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Protein chemistry of triose phosphate isomerase Burgess, Helen Diana 1976

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PROTEIN CHEMISTRY OF TRIOSE PHOSPHATE ISOMERASE  by  HELEN DIANA BURGESS B.A., U n i v e r s i t y o f L e t h b r i d g e , 1973  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  The Department o f Chemistry We accept  t h i s t h e s i s as conforming  to the r e q u i r e d  THE UNIVERSITY OF ERITI3H COLUMBIA June, 1976  standard  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y s h a l l I  f u r t h e r agree  in p a r t i a l  fulfilment of  the requirements f o r  the U n i v e r s i t y of B r i t i s h Columbia,  make i t  freely available  that permission  for  I agree  that  r e f e r e n c e and study.  for extensive copying of  this  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s of  this  written  representatives. thesis  It  for financial  i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n gain s h a l l  permission.  Department o f  /Ul  O^nj^M^  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 Vancouver, Canada V6T 1WS  It  Columbia  not be a l l o w e d without my  II.  ABSTRACT t  The  p r o t e i n , t r l o s e phosphate isomerase (TIM)  i s o l a t e d from f r e s h c h i c k e n b r e a s t muscle and  has  been  p u r i f i e d by  an-  i o n exchange chromatography on DSAE Sephadex A50..column.  Fur-  ther p u r i f i c a t i o n proceeded v i a B i o g e l A DEAE r e s i n .  TIM  f r a c t i o n s o f b o t h chromatographies were contained cent p r o t e i n peaks, A and The isozymic  separation  i n two  d i f f e r e n c e s i n the TIM  peaks was  found to be based upon  active protein;  g e l , showed one  Isoelectric  isozyme i n the Peak A  p r o t e i n w i t h pl=7.65 w h i l e Peak B p r o t e i n contained zymes of p i 7.56  and  7.49.  Focusing  two  No  Isozymic  separation  observed w i t h d i s c g e l e l e c t r o p h o r e s i s a t pH 8.5.  a c i d a n a l y s i s which was  Amino  c a r r i e d out on p u r i f i e d Peak A p r o t e i n  showed s u b s t a n t i a l d e v i a t i o n s from l i t e r a t u r e The  Iso-  of o l d peak B p r o t e i n  y i e l d e d a f o u r t h isozyme w i t h pl=7»62. was  adja-  B.  of the two  f o c u s i n g , b o t h column and  The  Peak A isozyme was  modified  values.  v i a the r e a c t i o n of  the  t h i o l of c y s t e i n e w i t h the maleimide N-ethylmalelmide (NEM), o r t r l f l u o r o N-ethylmaleimide (FEM), as w e l l as w i t h the f i d e s 4,4»  d i t h i o p y r l d l n e (4--PDS) or  zoic acid)  (DTNB)i  Two  equivalents  5*5'-dithiobis(2-nitrobenof reagent per molecule r e -  a c t e d but a k i n e t i c noh-eqiiiVaienee-of 19 was  observed. The  F NMR  of the FEM  disul-  the'two s i t e s to  l a b e l e d p r o t e i n was  modification  performed.  f i n a l chapter of t h i s t h e s i s d e a l s w i t h the k i n e t i c  a n a l y s i s a t s e v e r a l temperatures of the m o d i f i c a t i o n of TIM k-FD3 and  DTNB both i n the presence and  In the absence of  by sub-  iii.  s t r a t e g l y c e r a l d e h y d e 3-phosphate.  Blphasic Arrhenlus plots  w i t h a break a t approximately 2 5 ° C were observed f o r the PDS  modification;  energy f o r  T>25°C  50.0 kcal/mole. energy f o r  T>25°C  4-  In the presence of s u b s t r a t e , the a c t i v a t i o n was  7»2  kcal/mole w h i l e f o r  In the absence was  4.4  T<J25°C  i t was  of s u b s t r a t e , the a c t i v a t i o n  kcal/mole w h i l e f o r  T<T25°C,  i t was  39.9 k c a l / m o l e . L a s t l y , a segment d i s c u s s i n g the importance  of the r e s u l t s i-  d e s c r i b e d i n t h i s t h e s i s , i n terms of the c u r r e n t T I M l e t e r a t u r e . is  included.  iv  TABLE OF CONTENTS  CBAPTER I.  II.  Till.  IV.  V.  PAGE GENERAL INTRODUCTION•  .  1  1.1  P u r i f i c a t i o n and C h a r a c t e r i z a t i o n .  3  1.2  Enzymatic I n h i b i t i o n and the C a t a l y t i c Process of TIM...................ii..........5  1*3  S t r u c t u r a l Aspects  .12  ISOLATION AND PURIFICATION 2.1  Materials  ; i  2.2  Methods  i.i  2.3  Results.......  19 . . i i i  20 26  CHARACTERIZATION OF PEAK A AND . PEAK . B 3.1  Methods  3*2  Results  .;  .39 tjr?  PROTEIN MODIFICATION 4.1  Introduction., . i i  4.2  Methods  4.3  Results  i .i.....  i  75 84  i . .  ....90  KINETICS OF DISULFIDE MODIFICATION OF TIM 5.1  Methods  5.2  Results.  105 i  CONCLUSIONS REFERENCES .  112 122  •  •.  125  V.  LIST OF TABLES  TABLE L  II  . PAGE D e t e r m i n a t i o n of a c t i v i t y u n i t s f o l l o w e d throughout a t y p i c a l l i v e chicken prepa r a t i o n of chicken b r e a s t muscle...... Stock s o l u t i o n s and b u f f e r s f o r SDS gel electrophoresis  III IV V  28  I s o e l e c t r i c focusing  .41 solutions f o r gels  Amino a c i d a n a l y s i s o f Peak A T I M . . . i i . . M o d i f i c a t i o n w i t h 4-PDS M o d i f i c a t i o n w i t h DTNB  .......... .  .52 73 112 12°  vi. LIST OF FIGURES FIGURE  PAGE  1  U l t r a v i o l e t s p e c t r a o r NAD  2  I n i t i a l DEAE Sephadex A 5 0 Chromatography  27  3  I n i t i a l DEAE Sephadex A50 Chromatography of a L i v e Chicken P r e p a r a t i o n  .30  4 5 6  +  and NADH.....*  24  Rechromatograr>hy o f TIM F r a c t i o n s on DEAE Sephadex A50  33  Rechromatcgraphy o f peak A and Peak B TIM F r a c t i o n s on DEAE Sephadex kjO  35  Rechromatography of Peak A and Peak B TIM F r a c t i o n s on DE52 C e l l u l o s e  36  7  Rechromatography o f TIM F r a c t i o n s on B i o g e l A DEAE  38  8  E l e c t r o p h o r e s i s Apparatus  40  9 10  SDS G e l E l e c t r o p h o r e s i s o f P r o t e i n Standards A Sketch o f the I s o e l e c t r i c F o c u s s i n g (IEF) Column  44 47  11  Disc G e l Electrophoresis Using Fresh Protein from Peak A (A) and Peak B ( B ) , and O l d P r o t e i n from peak B (B* ) ;  _ 5  9  12  Column I E F o f Rechromatographed Peak A.....  62  13  Column I E F o f Rechromatographed Peak B...  64  14  Column I E F o f Rechromatographed Peak A and Peak B  65  15  G e l i s o e l e c t r i c F o c u s s i n g o f Peak A and Peak B  16  Sample G e l s o f G e l I s o e l e c t r i c F o c u s s i n g o f Peak A and Peak B  17  ...67 68  G e l I s o e l e c t r i c F o c u s s i n g o f F r e s h P r o t e i n from Peak A and O l d P r o t e i n from peak B  7 i 1  18  U l t r a v i o l e t S p e c t r a o f NEM and FEM  19  Pseudo F i r s t Order A n a l y s i s o f the M o d i f i c a t i o n of TIM w i t h NEM  80 01  vii.  FIGURE 20  PAGE 1  ^ F NMR o f FEM M o d i f i e d Phosphate Isomerase.  Triose .95.  21 - Pseudo F i r s t Order A n a l y s i s of the Cyanide Displacement o f TNB" from the DTNB M o d i f i e d peak A P r o t e i n « 22  ••100  A T y p i c a l Example o f Monophasic K i h e t i c s 4-PDS M o d i f i c a t i o n o f TIM a t 2 2 C i n the Absence o f G l y c e r a l d e h y d e 3-Phosphate.  109  A T y p i c a l Example o f B i - p h a s i c K i n e t i c s 4-PDS M o d i f i c a t i o n o f TIM a t 25°C i n t h e Absence o f G l y c e r a l d e h y d e 3-Phosphate.  110  A r r h e n i u s P l o t f o r the 4--PDS M o d i f i c a t i o n o f TIM i n the Presence of G l y c e r a l d e h y d e 3-Phosphate .  