o f lysozyme. The y i e l d o f l y s o z y m e was stage.  Table  I I I shows t y p i c a l r e s u l t s o f t h e &28O  a p p l i e d and s u b s e q u e n t l y gel.  u  n  i t s  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  polyacrylamide  substantial at this  &  Assuming E ^ . m  2  go  nm  up t o 25 mg. p e r e g g w e r e p u r i f i e d by t h i s  =  ^6.3 ( 2 ) ,  method.  Table I I .  Typical yields of A  2 g 0  m a t e r i a l f r o m egg w h i t e by  CM-52 method o f Chance a n d C l a r k  Wash  PH  ph 9.0'  ph 9.2'  number  280/ml  modified  (2) .  Volume (ml)  Total A  1  13.64  81  1105  2  3.2  72  230  3  0.62  74  46  4  0.18  75  13  1  2.0  49  98  2  0.9  54  49  3  0 . 38  51  19  4  0.23  52  12  Carboxymethyl c e l l u l o s e i s c e n t r i f u g e d -75 m l b u f f e r A.  280  (4080 x G, 20 m i n ) a n d r e s u s p e n d e d i n  2  pH 9.0 w a s h i n g s 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  proteins.  3  The ph 9.2 w a s h i n g s a r e p o o l e d a n d c o n c e n t r a t e d i n a n u l t r a f i l t r a t i o n in preparation f o rpreparative electrophoresis. This i s t h e lysozyme fraction.  device enriched  Table I I I .  Typical recovery (7 h r . d u r a t i o n )  data 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 o f CM-52 i s o l a t e d l y s o z y m e p r e p a r a t i o n . Absorbance*  2 80 nm A.U^gQ** m a t e r i a l e x t r a c t e d from  N a t u r e o f Sample Applied CM-52 l y s o z y m e  228  Background ( s u c r o s e i n B u f f e r E)  0.25  Upper Reservoir  Lower first second third Reservoir extract, extract, extract. 60  193  84  polyacrylamide g e l  3.5  3.4  28  10  2.5  1 mg l y s o z y m e h a s 2 80 ~ 2.0. A  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 h a s an a b s o r b a n c e 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 w a v e l e n g t h o f 280  - 30 -  A l t h o u g h up t o 5 0 mg w e r e e x t r a c t e d f r o m t h e g e l , l a r g e losses which occurred during the d e s a l t i n g steps prevented quantitative recovery. Y i e l d s o f 5-25 mg. typical.  o f p u r i f i e d m a t e r i a l were  L y s o z y m e p r e p a r e d by t h i s m e t h o d 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 b a n d on SDS d i s c g e l s  (Fig.5).  T h i s l y s o z y m e was f o u n d t o c o n t a i n  contaminants  a f t e r h a v i n g b e e n 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 t h e s e c o n t a m i n a n t s w e r e n o t  characterized.  I t was n o t e d h o w e v e r , t h a t t h e m a j o r  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 a c r y l a m i d e g e l s as d i d t h e m a j o r of  commercial  and c o u n t e d  lysozyme.  (inProtosol  scintillation  7.5%  contaminant o f a sample  When t h e s e b a n d s w e r e c u t o u t & Econoflour) i n a  liquid  counter, a l ldetectable r a d i o a c t i v i t y  found i n the lysozyme  band.  was  r  HIS l l l l I I 00 1—'  / 2 3 ? PH?.O WASH  Fig.  5.  5 6 ? />// * 2  1  8  to p#?2 *£S.  II  /a  AES.  STMT).  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 the lysozyme i s o l a t i o n procedure. See a l s o T a b l e I I . L o w e r r e s . and u p p e r r e s . r e f e r t o t h e u p p e r 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 the g e l ) of 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 .  - 32 -  D.  SUMMARY  An i n e x p e n s i v e ,  simple  preparative  polyacrylamide  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 observation  lysozyme.  T h i s m e t h o d i s b a s e d on t h e  t h a t lysozyme appears t o be t h e o n l y  c h a r g e d p r o t e i n i n egg w h i t e apparently  p u r e l y s o z y m e was i s o l a t e d  Some c o n t a m i n a t i o n exceeding  a t pH 7.6.  positively  Up t o 25 mg.  with this  of  technique.  was f o u n d i n s a m p l e s s t o r e d f o r p e r i o d s  one month.  observed i n commercial  However, t h i s c o n t a m i n a t i o n preparations.  i s also  - 33 -  PART I I .  PREPARATION OF RADIOACTIVE LYSOZYME IN LAYING HENS  A.  INTRODUCTION.  A b r i e f survey o f the l i t e r a t u r e d i d not suggest an optimum time a t which to i n j e c t a "^C-amino a c i d h y d r o l y s a t e i n t o l a y i n g hens i n o r d e r to get maximum i n c o r p o r a t i o n of l a b e l i n t o egg white lysozyme. Oades and Brown (17) found t h a t the o v i d u c t o f a mature l a y i n g hen c o n t a i n s s u f f i c i e n t water oviduct proteins  (WSOP) f o r about two eggs.  soluble Most of i t  appears to be formed i n the magnum d u r i n g the i n t e r v a l s between the passage o f s u c c e s s i v e eggs down the o v i d u c t (18, 19-21).  S e c r e t o r y g o b l e t c e l l s o f the magnum  discharge t h e i r contents computely d u r i n g the passage o f an egg and are r e p l e n i s h e d a few hours l a t e r  (22, 23);  a s i m i l a r d i s c h a r g e from the t u b u l a r gland c e l l s has a l s o been r e p o r t e d  (21, 24).  The r a t e a t which these c e l l s are recharged w i t h different proteins varies.  Mandel and Ducay (18) found t h a t 14  birds injected  (i.v.) with  C l a b e l l e d ammo a c i d s d i d not  l a y a r a d i o a c t i v e egg u n t i l the second day a f t e r  injection.  They a l s o found t h a t ovalbumin and conalbumin were 50% more r a d i o a c t i v e than lysozyme.  These workers d i d not 14 s t a t e the exact time o f i n j e c t i o n o f the C labelled  - 34 -  amino a c i d s  they  used.  