115  A r r h e n i u s P l o t f o r the 4-PDS M o d i f i c a t i o n of TIM i n the Absence o f G l y c e r a l d e h y d e 3-Phosphate •  116  C  23  24  25  ACKNOWLEDGEMENTS I would l i k e t o express my a p p r e c i a t i o n t o Dr. D.G. C l a r k whose enthusiasm and knowledge g r e a t l y a i d e d s u p e r v i s i o n o f my work. I wouj.a xiKe Department  t o thank Josepn Durgo o f the Biochemistry  (UBC) f o r t e c h n i c a l a s s i s t a n c e w i t h the amino a c i d  a n a l y s i s as w e l l as Dr. A.G. M a r s h a l l f o r h i s a s s i s t a n c e  with  19 the  F. NMR and D r . R.E. P l n c o c k f o r the use o f h i s  spectro-  photometer. I would l i k e to thank L e s l i e deBruyn and Monica E . Rosenberg f o r t h r i r help i n the p r o d u c t i o n  of t h i s t h e s i s .  F i n a l l y , I am very g r a t e f u l f o r the generous support o f a N a t i o n a l Research C o u n c i l Postgraduate F e l l o w s h i p 197*0 and a H.R. MacMillan Family study a t UBC.  Fellowship  (1973-  (1974-1975) t o  CHAPTER I GENERAL INTRODUCTION The  purpose of t h i s i n t r o d u c t i o n i s to p r o v i d e ,  e r a t u r e b a s i s f o r the present  isomerase (TIM)  2 ) the mechanism of the  chemical and  s t r u c t u r a l aspects  from d i f f e r e n t  c a t a l y t i c process, of the enzyme.  the work p r e s e n t e d i n t h i s t h e s i s was muscle;  lit-  u n d e r s t a n d i n g of the presence'of m u l t i -  enzyme forms o f t r l o s e p h o s p h a t e sources,  l ) the  The  and  3 ) some  TIM  used f o r  I s o l a t e d from chicken  :  breast  however much of the e a r l y i n v e s t i g a t i o n s have i n v o l v e d  enzyme from r a b b i t muscle ( 4 ) and  yeast  (5)«  In a d d i t i o n d e f i n i -  t i v e s t u d i e s have been preformed on both human.(6) and b a c t e r i a l (7) t r i o s e phosphate isomerase. sources,  These s t u d i e s from the v a r i o u s .  formed the background f o r the I s o l a t i o n and  of the p r o t e i n , as w e l l as the c h a r a c t e r i z a t i o n and i f i c a t i o n s which were performed and The  reported  purification the chemical mod-  in this thesis.  enzyme, t r i o s e phosphate isomerase (TIM)  p l a y s a cen-  t r a l r o l e l n g l y c o l y s i s , as w e l l as l n g l u c n e o g e n e s i s and Of  the h i g h e s t  has  one  c a t a l y t i c r a t e s of a l l the g l y c o l y t i c enymes.  c a t a l y z e s the r e v e r s e  a l d o s e k e t o s e l s o m e r i z a t i o n of g l y c e r a l d e -  hyde 3-phosphate ( G 3 P ) and  dlhydroxyacetone phosphate (DHAP).  T h i s b l f u n c t i o n a l p r o t e i n has been c o n s i d e r e d  to be an u n l i k e l y  p o i n t f o r m e t a b o l i c r e g u l a t i o n s i n c e i t s i t s high c a t a l y t i c r a t e a b i l i t y and  It  i n vivo concentration  has  t h a t i t i s not m e t a b o l l c a l l y r a t e l i m i t i n g . evidence ( l , 2 ) of g e n e t i c a l l y t r a n s m i t t e d  l e d to the  turnover  concept  However, there i s isomerase d e f i c i e n c i e s  l n humans which l e a d s to 5 - 2 0 $ the normal l e v e l s and  can  result  2.  i n a severe metabolic b l o c k , even though the a n t i c i p a t e d l e v e l s of TIM would suggest The  that i t s t i l l  should not be r a t e l i m i t i n g .  lsomerase has been shown by v a r i o u s groups to be composed  of s e v e r a l isozymes (3)»  A few examples ( 6 ) o f the TIM isozymic  content o f v a r i o u s s p e c i e s a r e : Species Human Erythrocytes S k e l e t a l muscle Brain Liver C a r d i a c muscle Spleen Rhesus Bovine Porcine Dove Turtle Prog Catfish Muscle Liver Kidney Brain Heart Crab Lobster Shrimp Beetle Cricket Grasshopper Clam Snail Squid A s c a r l s suum Sea Anemonae Euglena g r a c i l i s Escherichia c o i i Bacillus s u l l t l l i s pseudomanas a e r u g i n o s a Staphylococcusaureus  Number o f Isozymes 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2  1 1 1 1 1 1 1 1 1 1 1 1 1  3. 1*1  P u r i f i c a t i o n and C h a r a c t e r i z a t i o n In r e c e n t years I n t e r e s t has centered on the TIM o b t a i n e d  from r a b b i t muscle and c h i c k e n muscle.  Preparation  procedures  f o r the r a b b i t muscle enzyme have been d e s c r i b e d (9»10) as w e l l as f o r the c h i c k e n TIM ( l i ) .  C o n s i d e r a b l e evidence  has been p r e -  sented to show t h a t the n a t i v e enzyme i s composed o f two s u b u n i t s (9-16). TIM  V a l u e s f o r the n a t i v e dimer molecular weight of r a b b i t  o b t a i n e d p r i o r to 1970, range from 4300u-6uuuu (summarized by  Norton et a l (3)) although l a t e r molecular weights were c a l c u l a t e d to be approximately  53*000 (3,11).  Chicken muscle TIM was  found  to be 48,400 (11) but more r e c e n t amino a c i d sequence and x-ray; c r y s t a l l o g r a p h i c r e s u l t s p l a c e i t nearer t o t h a t o f the r a b b i t muscle w i t h a molecular weight o f 54,400 (17,18).  I t has been  demonstrated t h a t there a r e no d i s u l f i d e b r i d g e s l i n k i n g the two s u b u n i t s , or w i t h i n a s i n g l e subunit "(5). The  h e t e r o g e n e i t y o f t r i o s e phosphate isomerase  r e p o r t e d f o r a wide v a r i e t y o f animal (3*4).  •  Suggestions  has been  (19,20) and human t i s s u e s  f o r the m u l t i p l e nature o f the enzyme have  i n c l u d e d i t b e i n g the r e s u l t of an a r t e f a c t o f the i s o l a t i o n cedure (23)> the presence  pro-  o f c o n f o r m a t i o n a l isoenzymes (13) or  the e x i s t e n c e o f n o n - I d e n t i c a l subunits (5,24).  The isozymes t o  date have been i d e n t i f i e d through p o l y a c r y l a m i d e  (5»25) and  s t a r c h g e l e l e c t r o p h o r e s i s (5»20). In a d d i t i o n , a chromatographic  s e p a r a t i o n f o r the isozymes  o f r a b b i t muscle TIM has been r e p o r t e d by K r l e t s c h et a l  (16).  A rechromatography of p u r i f i e d r a b b i t muscle TIM by a D E A E - c e l l u l o s e column e l u t e d by a v e r y shallow g r a d i e n t succeeded i n sep-  a r a t i n g the m a t e r i a l i n t o t h r e e major forms? ^ , p,. and  two  minor components, d and £  showed the d and t i v e l y and  ,(3%)*  (97%)  and  Hybridization studies  Isozymes to he homodimers AA and BB r e s p e c t -  § the heterodimer  t h e r e f o r e found  AB.  The  h e t e r o g e n e i t y of r a b b i t  TIM  was  two  c h e m i c a l l y n o n - i d e n t i c a l s u b u n i t s A and B.  the 6 and £ forms was  to be l a r g e l y due  to the combination  of  H y b r i d i z a t i o n of  more complex w i t h r e a s s o c i a t l o n r e s u l t i n g  in  the f o r m a t i o n of a l l f i v e bands as w e l l as two  of  £ ) or f o u r ( i n the case of £ ) a d d i t i o n a l more e l e c t r o p h o r -  e t i c a l l y mobile bands.  ( l n the  case  Amino a c i d a n a l y s e s of the three major .  isozymes were a b l e to r e v e a l s m a l l d i f f e r e n c e s but the t i o n of t h i o l s w i t h 5 , 5 - d i t h i o b l s - ( 2 - n i t r o b e n z o a t e ) ,  titra-  (DTNB)  gave i d e n t i c a l numbers f c r the number of s u l f h y d r y l ( c y s t e i n e ) residues. A degree of ambiguity in  concerning TIM  the l i t e r a t u r e f o r some time.  separated commercial r a b b i t TIM  isozymes has e x i s t e d  For example Lee and  Snyder  i n t o f i v e bands i n acrylamide  e l e c t r o p h o r e s i s (26) w i t h a p a t t e r n of r e l a t i v e a c t i v i t i e s p r o t e i n c o n c e n t r a t i o n s of 7(d)i 90$  of the a c t i v i t y and p r o t e i n l n the f i r s t  JC; however Burton and Waley (23) bands w i t h 85$ in  15(p)s 6 ( 5 ) j 2(S)t  the second (p)  with  3 bands o<, p , • and  found only the t h r e e ^, j3 and  a c t i v i t y l n the slower m i g r a t i n g <A band and and  third  Coulson and Knowles (25) polyacrylamide  1(£)  and  (t)  15$  f a s t e r m i g r a t i n g forms, but  c o u l d d e t e c t j u s t one  sharp zone i n the  e l e c t r o p h o r e s i s of commercial enzyme.  The  i m e n t a l c o n d i t i o n s of e l e c t r o p h o r e s i s , most n o t a b l y pH,  exper-  were  p r o b a b l y the  c o n t r o l l i n g f a c t o r s since Krietsch  (16)  observed  that i n polyacrylamide electrophoresis with a 1 g e l  at pH  i n the presence of mercaptoethanol TIM  single  band, w h i l e a t pH  6.6  the p r o t e i n showed the same  p a t t e r n as i n s t a r c h g e l Scopes (20)  migrates as one  9*5  heterogeneity  electrophoresis.  observed a s i m i l a r phenomenon w i t h chicken  mus-  c l e TIM,  namely a s i n g l e band r e s u l t e d upon a c r y l a m i d e e l e c t r o -  phoresis  (pH  separate out  8.5)  w h i l e a minor contaminating band was  on s t a r c h g e l e l e c t r o p h o r e s i s .  These r e s u l t s have  encouraged the b e l i e f t h a t the p r o t e i n from chicken f r e e from Isozymes.  Thus, much of the most r e c e n t  notably  x-ray c r y s t a l s t r u c t u r e s t u d i e s , has  chicken  muscle enzyme. The  observations  reported  seen to  is relatively work, most  c e n t e r e d on  the  i m p l i c a t i o n s of t h i s i n l i g h t of  i n t h i s t h e s i s , w i l l be d i s c u s s e d  in  the the  main body of t h i s work. .A t h i r d source of TIM has been c h a r a c t e r i z e d  f o r which the enzyme  i s human e r y t h r o c y t e s  have been found, to e x i s t f o r the human TIM AB  and  BB  d i s t r i b u t i o n of dlmers.  (6).  heterogeneity Three isozymes  as a r e s u l t of an  Amino a c i d a n a l y s e s and  t i d e f i n g e r p r i n t s have been a b l e  to i n d i c a t e t h a t the two  of s u b u n i t s are very s i m i l a r but  contain  t h e i r primary s t r u c t u r e s . and 1.2  AA,  peptypes  several differences i n  They show s i m i l a r c a t a l y t i c  properties  are found In a l l human t i s s u e s . Enzymatic I n h i b i t i o n and Characterization  chemical m o d i f i c a t i o n  the C a t a l y t i c Process of  of an enzyme's a c t i v e s i t e may  TIM  be a i d e d  by  i f the reagent's s p e c i f i c i t y a l l o w s i n t e r -  6.  p r e t a t i o n w i t h r e s p e c t to the r e a c t i o n s p e c i f i c i t y m o d i f i e d ) and t o p o g r a p h i c a l s p e c i f i c i t y ; t i o n s p e c i f i c i t y may  (amino a c i d  The problem of r e a c -  be s o l v e d by t a k i n g advantage  of known r e -  a c t i o n types w h i l e the problem of t o p o g r a p h i c a l s p e c i f i c i t y  may  be r e s o l v e d by the procedure of " a f f i n i t y l a b e l i n g * i n which a p r o t e i n reagent i s designed to resemble  the s u b s t r a t e and  have an a f f i n i t y f o r the s u b s t r a t e b i n d i n g s i t e . t i o n of t r i o s e phosphate phosphates  and.epoxides  The  thus  inactiva-  lsomerase has been a c h i e v e d by  haloacetal  ( g l y c i d o l phosphate) (25,27) which are  r e a c t i v e analogs of the s u b s t r a t e dihydroxyacetone phosphate have been proven to be a s u c c e s s f u l example of a f f i n i t y  and  labeling.  A h i g h l y s e l e c t i v e m o d i f i c a t i o n of o n l y one r e s i d u e was served w i t h l o s s of a c t i v i t y p r o c e e d i n g pseudo f i r s t a d d i t i o n of h i g h reagent to enzyme molar r a t i o s .  ob-  o r d e r upon  I t has been  found t h a t ^ - g l y c e r o p h o s p h a t e , a c o m p e t i t i v e i n h i b i t o r of  TIM,  p r o t e c t s the enzyme a g a i n s t i n a c t i v a t i o n which suggests a competi t i o n of h a l o a c e t o l phosphates active  and glycerophosphate f o r the same  site.  The i n a c t i v a t e d enzyme was  found to c o n t a i n  1 mole of co-  v a l e n t l y bound reagent per mole of c a t a l y t i c subunit (15)» f o r e i t was  p o s s i b l e f o r the f i r s t  time t o determine w i t h some  c e r t a i n t y the presence of 2 a c t i v e s i t e s  (one/monomer) f o r TIM.  The I n i t i a l m o d i f i c a t i o n of the p e p t i d e chains was proceed v i a an e s t e r i f l c a t l o n of g l u t a m i c a c i d ; c o n t a i n i n g the h a l o a c e t a l phosphate  There-  A  found to  hexapeptlde  has been i s o l a t e d from both  c h i c k e n (28) and r a b b i t muscle (52) w i t h the Ala-Tyr-Glu*-Pro-Val-Trp  sequence:  7.  TIM  from a wide v a r i e t y o f s p e c i e s has been found t o be i n a c t i -  vated by h a l o a c e t o l phosphates, which I n c r e a s e s the l i k e l i h o o d t h a t a common e s s e n t i a l r e g i o n i s the s i t e of m o d i f i c a t i o n . the g l u t a m y l c a r b o x y l a t e  If  i s f u n c t i o n a l i n c a t a l y s i s , i t may be  expected t o be an I n v a r i a n t f e a t u r e among t r i o s e phosphate i s o merases. served  The above mentioned hexapeptlde was found t o be con-  during evolution  to f u n c t i o n .  (30) and i s t h e r e f o r e probably  critical  A comparison o f yeast and r a b b i t muscle TIM was  chosen as an i n d i c a t i o n o f t h e constancy o f the a c t i v e s i t e g l u tamyl r e s i d u e  (and adjacent  a wide e v o l u t i o n a r y  amino a c i d sequence) s i n c e there i s  s e p a r a t i o n between the two organisms.  hexapeptlde c o n t a i n i n g the a c t i v e s i t e glutamic be  i d e n t i c a l i n both The  The  a c i d was found t o  species.  problem e x i s t s as t o whether the enzymatic i n a c t i v a t i o n  r e s u l t s from m o d i f y i n g a c a t a l y t i c a l l y f u n c t i o n a l r e s i d u e or merely from p r e v e n t i n g servations catalysis.  substrate binding.  However, s e v e r a l ob-  suggest that the g l u t a m y l r e s i d u e  i s functional i n  F o r example, the o n l y f u n c t i o n a l group i n model com-  pounds f o r p r o t e i n s which r e a c t s w i t h h a l o a c e t o l phosphates i s the s u l f h y d r y l f u n c t i o n a l i t y (27)» but the group i n TIM t h a t i s a carboxyl. if  The r a t e o f e s t e r i f i c a t i o n i s r a p i d .  reacts  In a d d i t i o n ,  the e s t e r i f i e d glutamyl r e s i d u e o f r a b b i t TIM i s not c a t a l y t -  i c a l l y e s s e n t i a l b u t merely l o c a t e d l n t h e a r e a o f the a c t i v e s i t e , i t would be expected t h a t i n some s p e c i e s , r e s i d u e s not s u s c e p t i b l e t o e s t e r i f i c a t i o n would occupy the c o r r e s p o n d i n g position.  However, the f i n d i n g s t h a t TIM from e v o l u t i o n a r l l y d i -  fc$.  