Preliminary  work f o r t h i s t h e s i s  showed  that  14 l.v.  i n j e c t i o n with  directly  5 jaCi o f a  C amino a c i d  after oviposition resulted  detectable r a d i o a c t i v i t y . possibility  that  hydrolysate  i n lysozyme with  zero  These r e s u l t s s u g g e s t e d t h e  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 h e n s k e p t a t t h e U.B.C.  Farm were used.  A commercial l a y e r  Poultry-  r a t i o n and w a t e r were  s u p p l i e d ad libitum-. The c l u t c h only birds were  length of 6 birds  was r e c o r d e d a n d  i n the beginning or middle o f t h e i r  clutch  used. On t h e day o f i n j e c t i o n t h e e x a c t t i m e o f o v i -  position  was d e t e r m i n e d a n d t h e b i r d was i n j e c t e d  i . v . with  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  oviposition.  The b i r d was s a c r i f i c e d b y c e r v i c a l  dislocation of  albumen  after  f o u r eggs h a d been c o l l e c t e d .  (0.25 m l ) w e r e c o u n t e d f o r r a d i o a c t i v i t y  (PART I , A, v i ) a n d t h e " h o t t e s t " subsequent  Aliquots  lysozyme i s o l a t i o n .  eggs were used f o r  The m e t h o d o f i s o l a t i o n  was t h a t a s d e s c r i b e d i n PART I , A. In a l l c a s e s , t h e second egg l a i d i n j e c t i o n had t h e " h o t t e s t " and t h i r d  albumen f o l l o w e d by t h e second  eggs. In t h e i n i t i a l  hen was i n j e c t e d lysozyme i s o l a t e d  e x p e r i m e n t d e s c r i b e d above, t h e  a t 0.75 h r . a f t e r o v i p o s i t i o n .  The  f r o m t h e s e c o n d a n d t h i r d e g g s was n o t  c o n s i d e r e d " h o t " enough f o r t h e c u l t u r e *  following  NEN C a n a d a L t d .  e x p e r i m e n t s t o be  s e e a p p e n d i x 10.  - 36 -  c o m p l e t e d i n PART I I I .  In efforts  to achieve "hot"  l y s o z y m e , t h e d o s a g e 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 a n d t h e b i r d was i n j e c t e d A t h i r d a t t e m p t was a l s o made.  3 hrs.  The d o s a g e  c o n s t a n t b u t h e b i r d was i n j e c t e d  \  postoviposition.  5 hrs.  remained postoviposition.  - 37 -  C.  RESULTS AND DISCUSSION  A supply o f r a d i o a c t i v e lysozyme was r e q u i r e d to complete the experiments d e s c r i b e d i n PART I I I .  Initially  i t was thought t h a t i . v . i n j e c t i o n o f 5 jaCi o f an amino a c i d h y d r o l y s a t e i n t o a l a y i n g hen 45 min. a f t e r  oviposition  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 o f the egg l a i d  the f o l l o w i n g day.  T h i s was based  on the assumption t h a t the m a j o r i t y o f the egg white lysozyme was s y n t h e s i z e d and l a i d down i n the albumen as the  y o l k passed through the magnum.  I t was found t h a t  a dosage o f 75 uCi i n j e c t e d a t 3 or 5 hours p o s t o v i p o s i t i o n -was  r e q u i r e d t o produce lysozyme w i t h a s p e c i f i c  activity  g r e a t e r than 2 x l O ^ dpm/mole i n the second egg l a i d 1  following ovposition. of  T h i s data suggests that the bulk  the lysozyme f o r an egg i s s y n t h e s i z e d up t o 44 h r s .  p r i o r t o o v i p o s i t i o n , and not as the y o l k passes through the  magnum. Table IV summarizes the r e s u l t s o b t a i n e d i n  attempts t o 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 o f a  C-amino a c i d h y d r o l y s a t e 3 h r s .  p o s t o v i p o s i t i o n r e s u l t s i n lysozyme c a . 6 times " h o t t e r " than when a 0.75 h r . i n j e c t i o n p o s t o v i p o s i t i o n was made. I n c r e a s i n g t h e i n t e r v a l t o 5 h r s . r e s u l t s i n an i n c r e a s e  /  - 38 -  Table IV.  In vivo  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 14 a C-ammo a c i d h y d r o l y s a t e .  Time p o s t oviposition  Lysozyme s p e c i f i c Egg  injection  Hrs.  2  activity  1  Number 3  2-5'  1.18  0  0.76  .75  0.36"  3.0  2.22  0.45  5.0  2.60  1.10  S p e c i f i c a c t i v i t y , DPM/mole x 1 0 " ^ ; assume m o l e c u l a r w e i g h t o f 14,300 gm/mole o f l y s o z y m e . Egg l a i d a f t e r f i r s t d a y p o s t i n j e c t i o n , a f t e r f i f t h day. O n l y 50 j i C i  Id "C-ammo a c i d h y d r o l y s a t e  i  but not .. injected.  - 39 -  i n a c t i v i t y o f c a . 1.2 o v e r Fig.  that of the 3 hr. injection.  6 d e p i c t s t h e 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  14 ( *C l a b e l l e d )  (75  a t various times a f t e r o v i p o s i t i o n .  i s o l a t e d from t h e second egg 14 of 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 jiCi)  activity  The  seems t o h a v e p l a t e a u e d a t a b o u t 5 h r s . p o s t 14  o v i p o s i t i o n , which suggests  that i n j e c t i n g the  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  not r e s u l t i n a s i g n i f i c a n t increase i n the 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 l y s o z y m e . 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 .  This  This would  suggestion 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 activity  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  lysozyme.  specific  Since the nature of the i n c o r p o r a t i o n  14 of the  C-amino a c i d s i n t o l y s o z y m e was n o t  determined,  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  *  0  ]  2  - *  3  A  4  •  "  5  *  6  T  n  hrs  7  -  8 14  Fig.  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 l y s o z y m e ( C l a b e l l e d ) i s o l a t e d f r o m t h e s e c o n d egg o f b i r d s i n j e c t e d i . v . w i t h a 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 after oviposition. (Note: the f i r s t p o i n t i s the r e s u l t o f 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 points). 