v e r s e s p e c i e s a r e i n a c t i v a t e d by h a l o a c e t o l phosphates  with i n -  a c t i v a t i o n p r o c e e d i n g a t s i m i l a r r a t e s , i s c o n s i s t e n t w i t h modi f i c a t i o n of a c a t a l y t i c a l l y functional, invariant residue. The p o s s i b i l i t y o f the c a t a l y t i c a l l y important r e s i d u e bei n g a glutamate  f i t t e d w e l l w i t h p r e v i o u s mechanism which were  p o s t u l a t e d as t h e chemical pathway f o r t h e a l d o l - k e t o isomerism. The mechanism which was f i r s t f o r m u l a t e d by Rose {31,3'd)  involved  the a b s t r a c t i o n o f a proton from G-3 o f dihydroxyacetone  phosphate  to g i v e an e n e d i o l a t e anion which i s a b l e t o p i c k up a proton a t C-2  to y i e l d glyceraldehyde-3 H H |,0H" H  C-=0  • 0® r, CH 2  Any  phosphates r. *'  H  B-H^j  H  0 H  C—0"  "  CH 0 ^ (?) 1  2  N  ^0  H — C —OH •  I ^ CH 0 Q) 2  conjugate base, i n c l u d i n g a glutamyl ^ - c a r b o x y l a t e , c o u l d  f u n c t i o n i n t h i s proton t r a n s f e r .  I t has been e s t a b l i s h e d t h a t  c a r b o x y l a t e s can promote e n o l i z a t i o n v i a g e n e r a l base  catalysis,  s i m i l a r t o the mechanism above ( 3 3 ) * Prom t h e pH dependence of the r a t e o f I n a c t i v a t i o n o f yeast TIM, the apparent pKa o f the a c t i v e - s l t e c a r b o x y l group was estimated by Hartman t o be 3*9-0.1 (34). A f f i n i t y l a b e l i n g o f TIM was c a r r i e d out f u r t h e r by the prepa r a t i o n o f a reagent designed to mimic the c i s - e n e d i o l which i s the p o s t u l a t e d Intermediate I n the r e a c t i o n c a t a l y z e d by t r i o s e phosphate isomerase.  T h i s reagent i s g l y c i d o l phosphate ( 2 , 3 -  9. epoxypropanol)  which was  found to l a b e l the g l u t a m y l group l n  the I s o l a t e d hexapeptldet A l a - T y r - G l u * - P r o - V a l - T r p ( 3 5 ) . T h i s i s the same r e s i d u e t h a t was The  l a b e l e d by the h a l o a c e t o l phosphates.  l a b e l i n g of TIM by t h i s reagent s t r o n g l y supports the  g e n e r a l base mechanism as proposed  f o r the a l d o s e - k e t o s e  single isomer-  i z a t i o n of TIM ( 3 6 ) . The most powerful c o m p e t i t i v e i n h i b i t o r of TIM  i s 2-phos-  p h o g l y c o l l a t e whose s t r u c t u r e i s g i v e n below:  0 II  y°\ I  0  The  i n h i b i t o r s s t r u c t u r a l s i m i l a r i t y to the e n e - d i o l and i t s  a n i o n i c c e n t r e i s important f o r t r a n s i t i o n s t a t e b i n d i n g . ultraviolet  spectrum of TIM  i n the 280nm r e g i o n may  be  changed  by b i n d i n g of 2 - p h o s p h o g l y c o l l a t e and 2-glycerophosphate reagent which i n h i b i t s due  The  (another  t o i t s a b i l i t y t o b i n d t o the a c t i v e  s i t e ) but not by the a n i o n i c i n h i b i t o r s s u l f a t e or i n o r g a n i c phosphate ( 3 7 ) . The  s h i f t , which was  •'region which corresponded was  most marked i n the 280nm  to t y r o s i n e and tryptophan absoiXbtlons  r e l a t e d to the presence of each of these amino a c i d s i n the  hexapeptlde which was  found to be e s t e r i f i e d upon i n a c t i v a t i o n  of TIM by g l y c l d o l phosphate ( 3 7 ) . In a d d i t i o n to the change i n the U.V.  spectrum,  there was  observed t o be a change i n the  crys-  t a l s t r u c t u r e of the enzyme upon b i n d i n g of g l y c l d o l phosphate  10.  which was  a b l e to suggest  t h a t t h e r e are two  or more  s i t e s of b i n d i n g . . The b i n d i n g of the i n h i b i t o r and s t a t e analogue was  2-phosphoglycollate  important transition  to the r a b b i t muscle enzyme  a b l e to e f f e c t a s i m i l a r s o r t of change as observed  h% c o n t r a c t i o n i n the c r y s t a l s t r u c t u r e .  by  the  At the same time,  &$)  6% c o n t r a c t i o n has been noted f o r b i n d i n g of e i t h e r t r i o s e subs t r a t e to r a b b i t or chicken muscle TIM  (37,38).  I t appears  l y then t h a t the magnitude of the change upon b i n d i n g of i t o r , r e p r e s e n t s a s t r u c t u r a l change i n the p r o t e i n  like-  inhib-  conformation  which mimics the c o n f o r m a t i o n a l change which occurs upon b i n d i n g of  the two  s u b s t r a t e s dlhydroxyacetone  hyde 3-phosphate. conformation  The  phosphate and g l y c e r a l d e -  c a t a l y t i c r o l e of t h i s  change i s unknown and  substrate-induced  our understanding  of  this  phenomonen i s a l o n g term g o a l of t h i s l a b o r a t o r y . The  dependence of the r a t e of enzyme-catalyzed  pH i s an important  r e a c t i o n s on  parameter t h a t must be accommodated by any  p l e t e p r o p o s a l of mechanism.  There Is fundamental i n f o r m a t i o n to  be gained from a knowledge of the pH-dependence of the k i n e t i c parameters.  com-  They must be c o n s i s t e n t w i t h the  individual mechanistic  p r o p o s a l s and be i n t e r p r e t a b l e w i t h r e s p e c t to the knowledge of the t h r e e - d i m e n s i o n a l  s t r u c t u r e of the enzyme and the  functional  catalytic residues. The  most comprehensive s e r i e s of pH r a t e s t u d i e s were per-  formed by p l a u t and Knowles (39). was  One  the d e t e r m i n a t i o n of the s t a b i l i t y  zyme as a f u n c t i o n of pH.  of the most u s e f u l s t u d i e s of the chicken muscle  I n c u b a t i o n of enzyme i n b u f f e r s of  en-  11  v a r i o u s pH's  was  c a r r i e d out a t 3 8 ° C f o r 6 hours b e f o r e  a t i o n of the enzymatic  activity.  The r e s u l t s showed a p l a t e a u  ( 1 0 0 $ a c t i v i t y ) between pH 6.5 and  7.5 w i t h a f a i r l y r a p i d  crease i n s t a b i l i t y a t lower or h i g h e r pH's. c o n t r a s t to t h i s was p h i l i c organism  determin-  observed i n the TIM  An  interesting  I s o l a t e d from a p s y c h r o -  (optimum growth below 2 0 ° C ) , C l o s t r i d i a ,  a 70% r e d u c t i o n In enzyme a c t i v i t y w i t h a h a l f hour heat ment of c e l l - f r e e e x t r a c t s a t 32*C was  de-  seen  in'which treat-  (pH 7 - 3 ) ( 4 0 ) .  This  can p r o b a b l y be e x p l a i n e d by the e v o l u t i o n a r y a d a p t a t i o n of t h i s microorganism. Other pH s t u d i e s  showed a pH dependence of k„  ./K  CeLu  which DO.  a l l o w e d c a l c u l a t i o n of the pKa o f two k l n e t i c a l l y important t i o n a l i t i e s : v a l u e s o f 6 . 0 5 and  func-  9*05 were d e r i v e d w i t h d i h y d r o x y -  acetone phosphate (DHAP) as s u b s t r a t e and 6.0 and 9.2 when g l y c eraldehyde 3-phosphate (G3P) in k  C a t  /K  m  may  was  used.  The apparent pKa  values  r e l a t e to i o n i z a t i o n s i n the f r e e enzyme or i n the-  free substrate.  Under the e x p e r i m e n t a l c o n d i t i o n s , the pKa  ues of s u b s t r a t e alone a r e 6.0 f o r DHAP and  6.3 f o r G 3 P .  val-  There-  f o r e , t h e r e i s a p o s s i b i l i t y t h a t the lower observed pKa*s of the k ,/K p r o f i l e s have a r i s e n from i o n i z a t i o n of s u b s t r a t e s , cat m The upper pKa  v a l u e o f about  9 cannot be r e a d i l y a s s i g n e d but  p o s s i b i l i t i e s a r e l y s i n e or a r g l n i n e r e s i d u e s . t h e r e i s the p o s s i b i l i t y  On the other hand,  t h a t the i o n i z a t i o n a t pH 9 governs  a  l a r g e - s c a l e conformation change r e s u l t i n g In l o s s o f enzyme a c t i v i t y owing to a l o s s o f the s t r u c t u r a l i n t e g r i t y or the a c t i v e site ( 3 9 ) .  