1 4  - 41 -  D.  SUMMARY  Radioactive l a y i n g hens  l y s o z y m e was i s o l a t e d f r o m e g g s o f  (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 of  C-amino a c i d h y d r o l y s a t e s .  The s e c o n d e g g  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 the highest  specific activity  lysozyme.  contained  The optimum t i m e  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 i n l y s o z y m e w i t h a n a c t i v i t y o f 2.6 x  laid  resulted  lO^dpm/mole.  14 Injecting the  C-amino a c i d h y d r o l y s a t e  5 hrs. postoviposition lysozyme.  resulted  less  than  i n lower s p e c i f i c a c t i v i t y  42 -  PART I I I .  I N VITRO CULTURES WITH RADIOACTIVE LYSOZYME  A,  INTRODUCTION  These e x p e r i m e n t s aspects  o f a new t h e o r y  laboratory  (PART I I I ) w i l l  conceived  and d e v e l o p e d  ( P o u l t r y Embryology Laboratory,  the l a s t s i x years  (25) .  i n this  U.B.C.)  This theory attempts  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 an immune  d e a l w i t h some  over  to describe  diversity  during  response. B r i e f l y s t a t e d , t h e theory 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 enters the host i t i s immediately e n g u l f e d by p h a g o c y t i c  cells  (most l i k e l y K u p f e r  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). 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  Some o f  t o g i v e an RNA  a n t i g e n c o m p l e x h e r e a f t e r r e f e r r e d t o a s RNA-Ag. complex i s p r o c e s s e d  cells,  This  b y v a r i o u s p r o t e o l y t i c and RNase  enzymes s o t h a t t h e 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 a s d e s c r i b e d b y many r e s e a r c h e r s According  to Fitzsimmons  acts to prevent determinant  (elsewhere  review).  ( 2 5 , 2 6 ) , t h e RNA-Ag c o m p l e x  excessive hydrolysis of the antigenic  a n d a s s o c i a t e d RNA m o l e c u l e ,  p r o t e c t i o n occurs  through  from excess  i . e . mutual  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. the o t h e r  this  processing.  Each p r o t e c t s  We now h a v e a  - 43 -  p r o c e s s e d RNA-Ag c o m p l e x .  T h i s complex  i s transferred  to 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 f r o m t h e m a c r o p h a g e t o be  p i c k e d up b y r e c e p t i v e l y m p h o c y t e s . lymphocyte  One i n s i d e t h e  t h e RNA may e i t h e r be ( i ) r e p l i c a t e d a n d t h e n  reverse transcribed  ( 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 s e q u e n c e s , w h i c h may t h e n be i n t e g r a t e d i n t o t h e l y m p h o c y t e genome a t t h e h y p e r - v a r i a b l e or  (ii)  reverse transcribed directly  and i n t e g r a t e d i n t o t h e genome. RNA c o d e s  a s DNA  sites;  fragments  In both cases, the  ( t h r o u g h DNA) f o r a n amino a c i d s e q u e n c e  which  w o u l d b e i d e n t i c a l t o t h a t o f one o r more o f t h e h y p e r v a r i a b l e r e g i o n s i n an i m m u n o g l o b u l i n m o l e c u l e . i m m u n o g l o b u l i n s p r o d u c e d would be s p e c i f i c antigen which i n i t i a t e d  this  sequence  The  f o r the  of events.  RNA h a s b e e n i m p l i c a t e d t o h a v e t h e r o l e above  (Cl  t  s i n c e Garvey and Campbell  RNA c o m p l e x e d  (27) f i r s t  discussed  observed  w i t h f r a g m e n t s 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 o n t h e r o l e o f RNA i n t h e 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 s u g g e s t  t h a t t h e RNA a c t s s i m p l y a s a n a d j u v a n t f o r t h e A g , h e n c e 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 these complexes are a r t i f a c t u a l .  later  paper  (.30)  In  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  the  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 c h a r g e and to t o t a l c e l l u l a r their  RNA,  derived  a  from macrophages.  size From  r e s u l t s t h e y c o n c l u d e d t h a t a l l m a c r o p h a g e RNA-Ag  c o m p l e x e s a p p e a r t o be m a g n e s i u m i o n c o m p l e x e s , negative  g r o u p s on  the polypeptide  c h a r g e d p h o s p h a t e g r o u p s on C29) q u e s t i o n e d  the  chain with  RNA.  these conclusions  and  negatively  G o t t l i e b and  Schwartz  s u b s e q u e n t l y >{  d e m o n s t r a t e d t h a t s p e c i f i c i t y may  o r may  d e p e n d i n g on v a r i o u s  o f w h i c h was  factorsone  joining  not  be  shown incubation  conditions. Little formation. active  By  i n c u b a t i n g macrophage c u l t u r e s w i t h  lysozyme  times a f t e r  i s known o f t h e m e c h a n i c s o f RNA-Ag  (antigen)  initial  for  e x t r a c t i n g RNA  e x p o s u r e , i t was  a t w h i c h RNA-Ag f o r m a t i o n Once t h i s was  and  was  at  various  hoped t h a t the  maximum c o u l d be  a l l o w more i n t e n s i v e s t u d y RNA-Ag c o m p l e x .  o f RNA-Ag c o m p l e x e s . o f t h e RNA  time  determined.  d e t e r m i n e d , a m e t h o d c o u l d t h e n be  the r o u t i n e i s o l a t i o n  radio-  established This  involved i n  the  would  - 45  B.  METHODS  1.  Isolation of  -  lymphocytes  W h o l e b l o o d (1 ml) was v e i n o f mature b l o o d ) was w i t h 0.25  c o l l e c t e d from the wing  w h i t e l e g h o r n hens.  