12  1.3  S t r u c t u r a l Aspects A.  The  Subunit Monomer  When s t u d y i n g  the r e l a t i o n s h i p between the q u a t e r n a r y s t r u c -  t u r e of a m u l t i - s u b u n i t  enzyme and  its activity,  i t i s o f t e n of  i n t e r e s t to know the enzymatic a c t i v i t y of the monomer, i f i n deed a s s o c i a t i o n of the s u b u n l t s ability.  i s not necessary f o r c a t a l y t i c  A u s e f u l approach i s to a t t a c h the enzyme to a  Support v i a a s i n g l e subunit, and  solid  to remove the o t h e r ( s ) so  the p r o p e r t i e s of the i s o l a t e d subunit may  be  that  s t u d i e d under con-  d i t i o n s where r e a s s o c l a t i o n i s not p o s s i b l e . Such a procedure was (43).  performed r e c e n t l y by F e l l and  White  They found t h a t the k i n e t i c p r o p e r t i e s of chicken TIM  a l t e r e d by resented  immobilization  on Sepharose.  The  t h a t of the bound monomer gave a K  of the s o l u b l e enzyme was  r e p o r t e d to be  i n g w i t h guanidine h y d r o c h l o r i d e  and  m  a c t i v i t y , which o f 1.7mM while  0.35^M.  o n s t r a t e t h a t the 3 tween these two  2  d i f f e r e n c e s i n amino amino a c i d sequence  Interface region  not  i n such experiments;  one  spacer  subunit was  arm  be-  significantly a l -  (44). evidence  For example, a c o n t r a s t i n g r e p o r t  o f the p o s s i b i l i t y of d i r e c t l y l i n k i n g TIM ( l e . no  rabbit  I t i s t h e r e f o r e p o s s i b l e to dem-  I t i s Important to e x e r c i s e c a u t i o n when e v a l u a t i n g obtained  that  subsequent r e h y b r i d i z a t i o n  enzymes (86$ homology) has  t e r e d the subunit  rep-  Upon denatur-  w i t h r a b b i t muscle enzyme, an a c t i v e h y b r i d of chicken and muscle enzyme c o u l d be formed.  were  to an agarose  between enzyme and agarose was  support  inserted) v i a  r e p o r t e d by Sawyer and Gracy (45) when they  ob-  13.  served and  t h a t both subunits  o f human TIM were l i n k e d t o t h e matrix  t h a t t h e double l i n k a g e was p r e v e n t i n g  dissociation.  t r a s t , t h e p r o t e i n bound t o t h e agarose v i a an  I n con-  acetamidoethyl  l i n k a g e had k i n e t i c as w e l l as s t a b i l i t y p r o p e r t i e s c l o s e r t o the n a t i v e enzyme.  This data challenges  the v a l i d i t y o f the ex-  periments of P e l l and White who used d i r e c t l i n k a g e of the p r o t e i n to the support. The  p o s s i b i l i t y t h a t t h e monomer o f TIM i s i n a c t i v e has been  suggested by Waley (46) who performed experiments i n which r a b b i t or c h i c k e n muscle TIM was denatured by g u a n i d i n i i u m  hydrochloride  and  H i s scheme  then f o l l o w e d the k i n e t i c s o f the r e n a t u r a t i o n .  i n v o l v e d f i r s t o f a l l the r e f o l d i n g o f monomer and then the a s s o c i a t i o n o f t h e two f o l d e d monomers t o form a dimer. concentrations  d i m e r i z a t i o n was a r a t e - d e t e r m i n i n g  step  found t o be second order a t low enzyme c o n c e n t r a t i o n s )  A t low (kinetics and s i n c e  the reappearance o f dlmers was f o l l o w e d by the i n c r e a s e i n a c t ivity,  t h i s was taken t o i n d i c a t e t h a t t h e monomers showed  or no a c t i v i t y .  I f t h i s indeed  i s t r u e , i t would mean t h a t t h e  a c t i v e s i t e enzyme conformation induced is  by t h e subunit a s s o c i a t i o n  e s s e n t i a l f o r the c a t a l y t i c a c t i v i t y o f TIM. It  any  little  i s c l e a r t h e r e f o r e t h a t t h i s i s s u e i s s t i l l u n r e s o l v e d and  probe o f the i n t e r - s u b u n i t I n t e r f a c e when r e l a t e d t o enzymatic  a c t i v i t y w i l l be o f c o n s i d e r a b l e B.  value.  Protein Modifications  There e x i s t s very l i t t l e amino a c i d r e s i d u e s , other  i n f o r m a t i o n about the p a r t i c u l a r  than the c a t a l y t i c s i t e hexapeptide,  14.  which are most I n t i m a t e l y therefore studies  I n v o l v e d i n the TIM  e s s e n t i a l to enzymatic c a t a l y s i s .  have i n v e s t i g a t e d  there are  the  c o n f l i c t i n g reports  r o l e of the i n the  active site  However, r e c e n t  s u l f h y d r y l groups  l i t e r a t u r e concerning  e f f e c t of t h i o l - s p e c i f i c reagent on enzymatic a c t i v i t y One was  and  and the  (23,29).  o f the more comprehensive surveys of t h i o l r e a c t i v i t y  p r e s e n t e d by D a v i s et a l (47).  Investigation of O  SH r e a c t i v i t y  by r e a c t i o n w i t h malelmldes r e s u l t e d i n a t h r e e f o l d g r e a t e r a e l i s constant f o r the m o d i f i e d r a b b i t enzyme. mole enzyme r e a c t e d . a greater  Three  thiols/  However, m e r c u r i a l s were found to  change l n K  m  and  Mich-  cause  w i t h 6 SH's/mole r a b b i t  reacted  en-  zyme. I t was  r e p o r t e d t h a t the r a b b i t muscle and  pear to have s i m i l a r p r o p e r t i e s i s less reactive  (47).  The  but  yeast  l i v e r enzyme  ap-  t h a t the c h i c k e n muscle enzyme  enzyme does not become i n a c t -  i v a t e d upon s u l f h y d r y l m o d i f i c a t i o n .  The  enzyme from c h i c k e n  been found (48)  one  t h i o l group per  muscle has that  to c o n t a i n  i s more r e a c t i v e than the o t h e r s to the reagent  b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB). c e s s i b l e t h i o l group was of the  enzyme (48)  ed i n t h i s t h e s i s .  I t was  t h i o l per  DTNB w i t h a 5 0 $ l o s s of a c t i v i t y and  to r e s u l t s  i s inaccessible  report-  been suggested t h a t f o r dimer can be m o d i f i e d  the by  t h a t a second e s s e n t i a l  group which corresponds to the o t h e r a c t i v e s i t e i s s t i l l t i o n a l l y i n t a c t but  ac-  catalytic activity  which i s i n d i r e c t c o n t r a s t  r a b b i t muscle p r o t e i n , one  5»5"-dlthlo-  observed t h a t the  unnecessary to the  However, i t has  subunit  SH  frac-  through the a s s o c i a t i o n  of  15.  the two  s u b u n i t s (16),  However, t h e r e i s a r e a l d e f i c i e n c y of  knowledge r e g a r d i n g the r e a c t i v i t y of the  t h i o l s of TIM  and  i t is  i n t h i s a r e a t h a t the r e s u l t s of t h i s t h e s i s attempt to expand "the knowledge of the m o d i f i e d p r o t e i n . C.  Amino A c i d Sequence and X-Bay C r y s t a l S t r u c t u r e of  TIM  The amino a c i d sequence of the r a b b i t muscle enzyme f i r s t r e p o r t e d by Corran and Waley (4-9) who chain had 248 amino a c i d r e s i d u e s and of the dlmer was c l e TIM was  53s257.  The  found  weight  t r y p t i c p e p t i d e s of the r a b b i t mus-  the c h i c k e n has 24-7 amino a c i d r e s i d u e s and  {86% homology).  the p o l y p e p t i d e  t h a t the molecular  compared w i t h t h a t of c h i c k e n ( 5 1 ) .  i n each c h a i n .  was  Each c h a i n of  t h e r e i s one  deletion  Apart from these gaps, t h e r e are 3 2 d i f f e r e n c e s 22 of the 32 i n t e r c h a n g e s are c o n s i s t e n t w i t h  a change of one n u c l e o t i d e i n the DNA  codon.  In a d d i t i o n , the  amino a c i d sequence of the 1 5 r e s i d u e t r y p t i c p e p t i d e t h a t cont a i n s the a c t i v e - s i t e g l u t a m y l r e s i d u e as determined and Waley ( 5 3 ) was  by  Corran  found to be d i f f e r e n t than the analogous r a b -  b i t p e p t i d e r e p o r t e d by Hartman ( 5 2 ) i n t h a t v a l i n e i n c h i c k e n was  s u b s t i t u t e d f o r tryptophan  in rabbit.  The amino a c i d sequence data f o r the c h i c k e n b r e a s t muscle was  published i n 1975 ( 5 4 ) .  The  sequence d a t a f a c i l i t a t e d  the  d e t e r m i n a t i o n of the 2.5 A r e s o l u t i o n c r y s t a l s t r u c t u r e which r e p o r t e d i n the same paper. the c h i c k e n muscle TIM  I t was  found  was  t h a t each subunit of  i s composed of a l t e r n a t e segments of p o l y -  p e p t i d e c h a i n i n the <*.- and  ^-conformations  t h a t a r e arranged  form an i n n e r c y l i n d e r of p a r a l l e l - p l e a t e d sheet and a l a r g e l y  to  16  h e l i c a l outer s h e l l .  They were a l s o a b l e  dues p a r t i c i p a t i n g i n the subunit making up the a c t i v e s i t e . two  fold axis.  t o i n d i c a t e the r e s i -  I n t e r f a c e as w e l l as those  The two s u b u n i t s  were r e l a t e d by a  I n t e r a c t i o n between the s u b u n i t s  involves, i n  p a r t , the l o o p s 70-80 which form hydrophobic pockets around the Met  14 r e s i d u e  from a d j a c e n t s u b u n i t s .  edges o f the I n t e r f a c e . of r e s i d u e s  The pockets l i e on the  The a c t i v e s i t e s o f t h e p r o t e i n  from both s u b u n i t s  consists  a s has been found f o r another  g l y c o l y t i c enzyme G3PD (89)« T h i s f i n d i n g i s p a r t i c u l a r l y i n j  t e r e s t i n g i n l i g h t of the p r e v i o u s l y mentioned o b s e r v a t i o n  that  the monomer o f TIM i s i n a c t i v e . The larly has  x-ray s t r u c t u r e as r e p o r t e d  by Banner e t a l i s p a r t i c u -  i n t e r e s t i n g l n l i g h t o f the s t r i k i n g resemblance which i t  t o other g l y c o l y t i c enzymes which a r e composed l a r g e l y o f  a l t e r n a t i n g segments o f d - and ^ - s t r u c t u r e which a r e f o l d e d simllarlly.  There i s the s u g g e s t i o n by Rao and Rossman t h a t s i m i l a r  super-secondary s t r u c t u r e s may be found l n many p r o t e i n molecules w i t h w i d e l y d i f f e r e n t amino a c i d sequence as a r e s u l t o f convergent  e v o l u t i o n -(55)• The  gap between c r y s t a l l o g r a p h y  p a r t a t l e a s t , be b r i d g e d of techniques such as NMR. being able  and k i n e t i c s can o f t e n , i n  by s p e c t r o s c o p i c  s t u d i e s which make use  NMR has the p a r t i c u l a r advantage o f  t o observe i n d i v i d u a l r e s i d u e s ; a l s o changes i n con-  f o r m a t i o n can o f t e n be observed and c h a r a c t e r i z e d  in detail.  A paper published, by Browne e t a l i n 1976 (56) has made cons i d e r a b l e p r o g r e s s towards a s s i g n i n g r a b b i t and chicken  the h i s t i d i n e resonances i n  TIM t o i n d i v i d u a l r e s i d u e s  and towards c h a r -  17.  a c t e r i s i n g the change i n conformation work, which was  when l i g a n d s b i n d .  made p o s s i b l e through knowledge of the  This  x-ray  s t r u c t u r e of Banner et a l (75) > weus a b l e to observe the  proton  resonances of f i v e h i s t i d i n e s i n the c h i c k e n muscle enzyme and one  h l s t l d i n e i n the r a b b i t enzyme which were observed  to  titrate  i n the pH 5 « 4 t o 9 range. The  extreme s e n s i t i v i t y of NMR  techniques  as observed  by  19 Browne e t a l ( 5 6 ) i s supported iments presented  by the p r e l i m i n a r y P  in this thesis.  We  NMR  exper-  have been a b l e to c o l l a b o r -  a t e the a b i l i t y o f magnetic resonance to d e t e c t changes i n the 19 environment of the observed  nucleus  'F when the two  most r e a c -  t i v e c y s t e i n e s of c h i c k e n TIM were m o d i f i e d by a t r i f l u r o maleimlde l a b e l  (FEM)  and  of the a d d i t i o n product  N-ethyl-  compared to a model compound c o n s i s t i n g  of FEM  and N - a c e t y l c y s t e i n .  Clues to  the p o s i t i o n of the s i t e of m o d i f i c a t i o n of the c h i c k e n TIM FEM  are g i v e n by examination  by  of the c r y s t a l s t r u c t u r e of Banner  et a l ( 5 5 ) . The x-ray  s t r u c t u r e p l a c e s the two  most r e a c t i v e c y s t e i n e s ,  to 2 - c h l o r m e r c u r i - 4 n l t r o p h e n o l , a t r e s i d u e 2 1 7 which would t h e r e f o r e be a l i k e l y s i t e f o r the FEM  m o d i f i c a t i o n of TIM.  Cys  i s s i t u a t e d on the o u t e r s u r f a c e o f the enzyme as p a r t of mainly  the  h e l i c a l segments of p o l y p e p t i d e c h a i n which form the  i n d r i c a l outer s u r f a c e of each s u b u n i t .  217  cyl-  I t i s 5 amino a c i d r e s -  idues away from the a c t i v e s i t e V a l 2 1 2 and  thus i s about  complete t u r n s of the d - h e l l x away from the a c t i v e s i t e .  1.4 There-  f o r e Cys 2 1 7 i s not i n the i n t e r s u b u n l t a r e a of c o n t a c t nor i n  18.  the immediately adjacent  active s i t e area.  However, Cys  c l o s e enough to the a c t i v e s i t e t h a t i t i s p o s s i b l e to  217  envision  the p o s s i b i l i t y of i t s chemical m o d i f i c a t i o n a f f e c t i n g the a l y t i c a b i l i t y of the a c t i v e s i t e i f a c o n f o r m a t i o n a l curs upon r e a c t i o n of the t h i o l .  is  cat-  change  oc-  However, s i n c e Banner et a l  a l s o observed m o d i f i c a t i o n of a second l e s s r e a d i l y a c c e s s i b l e p a i r of t h i o l s a t Cys  41 when u s i n g  the s m a l l e r and  more hydro-  phobic e t h y l mercury phosphate, i t cannot be assumed w i t h t a i n t y t h a t FEM  m o d i f i c a t i o n of c h i c k e n TIM  proceeds a t Cys  Cys 41 i s l o c a t e d on the s u r f a c e o f the Inner c y l i n d r i c a l sheet s t r u c t u r e which i s not a d j a c e n t i n t e r s u b u n l t contact areas. the a c t u a l FEM concrete FEM  modification.  217.  p-pleated  to e i t h e r a c t i v e s i t e  or  I t i s f a i r l y c l e a r that studies  l a b e l e d p r o t e i n must be  assumptions may  cer-  c a r r i e d out b e f o r e  be made concerning  on  any  the a c t u a l s i t e  of  19. CHAPTER I I ISOLATION AND 2.1  PURIFICATION  Materials The e x t r a c t i n g b u f f e r made use of e t h y l e n e d i a m i n e t e t r a -  a c e t a t e , dlsodlum s a l t pany, c e r t i f i e d ACS Coleman and B e l l . Reagent  (EDTA-2Na ) from F i s h e r S c i e n t i f i c +  grade, and 2-mercaptoethanol  Com-  from Matheson,  B u f f e r s c o n t a i n e d Trisma Base from Sigma,  Grade, and sodium  c h l o r i d e , BDH A n a l a r grade.  