H e p a r i n (15 ug p e r ml.  used as t h e a n t i c o a g u l a n t . m l 0.9%  hypaque s o l u t i o n  The b l o o d was  s a l i n e and l a y e r e d o v e r 3 m l o f ( a p p e n d i x 9) i n a 11 x 100 mm  After centrifugation  f o r 20 m i n  (400 x G)  in  the  lymphocytes  The  s u s p e n s i o n w e r e s e t t l e d by c e n t r i f u g a t i o n  The p l a s m a was RPM1 in  t e s t tube.  1640  ficoll-  t e s t tube.  w h i c h had c o l l e c t e d a t t h e f i c o l l - h y p a q u e plasma were a s p i r a t e d i n t o a s t e r i l e  mixed  interface  lymphocytes (10 m i n 150 x G ) .  d e c a n t e d and t h e p e l l e t w a s h e d t w i c e w i t h  (appendix 11).  The  f i n a l p e l l e t was  suspended  10 m l o f RPMl 164 0 and c o u n t e d i n a h e m o c y t o m e t e r .  s u s p e n s i o n was cell  adjusted to 2 x 10  6  c e l l s / m l w h i c h was  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 Cell viability  e x c l u s i o n method  was  the.  cultures.  d e t e r m i n e d by t h e t r y p a n b l u e  ( a p p e n d i x 12)  and a d i f f e r e n t i a l  Leishmans  s t a i n completed a t t h i s p o i n t .  viability  was  detected.  The  with  In a l l cases,  > 97% and n o n - l y m p h o c y t i c c e l l s w e r e n o t  - 46  2.  In v i t r o  -  lymphocyte c u l t u r e w i t h  C  labelled  lysozyme  Sterile with  disposable  snap f i t t i n g  experiments.  0.4  mg  Two  per  t e s t tubes  caps were used t h r o u g h o u t t h e ml  Lysozyme of  plastic  of c e l l  ( i n 0.9%  s u s p e n s i o n was s a l i n e ) was  c u l t u r e tube.  The  t u b e was  the c e l l  incubated with  the  suspension.  a t 37°  w i t h the  incubator  centrifuged  hrs. The  A 0.25  ml  cap  followed for  ca.  by  s a m p l e was  1 min  c e n t r i f u g e tubes 10 min  (3090 x G)  phenol.*  *  by  '  a t 0,  d e c a n t e d and This  This biphasic  then t r a n s f e r r e d to (13 x 104 a 0.25  mm).  ml  aqueous phase f o r c o u n t i n g . DPM  was Gas  the channels r a t i o  2,  exchange  impossible. incubator  5,  8,  16  removed f o r s c i n t i l l a t i o n  added t o the p e l l e t .  and  rate  lysozyme mixed  therefore  x G)  tube.  and  samples were removed f r o m the  f o r 10 min. (150  2 ml  inverted  snapped i n p l a c e .  r e m a i n i n g s u p e r n a t a n t was  ( a p p e n d i x 13)  used per  completed c u l t u r e  e n v i r o n m e n t was  Duplicate and  The  2 ml  and  was  s y s t e m was  shaken  polypropylene  After centrifugation for  a l i q u o t was  removed f r o m  A l l c o u n t s were c o r r e c t e d  preservatives.  24  counting.  PBS  addition  method.  M a l l i n c k r o d t - no  mm)  culture  added a t the  s l o w l y r o t a t e d t o make s u r e t h a t a l l t h e with  (16 x 95  the to  - 47 -  c 3.  I s o l a t i o n o f g u i n e a p i g macrophage  cells  The m o d i f i e d m e t h o d o f S a b e t 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 I P w i t h a s t e r i l e , o f S e p h a d e x G-50  fine.*  3% s a l i n e  suspension  A p p r o x i m a t e l y 72 h r s . p o s t  injection  t h e g u i n e a p i g was s a c r i f i c e d b y 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 f r o m t h e r e c t a l a r e a t o a p o i n t b e t w e e n 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 t o t h e l o n g a x i s of the c u t .  The u n d e r l y i n g m u s c l e  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 . of s t e r i l e  s a l i n e was d e c a n t e d  T w e n t y - f i v e t o 30 m l  i n t o the peritoneum  aspirated against the peritoneal l i n i n g .  and  The r e s u l t i n g  cell  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  culture  and c e n t r i f u g e d f o r 10-15 m i n  supernatant  (150 x G ) .  The  was d e c a n t e d and t h e p e l l e t washed w i t h 5 m l o f RPM1 The r e s u l t i n g p e l l e t s w e r e r e s u s p e n d e d  tubes  1640.  i n RPMl 1640 a n d  p o o l e d t o a t o t a l v o l u m e o f 20 m l . A b s o l u t e a n d d i f f e r e n t i a l c o u n t s w e r e 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  m a c r o p h a g e / m l w h i c h was subsequent  *  the f i n a l  culture  experiments.  trademark  - Pharmacia  c o n c e n t r a t i o n used i n  Fine Chemicals.  - 48  C e l l v i a b i l i t y was b l u e e x c l u s i o n method > 97%  -  determined  (appendix  12).  by t h e t r y p a n  Cell viability  was  i n a l l cases. Cells  macrophage.  h a r v e s t e d by t h i s  The  t e c h n i q u e w e r e 40-50%  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  labelled  lysozyme Sterile  disposable plastic  w i t h snap f i t t i n g caps were used experiments.  Two  tube.  The  t u b e was  i n v e r t e d and  completed  c u l t u r e was  snapped i n p l a c e . was  Gas  (16 x 95  the  mm)  culture  used  u s e d a t t h e r a t e o f 0.4  make s u r e t h a t a l l t h e l y s o z y m e The  throughout  m l o f c e l l s u s p e n s i o n was  L y s o z y m e was culture  t e s t tubes  per  mg  tube.  per  slowly rotated  to  mixed w i t h the c e l l suspension.  i n c u b a t e d a t 37° w i t h t h e  exchange w i t h the i n c u b a t o r  cap environment  therefore impossible. 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, hrs.  10 min)  A.0.25 m l a l i q u o t was  a t 0, 2, 4, 6,  decanted  was  f o l l o w e d by 2 ml p h e n o l .  *  and  Aquasol  2 ml PBS  - NEN  &  24  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 s a m p l e t o 15 ml A q u a s o l * ) . was  8, 12  The  remaining  added to t h e p e l l e t .  supernatant  This  T h i s b i p h a s i c s y s t e m was  trademark  addition shaken  -  49  -  f o r c a . 1 min and t h e n t r a n s f e r r e d c e n t r i f u g e tubes.  to polypropylene  After centrifugation  (12,350 x G, 10 min)  a 0.25 m l a l i q u o t was r e m o v e d f r o m t h e aqueous p h a s e f o r counting.  