Special  Enzyme Grade ( u l t r a pure) ammonium s u l f a t e from Schwarz/Mann was  used i n the p r e c i p i t a t i o n s . DEAE Sephadex A50 from Pharmacia F i n e Chemicals and DE52  and B i o g e l A DEAE from Bio-Rad were the r e s i n s used i n the chromatography experiments. The assay made use o f the d i e t h y l a c e t a l barium s a l t of DLG l y c e r a l d e h y d e 3-Phosphate, g l y c e r o l - 3 phosphate Dowex-50 Hydrogen Form R e s i n , and  dehydrogenase,  nicatinamldeadeninedlnucleo-  t i d e , reduced form (NADH) from the Sigma Chemical Company.  20.  2.2  Methods A.  Extraction and I s o l a t i o n  A t y p i c a l protein preparation  used 5 0 0 gms  ed from the breasts of four chickens.  of muscle dissect  The fresh muscle was  ob-  tained either from a l o c a l butcher shop or from f r e s h l y k i l l e d chickens (Department of Poultry Science, UBC);  A l l procedures  o  were carried out at 4 C or on ice (unless otherwise noted) using a modified procedure of McVittie*s The defatted breast muscle was  (11).  minced i n a Waring blender  for 1 minute with'an extraction buffer which consisted of mercaptoethanol and 1.5mM  ethylenedlamlnetetraacetlc  0.2$  a c i d (to  pH 7.0 at 22°C with NaOH) using 1 ml of cold buffer per gram of muscle.  The  homogenate was  centrifuged i n the GSA  rotor of a  S o r v a l l BC-2B Centrifuge at 1200Xg f o r 4 5 minutes. was  The  pellet  resuspended l n 0.5 ml of extraction buffer per gram of  mu-  scle used, s t i r r e d f o r 3 0 minutes and recentrlfuged as above. The  combined supernatants were f i l t e r e d through cheese c l o t h  three times, and brought to 6 5 $ saturation with s o l i d (NH4) S0^. 2  4 3 0 . 4 grams of  (NEj^SO^ per l i t e r of supernatant ( 6 5 $ ) were  added slowly with gentle s t i r r i n g , a l l the while the buffer being packed i n i c e .  After standing  l e a s t 18 hours, the 6 5 $ (NHi^SO^ saturated fuged at 5 5 0 0 X g f o r 7 5 minutes;  extraction  i n the cold f o r at solution was  The p e l l e t was  discarded  the supernatant brought to 9 0 $ ( N H 4 ) S 0 ^ (182;5 gm/liter) 2  centrland sat-  uration.  Upon standing  f o r about 24 hours, the protein precip-  i t a t e was  spun down and  then dissolved i n a minimum amount of  20mM T r i s j H C l buffer, pH 7.2;  ( A l l buffers were adjusted  to  pH  21.  at  22°C and  then s t o r e d a t 4°C.)  The  s o l u t i o n was  dialyzed  a-  g a i n s t the T r i s b u f f e r w i t h a t l e a s t 8 changes over 3 days.and then a p p l i e d to a DEAE Sephadex A 5 0 column ( 5 X 7 0 cm) i b r a t e d w i t h the d i a l y s i s b u f f e r . k liter  l i n e a r gradient  The  column was  ( i e . 2 1 of low  s a l t and  pre-equll-  eluted with a 2 1 of  high  s a l t b u f f e r ) of 2 0 to HOmM T r i s pH7.2 a t a f l o w r a t e of more than 2 0 ml per  hour.  F r a c t i o n s were c o l l e c t e d on a the absorbance a t 2 8 0 nm  Produkter f r a c t i o n c o l l e c t o r and mined on a Z e i s s PMQ  not  II UV-Visible  a c t i v i t y a s s a y s were performed on  spectrophotometer. center  LKBdeter-  Initially*  p r o t e i n f r a c t i o n s from  A c t i v i t y assays were then performed a t 2 or  each e l u t e d peaki  3 f r a c t i o n i n t e r v a l s across  those p r o t e i n peaks found to have  t r i o s e phosphate isom'erase a c t i v i t y . B.  Rechromatography  The  f i r s t rechromatography experiments were performed w i t h  DEAE Sephadex A 5 0 i (Peak A) was pH  7-2  Initially,  the f i r s t TIM  rechromatographed u s i n g a 3 l i t e r  gradient  than 2 0 ml/hour;  on a 1 1 0 X 2 . 5 cm The  e l u t e d p r o t e i n was  atographed u s i n g a 3 l i t e r cm  The  6 0 to 9 0 mM  as  I s o l a t e d A and  The  5 0 to 70 mM  •B*  TIM  peak was  pH  7.2  T r i s gradient  pre-  rechromon  Treatment  before:'. B peaks from a d i f f e r e n t p r o t e i n p r e p -  a r a t i o n were p o o l e d t o g e t h e r means of a g r a d i e n t  Tris  assayed, p o o l e d and  column, f l o w r a t e l e s s than 2 0 ml/hour.  of e l u t e d p r o t e i n was  column  column w i t h a f l o w r a t e of l e s s  c i p i t a t e d f o r s t o r a g e as b e f o r e .  a 2.5X92  peak o f f the  of 0 to  and  rechromatographed, t h i s time by  35mM NaCl i n 5mM  T r i s pH  7.2  over  22 k l i t e r s on a 2:5X70 cm column, flow r a t e l e s s than 20 ml/hour. The  c o n s i d e r a b l e shrinkage  o f t h e g e l over t h i s s a l t  concentra-  t i o n range n e c e s s i t a t e d the I n t e r r u p t i o n o f the g r a d i e n t p e r i o d i c a l l y , i n o r d e r t o a l l o w the b u f f e r t o go down t o the top o f the bed,  f o l l o w e d by resumption o f the g r a d i e n t ;  The rechromatography o f s t i l l another p r o t e i n p r e p a r a t i o n u t i l i z e d DE52 c e l l u l o s e r e s i n i  The pooled A and B peaks were  r e s e p a r a t e d w i t h a 0-30mM NaCl g r a d i e n t i n 5niM T r i s pH 7.8 over 3»5  l i t e r s , on a 2;5X60 cm column w i t h a flow r a t e o f l e s s than  18 ml/hour;  D E 5 2 c e l l u l o s e had the advantage o f n o t s h r i n k i n g  w i t h an i n c r e a s e i n the s a l t c o n c e n t r a t i o n o f the b u f f e r .  How-  ever, i t s p r o t e i n b i n d i n g c a p a c i t y i s lower than t h e Sephadex anion exchanger;  A f a i r l y high  (7«8) pH was r e q u i r e d t o make  the t r i o s e phosphate isomerase adhere t o t h e column.  P l a u t and  Knowles (39) have demonstrated a decrease i n TIM's s t a b i l i t y i n b u f f e r s o f pH g r e a t e r than pH 7*5 w i t h t h e t r e n d i n c r e a s i n g even more s h a r p l y a f t e r pH 8,0-i With these f a c t o r s i n mind, a t h i r d r e s i n , B i o g e l A DEAE, was  u t i l i z e d i n the rechromatography experiments.  T h i s g e l has  the advantage o f the high c a p a c i t y o f the Sephadex r e s i n b u t does n o t s h r i n k o r expand w i t h s a l t c o n c e n t r a t i o n changes.  The  pH a t which the r e s i n c o u l d be used f o r the rechromatography (7-5)  a l s o makes i t a b e t t e r c h o i c e than the DE52 c e l l u l o s e . The pooled A peak was rechromatographed u s i n g a 3 1. 0 t o  20mM NaCl g r a d i e n t l n 5mM T r i s pH 7.5 on a 2.5X30 cm column, and flow r a t e o f about 18 ml/hour. ohromatographed.  The B peak was s i m l l a r l l y  re-  23.  C.  Assay  The t r l o s e phosphate  Isomerase assay, which was used In the  work d e s c r i b e d i n t h i s t h e s i s , i s based upon a c o u p l e d enzyme system.  A s u b s t r a t e o f t r i o s e phosphate  hyde-3 phosphate  (G3P) i s f i r s t  droxyacetone phosphate reduced by d-glycerol 1-phosphate.  isomeras6,  glyceralde-  converted t o d l h y -  (DHAP) by TIM; DHAP i s next e n z y m a t i c a l l y phosphate  dehydrogenase (GPD) t o g l y c e r o l  The coupled enzyme r e a c t i o n o f GPD proceeds v i a a  n i c o t i n a m i d e adenine d i n u c l e o t i d e coenzyme o x i d a t i o n w i t h concomitant decrease i n A - ^ Q .  (NADH-* NAD ) +  A s i m i l a r assay t o the G3P/GPD  system i s c o n v e r s i o n o f the s u b s t r a t e dihydroxyacetone  phosphate  by TIM t o G3P, f o l l o w e d by the enzymatic o x i d a t i o n o f G3P by the NAD r e q u i r i n g enzyme g l y c e r a l d e h y d e - 3 phosphate (G3PD).  dehydrogenase  The G3PD o x i d a t i o n o f G3P proceeds v i a the r e d u c t i o n o f  4.  