A l l c o u n t s were c o r r e c t e d t o DPM by t h e  channels 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  positions  14 was u s e d i n t h e m a c r o p h a g e c u l t u r e e x p e r i m e n t s . l y s o z y m e was u s e d i n l y m p h o c y t e 3 tritiated  lysozyme  D r . D. C l a r k  C labelled  c u l t u r e experiments.  The  ( 2 . 1 x 10 DPM/mg) was t h e g i f t o f  (U.B.C. C h e m i s t r y D e p t . ) .  According to the theory discussed i n the Introduction, c u l t u r e o f m a c r o p h a g e 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  exposure.  Since the antigen  u s e d 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 , b e t w e e n RNA a n d l y s o z y m e  this  association  s h o u l d b e d e t e c t a b l e i n t h e 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  cells. 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  experiments with 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 curve f o r t h e lymphocyte lymphocyte the  cultures  level  (Fig. 7). Since the  i s not expected to a c t i v e l y ingest the antigen,  f o r m a t i o n o f RNA-Ag c o m p l e x e s  r a d i o a c t i v i t y would n o t appear phenol  culture  would n o t o c c u r and hence  i n t h e aqueous p h a s e o f a  extraction. The  r e s u l t s o f t h e macrophage c u l t u r e s  s h o u l d have r e f l e c t e d p r o t e i n Ag u p t a k e .  ( F i g . 8)  The m a c r o p h a g e  s h o u l d h a v e i n g e s t e d l a r g e amounts o f l y s o z y m e  resulting  in radioactivity i n the nucleic acid population.  This  - 51,  130  -  f"'''  c u l t u r e medium f r a c t i o n  dpm 110  60 nucleic acid f r a c t i o n  20 8  16  12  20  24  irs  Fig.  7,  Radioactivity p r o f i l e for nucleic acid extraction of lymphocyte c u l t u r e s incubated w i t h l ^ C l a b e l l e d lysozyme. R e s u l t s of one experiment.  Fig.  8.  Radioactivity p r o f i l e for nucleic acid of macrophage c u l t u r e s incubated w i t h lysozyme. R e s u l t s of one experiment.  extraction labelled  - 52 -  would  have  r e s u l t e d i n an i n c r e a s e  background.  Whether  fallen  to background  is  again  questionable. this  of  complexes.  suggest  might  Figures The  hover  from  about  lysozyme period  remaining  and/or  associated  with  used in  and hence  was n o t " h o t " e n o u g h  likely  populations. activity  The s o l u t i o n here  lysozyme  i s required.  results. experiments. extraction probably  of radioactive  between  interiorized  lysozyme  been  could  the n u c l e i c acid, f r a c t i o n s o f these  i n vivo  sample  (or lysozyme  membrane). responsible f o r  one i s t h a t  and hence  + Ag.  o f each  surface  have  to the  An  are  a t t h e amount  the e x t e r i o r c e l l  could  RNA  o f the phenol  nucleic acid associated  The most  one  ^  different  not differentiate  factors could  breakdown  of the i n vitro  level look  a  are conjecture.  phase  then  plateau  according  > RNA-Ag  i n the c u l t u r e media  would  Various results.  could  have  at a  had formed  completely  t h e aqueous  One  but this  lysozyme  these  given  over  to background  suggest  o f complexes  statements  the.background  insignificant.  dropped  one c o u l d  + Ag  would  o r remained  I f a plateau  RNA  above have  then  7 and 8 a r e t h e r e s u l t s  counts  increase  levels  turnover  formula; The  system  rise,  a constant  following  or not this  i n radioactivity  Had t h e a c t i v i t y  following RNA-Ag  \  the lysozyme  n o t be  various  i s obvious;  detected  cell  a higher  specific  -  -  53  Since lysozyme i s a s o l u b l e a n t i g e n , the r a t e o f i n g e s t i o n by m a c r o p h a g e m i g h t n o t be as g r e a t as f o r m a t e r i a l s such molecules  as r e d b l o o d c e l l s ,  (e.g. hemoglobin, horse  I f the r a t e i s depressed,  b a c t e r i a and  radish peroxidase).  low a c t i v i t y  p o p u l a t i o n c o u l d be e x p e c t e d .  macro-  i n the n u c l e i c a c i d  This reasoning could explain  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 The is  s o l u t i o n here,  although quite s t r a i g h t  l e s s d e s i r a b l e i n terms of i n t e r p r e t a t i o n of  results. such  By  as -a CM  experiments.  experimental  a t t a c h i n g t h e l y s o z y m e t o some l a r g e r s e p h a d e x o r CM  w o u l d be p h a g o c y t i z e d  cellulose particle,  along w i t h the p a r t i c l e .  forward  carrier,  the  lysozyme  This  r e s u l t i n a l a r g e amount o f l y s o z y m e i n t e r i o r i z e d by m a c r o p h a g e ; e s p e c i a l l y i f t h e CM w i t h the antigen.  This technique  should the  p a r t i c l e s were s a t u r a t e d should i n c r e a s e the  amount  of 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 the n u c l e i c a c i d  fraction.  One  carrier  disadvantage  o f t h i s m e t h o d c o u l d be  t h a t the  p a r t i c l e might i n t e r f e r e w i t h the normal p r o c e s s i n g of antigen  ( i . e . lysozyme) w i t h i n t h e macrophage. A n o t h e r s o l u t i o n w o u l d be  a c t i v e macromolecule.  Experimental  however, t h a t t h i s molecule and  t o use  a larger radio-  design would  dictate  h a v e a known p r i m a r y s t r u c t u r e  a number o f known a n t i g e n i c  determinants.  v  -  Although  54 -  the r e s u l t s i n these two  experiments.  were i n c o n c l u s i v e , a number o f c o n s t r u c t i v e suggestions have been made.  Using these technique i n c o n j u n c t i o n  with very h i g h s p e c i f i c a c t i v i t y lysozyme, some  light  should be shed on the manner o f lysozyme p r o c e s s i n g and nucleic acid  interaction.  1  - 55  D.  -  SUMMARY  RNA  extracts of chicken  l y m p h o c y t e s and  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 lysozyme  (PART I I ) c o n t a i n e d  little  radioactive  radioactivity.  o p t i m u m t i m e o f RNA-Ag c o m p l e x f o r m a t i o n  was  established.  Reasons f o r t h e s e i n c o n c l u s i v e  are  s u g g e s t e d and  followed  which could  g i v e more p o s i t i v e  a d i s c u s s i o n of results.  The  therefore  not  by  guinea  results  experiments  - 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 c o n c e r n e d mainly high  with  the preparation  of m i l l i g r a m q u a n t i t i e s of  specific radioactivity  lysozyme.  B o t h p a r t s were s u c c e s s f u l i n t h a t a  simple  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 l O ^ d p m / m o l e suggestions increase  lysozyme.  Some  h a v e b e e n made (PART I I ) w h i c h c o u l d  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 prepared  from egg w h i t e .  These s u g g e s t i o n s ,  e x p e r i m e n t s o f PART I I I s h o u l d m e c h a n i c s o f RNA-Ag Further  when a p p l i e d t o t h e  s h e d some l i g h t on t h e  formation.  e x p e r i m e n t s i n t h e 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 of the  possibly  t h e i n t e r a c t i o n s between t h e n u c l e o t i d e  a t the nature  sequences o f  RNA a n d 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  continuing  s e r i e s of experiments should  t h e work o f S a x i n g e r  These w o r k e r s have p u b l i s h e d amino a c i d s  greater  and Ponnamperuma  (36).  a t a b l e o f i n t e r a c t i o n s o f two  ( t r p a n d g l y ) w i t h a number o f d i - and  trinucleotides. tryptophan  involve  One e n t r y  selectivity  f r o m t h i s t a b l e shows t h a t t h e  c o e f f i c i e n t f o r ApApUp was t e n t i m e s  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 w e r e 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 determinants  s t r u c t u r e of the a n t i g e n i c  o f l y s o z y m e a r e known ( e . g . l o o p p e p t i d e ) ,  work i n t h e s e amino a c i d s . *  i n t e r a c t i o n experiments  w h i c h make up  should include  loop peptide.  t r a n s c r i p t i o n and  By  tracing  t h i s code  t r a n s l a t i o n of the r e s u l t a n t  step of t h i s experiment  w o u l d be  hypervariable regions of antibody the a n t i g e n i c determinant  the  chains d i r e c t e d against  i n question.  I f t h e amino  i d e n t i c a l or very s i m i l a r to the  c o d e d f o r by  t h e o r i g i n a l RNA  f o r the theory  acid  sequence  which i n t e r a c t e d w i t h  ( i . e . RNA-Ag c o m p l e x ) ,  These e x p e r i m e n t s  theoretically).  to sequence  s e q u e n c e was  supporting evidence  DNA,  antibody  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  determinant  through  integration stages, followed  the p r o t e i n sequence f o r the a n t i loop p e p t i d e  final  a  s e q u e n c e s f o r t h e amino a c i d s  t h e r e v e r s e t r a n s c r i p t i o n and  The  those  This would r e s u l t i n the g e n e r a t i o n of  series of t r i - n u c l e o t i d e  by  -  t h i s w o u l d be  the  strong  (PART I I I ) .  are e n t i r e l y  plausible.  Methods a l r e a d y e x i s t w h i c h d e s c r i b e the methodology f o r i s o l a t i o n of 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, 10). P r o t e i n sequencing  m e t h o d s a r e r a t h e r l e n g t h y and  b u t have been s t a n d a r d i z e d .  The  expensive  m e t h o d o l o g y f o r amino  l o o p p e p t i d e i s a 23 amino a c i d l o n g s e q u e n c e ( 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 f r o m egg w h i t e l y s o z y m e ( 1 7 ) .  acid,  -  trinucleotide binding  58  studies  -  a r e numerous and  column chromatography, e q u i l i b r i u m d i a l y s i s nitrocellulose binding is  the  One  and  foreseeable  problem  lack of commercially 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 there  sequences a v a i l a b l e . detection the  studies.  include  are  This  approximately ten of  r e s t r a i n t could  these  prevent  the  of c e r t a i n important i n t e r a c t i o n s simply  t r i n u c l e o t i d e sequences were n o t  because  available.  A n o t h e r p r o b l e m , p o s s i b l y more s e r i o u s , i s 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 . A,  U,  This  G,  C r e s u l t i n 64  does n o t  exist.  include  There are  Four bases, i . e .  combinations of t r i p l e t  sequences.  the v a r i a t i o n s of c e r t a i n bases  2 0 odd  variations.  This  S a x i n g e r and  Ponnamperuma  amino a c i d s , a g a i n n o t  be  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 . (36)  have a l r e a d y  extended with  shown a  the m a t e r i a l s  a v a i l a b l e , a t y p e o f c o d e f o r RNA acids  could To  be be  this  currently  nucleotides  - amino  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  only  T h i s w o u l d be  certain antigenic  good.  As  v a l i d i t y of these  useful i n establishing further ideas.  mentioned  d e t e r m i n a n t s need  Once t h e s e i n t e r a c t i o n s w e r e c o n f i r m e d ,  r e m a i n i n g work be  tested.  might develop which would negate t e s t i n g  of c e r t a i n combinations.  tested.  If  formulated.  Certain patterns  previously,  that  including  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 . scheme c o u l d  the  the the  be  - 59 -  BIBLIOGRAPHY  1.  A l d e r t o n , G. a n d H.L. T e v o l d . 1 9 4 6 . 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 and some c r y s t a l l i n e s a l t s o f l y s o z y m e . J. Biol. Chem. 1 6 4 : 1 .  2.  C h a n c e , K.D. a n d D.G. C l a r k . 19 76. In vivo i n c o r p o r a t i o n o f i s o t o p i c a l l y l a b e l l e d amino a c i d s into vertebrate proteins: L - ( m e t h y l - H) m e t h i o n i n e i n c o r p o r a t i o n i n t o c h i c k e n egg w h i t e lysozyme. Submitted f o r p u b l i c a t i o n t o Can.J. B i o c . O c t . 4, 1 9 7 6 .  3.  M e t c h n i k o f f , E.  4.  F l e m m i n g , A. 1 9 2 2 . On a r e m a r k a b l e b a c t e r i o l y t i c element found i n t i s s u e s and s e c r e t i o n s . Proc. R o y a l S o c . 93:306.  5. i  J o l l e n s , J . , J . J o u r e q u i - A d e l l , I . B e r n i e r and P. J o l l e s . 1 9 6 3 . The c h e m i c a l s t r u c t u r e o f egg w h i t e l y s o z y m e . Biochemical Biophysica Acta. 78:668.  6.  C a n f i e l d , R.E. 1 9 6 3 . The amino a c i d s e q u e n c e o f egg w h i t e l y s o z y m e . J . B i o l . Chem. 238:2698.  7.  S o p h i a n o p o u l o s , A . J . a n d K.E. V a n H o l d e . 1 9 6 4 . P h y s i c a l s t u d i e s o f m u r a m i d a s e ( l y s o z y m e ) . I I pH dependant d i m e r i z a t i o n . J . B i o l . Chem. 2 3 9 : 2 5 .  8.  B e r g e f , L.R. a n d R.S. W e i s e r . 1 9 5 7 . The g l u c o s a m i n i d a s e a c t i v i t y o f egg w h i t e lysozyme. Biochemica Biophysica A c t a . . 26:517.  9.  M c K e l v e y , D. 1 9 7 5 . The i s o l a t i o n a n d a n a l y s i s o f chicken a n t i b o d i e s t o lysozyme. B . S c . T h e s i s , U.B.C.  1 8 9 9 . A n n . 1 s t . P a s t e u r . 17:37.  10.  C a n f i e l d , E.R. a n d A.K. L u i . 1 9 6 5 . The d i s u l f i d e bonds o f egg w h i t e l y s o z y m e . J . B i o l . Chem. 2 4 0 : 1997.  11.  N a k a i , S.  12.  P e t r i , W.H. 1972. D i s c o n t i n u o u s p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f RNA. A n a l . B i o c . 48:442.  1976.  Personal  communication.  - 60 -  F e e n e y , R.E. a n d R.G. A l l i s o n . 1967. E v o l u t i o n a r y Biochemistry of P r o t e i n s . Wiley Interscience. New York. M a r o n , E., E.. S h i o z a w a , R. A r n o n a n d M. S e l a . 1 9 7 1 . Chemical and Immunological C h a r a c t e r i z a t i o n o f a unique a n t i g e n i c region i n lysozyme. Biochem. 10: 763. A t a s s i , M.Z., J . K o k e t s u a n d A.F.S.A. Habeeb. 1 9 7 6 . Enzymic and immunochemical p r o p e r t i e s o f lysozyme. XIII Accurate d e l i n e a t i o n o f the r e a c t i v e s i t e a r o u n d t h e d i s u l f i d e 6-127 b y i m m u n o c h e m i c a l ' s t u d y o f B p r o p i o l a c t o n e lysozyme d e r i v a t i v e and o f s y n t h e t i c d i s u l f i d e peptides. Biochemica Biophysica Acta , 420:358. H j e r t e n , S., S. J e r s t e d t a n d A. T i s e l i u s . 1 9 6 9 . .Apparatus f o r l a r g e s c a l e p r e p a r a t i v e poly-acrylamide g e l e l e c t r o p h o r e s i s . A n a l y t . B i o c . 27:108. O a d e s , J.M. a n d W.O. B r o w n . 1965. A s t u d y o f t h e w a t e r - s o l u b l e o v i d u c t p r o t e i n s o f t h e l a y i n g hen and the female c h i c k t r e a t e d w i t h g o n a d a l hormones. Comp. B i o c h e m . P h y s i o l . 1 4 : 4 7 5 . M a n d e l e s , S. a n d E.D. Ducay. 1962. S i t e o f egg w h i t e protein formation. J . B i o l . Chem. 237:3196. C o n r a d , R.M. a n d H.M. S c o t t . 1 9 4 2 . The a c c u m u l a t i o n of p r o t e i n i n the oviduct o f the fowl. Poult. Sci. 21:81. S m i t h , A.H., G.N. H o o v e r , J.O. N o r d s t r o m a n d C M . W i n g e t . 1957. Q u a n t i t a t i v e changes i n t h e hens o v i d u c t a s s o c i a t e d w i t h egg f o r m a t i o n . P o u l t . S c i . 36:353. Wyburn, G.M., H.S. J o h n s t o n , M.H. D r a p e r a n d M.F. Davidson. 1 9 7 0 . Q. F l . E x p . P h y s i o l . 5 5 : 2 1 3 . t  Guzsal,  E.  1966. K i s e r l .  K o g l . 59:3.  Guzsal,  E.  1 9 6 8 . A c t a . V e t . Hung. 1 8 : 2 5 1 .  D a v i d s o n , M.F., M.H. D r a p e r , H.S. J o h n s t o n a n d G.M. Wyburn. 196 9. P r o t e i n s e c r e t i o n b y t h e magnum r e g i o n o f t h e o v i d u c t o f t h e hen. J . P h y s i o l . Lond. 205:23.  F i t z s i m m o n s , R.C.  1976.  P e r s o n a l communxcation.  F i t z s i m m o n s , R.C. 1977. P a p e r s u b m i t t e d t o D e v e l o p m e n t a l I m m u n o b i o l o g y Symposium, S c o t t i s h Immunology G r o u p , Aberdeen U n i v e r s i t y , Aberdeen, S c o t l a n d . G a r v e y , J . S . a n d D.H. C a m p b e l l . 1 9 5 7 . The r e t e n t i o n of 3 5 S - l a b e l l e d b o v i n e serum a l b u m i n i n n o r m a l and immunized l i v e r t i s s u e . J . E x p . Med. 1 0 5 : 3 6 1 . C o h e n , E.P. Ohio.  1976.  G o t t l i e b , A.A. interactions.  Immune RNA.  CRC P r e s s ,  and R.H. S c h w a r t z . 1972. C e l l Immunol. 5:341.  Cleveland,  Antigen-RNA  R o e l a n t s , G.E. and J.W. Goodman. 1969. The C h e m i c a l m a t u r e o f m a c r o p h a g e R N A - a n t i g e n c o m p l e x e s and t h e i r r e l e v a n c e t o immune i n d u c t i o n . J.Exp.Med. 130:557. R o e l a n t s , G.E., J.W. Goodman a n d H.O. M c d e v i t t . 1971. Binding of polypeptide antigen to ribonucleic acid f r o m m a c r o p h a g e , H e l a and E. C o l i c e l l s . J . Immunol. 106:1222. Goodman, J.W., G.E. R o e l a n t s and V.S. B y e n s . 197 3. RNA: a n t i g e n c o m p l e x e s : M e c h a n i s m o f f o r m a t i o n a n d t h e t e s t i n g o f a p o s t u l a t e d mode o f a c t i o n . A n n a l s New Y o r k A c a d . S c i . 207:288. J o h n s o n , A.G., K.E. S c h m i d t , K. M e r r i t and I . Han. 1969. E n h a n c e m e n t o f a n t i b o d y f o r m a t i o n b y n u c l e i c a c i d s and t h e i r d e r i v a t i v e s . Nucleic Acids i n Immunology. R o e l a n t s , G.E. and J.W. Goodman. 196 8. 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 e x u d a t e 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 i m m u n o g e n i c f o r m s . B i o c h e m . 7:1432. S a b e t , 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. a n d C. Ponnamperuma. 1974. Interactions b e t w e e n a m i n o 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 milieu. 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 m l d i H 0 2  adjust  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 C a r b o n a t e pH 9.2 76.8  gm Ammonium C a r b o n a t e  t o 2000 d i H 0 2  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% C o m p l e t e 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 Electrophoresis 12 m l s t o c k  acrylamide solution (3i)  7.5 m l 5 x c o n c . b u f f e r E ( 3 i i ) 12 m l d i H 0 2  0.025 m l Temed (N,N,N',N' t e t r a m e t h y l e t h y l e n e 0.375 m l Ammonium P e r s u l f a t e S o l u t i o n i.  Stock Acrylamide  Solution  Acrylamide & Bis-acrylamide Electrophoresis Purity 15  (3iii)  a r e BIORAD*  Reagents  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 0 t o 100 m l 2  filter  t h r o u g h Whatman #1 a n d s t o r e a t 4°  i n brown b o t t l e . Biorad  Laboratories,  Mississauga,  Ontario.  diamine  -  ii.  63 -  5X c o n c e n t r a t e d  buffer E  21.8 gm TRIS  (hydroxymethylamino  20.7 gm NaH PC> 2  1.85 diH 0 2  iii.  methane)  4  gm EDTA ( e t h y l e n e d i a m i n e  tetracetate-Na ) 2  t o 1000 m l .  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 m l d i H 0 2  prepared f r e s h . d a i l y . 4.  B u f f e r E: 17.44 gm TRIS 16.56 gm N a H P 0 2  66.3 gm N a H P 0  4  2  5.9 gm EDTA  diH 0  diH 0  pH  to 4,liters  2  7.6-7.8 Disc  Electrophoresis. 0.2 gm  S o d i u m D o d e c y l S u l f a t e (SDS)  9.8 m l  Buffer E  0.2 m l  2-mercaptoethanol  1 gm  Sucrose  Spatula  Complete  (BME)  t i p f u l l o f bromophenol  dye u n t i l  Disc  4  t o 16 l i t e r s  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 Gel  6.  69.8 gm TRIS  1.4 8 gm EDTA 2  5.  or  s o l u t i o n i s deep  tracking  blue.  Solution for Quantitative  Gels.  blue  Polyacrylamide  - 64  -  Q. "O  Volume  acrylamide  5.6  7.5  9.5  8  12.6  16.0  5  5.0  5.0  diH 0  8  7.4  4.0  Temed  .01  .01  .01  Ammonium P e r s u l f a t e  .25  .25  .25  Stock acrylamide 5X c o n e b u f f e r  s o l u t i o n 3(i)  E  2  7.  .25% C o o m a s s i e B l u e S t a i n i n g S o l u t i o n dissolve  1.25  gm C o o m a s s i e B r i l l i a n t B l u e i n 227  ml  Methanol add  45 m l A c e t i c  Acid  Make up t o 5 00 ml w i t h f i l t e r Whatman  diH 0 2  #1  S t o r e a t 4° i n b r o w n b o t t l e a l l o w t o come t o room t e m p e r a t u r e 8.  B u f f e r K:  0.066 M  9 gm  KH P0 .  H0  2  2  Ficoll  4  ml  a d j u s t t o pH 9.  use.  4  t o 1000  2  KH P0  before  6.24  w i t h NaOH*  - HYPAQUE:*  Ficoll  9 gms  30 m l Hypaque add  14.25  working  ml  diH 0 2  t o 100  ml.  solution diH 0 2  solution  10 p a r t s h y p a q u e 24 p a r t s  ficoll  3 ml w o r k i n g s o l u t i o n c a n s u p p o r t 3-5 Pharmacia  F i n e C h e m i c a l s , U p s a l a , Sweden.  ml d i l u t e d b l o o d .  -  10.  65  "  Amino A c i d H y d r o l y s a t e U s e d t o P r e p a r e Lysozyme  (Amersham S e a r l e ) :  Ammo a c i d  %  L-alanine  9.3%  L-arginine  6.3%  L-aspartic  acid  9.0%  L-glutamic  acid  11. 8%  glycine  4.6%  L-histidine  4.0%  L-leucine  11.8%  L-isoleucine  4.8%  L-lysine  5.1%  L-phenylalanine  6.7%  L-proline  5.6%  L-serine  4.8%  L-tyrosine  3.6%  L-valine  6.8%  Specific activity concentration 11.  Radioactive  2  2  Glucose  Culture  Medium  (Gibco): mg/L  Ca(N0 ) .4H 0 3  radioactive  50 u C i / m l .  R P M l 1640 T i s s u e Component  56 mCi/mAtom c a r b o n  100. 0 2000.0  MgSO .7H 0  100.0  KC1  400.0  4  2  - 66 -  Na^HPO, . 7H„0 2 4 2 NcCl L-'arginine  ( f r e e base)  1512.0 6000.0 200.0  L-asparagine  50.0  L-aspartic acid  20.0  L-cystine  50.0  L-glutamic  acid  L-glutamine  2 0.0 300.0  Glutathione  (reduced)  Glycine  1  L-histidine  ( f r e e base)  1.0 . 1 0 . 0 15.0  L-hydroxyproline  20.0  L-isoleucine  ( a l i o free)  50.0  (methionine free)  50.0  L-leucine  L - l y s i n e HCl  40.0  L-methionine  15.0  L-phenylalanine  15.0  L-proline  20.0  (hydroxy free)  L-serine  30.0  L-threonine  (allow free)  L-tryptophan  20.0 5.0  L-tyrosine  20.0  L-valine  20.0  Biotin  0.2  Vitamin  B  1 2  0.005  D-Ca p a n t o t h e n a t e  0.25  Choline  CI  3.0  acid  1.0  Folic  i - Inositol  35.0  Nicotinamide  1.0  Para-aminobehzoic a c i d  1.0  Riboflavin  0.2  Thiamine HCl  1.0  Phenol r e d NaHCO-,  -5.0 2000.0  -  12.  Trypan Cell  67  -  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  Viability 5  i.  t o 0.5 m l o f c e l l add  0.1 m l 0.4% t r y p a n b l u e  saline) ii. iii.  (1 x 10  t o 20 x 10 )  ( i n phosphate b u f f e r e d  and m i x t h o r o u g h l y ,  a l l o w t o s t a n d f o r 5-10 m i n . count v i a b l e c e l l s as % v i a b l e c e l l s  13.  suspension  5  i n a hemocytometer and e x p r e s s (dead c e l l s  Phosphate B u f f e r e d S a l i n e  are blue).  (PBS):  Na HP0 .7H 0  4.44 gm  NaH P0 .H 0  0.45 gm  2  4  2  4  2  2  NaCl Add  17.0 diH„0 t o ' 2 l i t e r s  gm  pH 7.4  

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