NAD;:; t o NADH.  A schematic r e p r e s e n t a t i o n o f the c e n t r a l r e a c -  t i o n s i n the two assays i s as f o l l o w s t  Thus, two assay procedures a r e p o s s i b l e , the f i r s t  b e i n g the  use o f G3P as s u b s t r a t e and GPD as the c o u p l i n g enzyme w i t h the a c t i v i t y o f TIM o b s e r v a b l e by the r a t e o f decrease i n the absorbance a t 340nm as NADH d i s a p p e a r s ; the second uses DHAP as subs t r a t e , G3PD as c o u p l i n g enzyme and the a c t i v i t y o f TIM i s f o l -  24.  lowed by an I n c r e a s e i n the absorbance ( a t 3^0nm) as the coenzyme NADH i s formed.  The s p e c t r a shown below demonstrates t h a t  a t 3^°nm, NADH i s a t i t s maximal absorbance w h i l e NAD interfering.  i s non-  T h e r e f o r e , the convenient wavelength 3^0nm, makes  i t p o s s i b l e to f o l l o w the disappearance o r appearance o f NADH and hence f o l l o w the i n i t i a t o r y t r i o s e phosphate isomerase reaction. FIGURE l l  U l t r a v i o l e t S p e c t r a o f NAD* and NADH  1.0- -  .•-.NAD"  Absorbc n e e  / / • /  0.5+  7  1  v . .  \".  \\ •-^  240  280  320 X  NADH  360  (nm)  In order t o a c c u r a t e l y determine the a c t i v i t y o f TIM, i t i s necessary that the c o u p l i n g system not be r a t e d e t e r m i n i n g , p l a u t and Knowles ( 3 9 ) , i n an e x c e l l e n t s e r i e s o f experiments, demonstrate the c o n d i t i o n s underwhich the o v e r a l l r e a c t i o n was l i m i t e d by the t r i o s e phosphate Isomerase c a t a l y s i s .  In order t o  check the v a l i d i t y o f the assays ( l e . t h a t the. observed r a t e s o f p r o d u c t i o n o r o x i d a t i o n o f NADH r e p r e s e n t s the r a t e o f the TIM r e a c t i o n ) , i t was n e c e s s a r y t o ensure the l i n e a r dependence o f  25. the observed i n i t i a l r a t e on the c o n c e n t r a t i o n of.TIM under the h i g h e s t s u b s t r a t e c o n c e n t r a t i o n s used ( f o r G3P the range o f 0.2l.OmM had been I n v e s t i g a t e d , w h i l e f o r DHAP the range was 0.22.0mM).  T h i s was accomplished by s t u d y i n g the i n i t i a l  f u n c t i o n o f lsomerase c o n c e n t r a t i o n  (at the h i g h e s t  r a t e as a  concentration  o f . s u b s t r a t e t o be u s e d ) . V a r i a t i o n i n the r a t e o f G3P o r DHAP i s o m e r i z a t i o n was s t u d i e d as a f u n c t i o n o f c o u p l i n g enzyme c o n c e n t r a t i o n .  The ex-  periments r u l e d out any p o s s i b i l i t y o f c o m p l i c a t i o n s a r i s i n g from enzyme-enzyme i n t e r a c t i o n s and, s t u d i e s , allowed  w i t h the evidence  o f the o t h e r  one to determine * s a f e * c o n c e n t r a t i o n s  species i n s o l u t i o n .  of a l l  I t was t h e r e f o r e p o s s i b l e t o e s t a b l i s h the  v a l i d i t y o f the assay f o r the c o n d i t i o n s used i n t h i s a c c o u n t . The  G3P/GPD system was used e x c l u s i v e l y i n the work presented i n  this  thesis.  Procedure s The  s u b s t r a t e , G3P, was prepared  by the h y d r o l y s i s o f the  d i e t h y l a c e t a l barium s a l t o f DL-glyceraldehyde 3-phosphate. u f a c t u r e r ' s p r e p a r a t i v e i n s t r u c t i o n s were f o l l o w e d * drogen Form R e s i n barium s a l t  Man-  Dowex 50 Hy-  (1.5 gm) was suspended i n water (6.0 ml); the  (100 mg) was added, mixed thoroughly,  and then the  mixture was p l a c e d i n t o a b o i l i n g water b a t h f o r 3 minutes.  The  G3P s o l u t i o n was c h i l l e d q u i c k l y by t r a n s f e r r i n g i t to an i c e bath and then i t was poured through a f u n n e l w i t h a g l a s s wool plug.  The r e s i n was washed w i t h 2 ml a l i q u o t s o f water u n t i l the  supernatant  f l u i d s measured 17.5 ml. The r e s u l t a n t s t o c k was  12mM i n s u b s t r a t e  (100/umoles o f e n z y m a t i c a l l y a c t i v e D-isomer)  26.  and had a pH of 2.4. 3torage I n v o l v e d d i v i d i n g the s o l u t i o n i n to separate 2 ml p o r t i o n s and f r e e z i n g u n t i l use.  The s u b s t r a t e  i s very s t a b l e when f r o z e n a t -20°C and c o u l d be kept f o r seve r a l months without any l o s s ; The f o l l o w i n g stock assay s o l u t i o n s were used i n a 3 ml cuvette t  2.6 ml o f lOOmM t r i e t h a n o l a m i n e pH 7.5 b u f f e r w i t h ad-  d i t i o n o f EDTA t o 5mM, 100jxl o f 6mM NADH, lOOyul o f 12mM G3P and lOOyul of d - g l y c e r o p h o s p h a t e  dehydrogenase.  The c o u p l i n g  enzyme was u s u a l l y p r o t e i n w i t h a s p e c i f i c a c t i v i t y o f about 200 units/mg  and s t o c k s o l u t i o n s f o r the assay had c o n c e n t r a t i o n s  of about 0.5 mg p r o t e i n p e r ml. in  the 3 ml volume werei  Therefore, f i n a l concentrations  lOOmM t r i e t h a n o l a m i n e pH 7.5, 5mM EDTA,  0.2mM NADH, 0.4mM G3P., and 17yug/ml GPDi t i a t e d by a d d i t i o n o f about 6 — 1 0 ume.  The r e a c t i o n was i n i -  o f TIM i n a 5 o r  vol-  The decrease i n absorbance a t 3^-Onm was f o l l o w e d f o r 1  minute and u s u a l l y d i d n o t exceed  0.1 absorbance u n i t s .  The  s m a l l volume i n c r e a s e d i d n o t s i g n i f i c a n t l y change absorbance so a c t i v i t i e s were c a l c u l a t e d upon a 3 ml volume;  A t the pH used,  there i s no d e t e c t a b l e decay o f NADH over one minute so the r e a c t i o n was u s u a l l y f o l l o w e d without a b l a n k . 2.3  Results High a c t i v i t y t r i o s e . phosphate Isomerase was e l u t e d from the  DEAE Sephadex A50 column i n two peaks a t approximately (fig.  2).  75mM T r i s  The r e s u l t s o f a t y p i c a l l i v e chicken p r e p a r a t i o n a r e  summarized i n Table I . The c o n s i d e r a b l e (36 X ,) p u r i f i c a t i o n , as determined the i n i t i a l  by s p e c i f i c a c t i v i t i e s , can be mainly a t t r i b u t e d to DEAE chromatography s t e p .  27.  FIGURE Zx  I n i t i a l DEAE Sephadex A50 Chromatography  Absorbance at 280  nm-o-K>w w v w i  FRACTION NUMBERS (-18 ml)  TABLE.It  Determination  o f a c t i v i t y u n i t s f o l l o w e d throughout a t y p i c a l l i v e c h i c k e n  prep-  a r a t i o n o f chicken b r e a s t muscle  For each k i l o o f t i s s u e : Fraction  OD's p r o t e i n  Uhits/OD  Total units  %  recovery  Purification  A.  Crude e x t r a c t  63,585  142.2  9,041,591  100 .  OX  B.  &5% P P t .  19,436  310.1  6,026,933  66.7  2.2X  C.  90% p p t .  10,064  340.8  3,429,661  37.9  2.4X  D.  Peak A+B o f i n i t i a l : 55.4  36.2X  DEAE Sephi A 5 0  971.7  (714.5mg)  5152.9  5,006,879  Peak A  898.3  (660.5mg)  5147  4,623,323  Peak B  73.4  (540.Omg)  5225  383,557  29.  In o r d e r to f u r t h e r p u r i f y and separate the two peaks, the p r o t e i n was rechromatographedI  The r e s u l t s a r e v a r i a b l e b u t d i d  r e s u l t i n a p r o t e i n up to twice as a c t i v e . p r o t e i n was observed as s e p a r a t i n g cates  t h a t the l o w e r i n g  chromatographed m a t e r i a l  The f a c t that no o t h e r  out i n subsequent steps  indi-  o f s p e c i f i c a c t i v i t y i n the one time i s p r o b a b l y due to s m a l l amounts o f i n -  h i b i t o r y p r o t e i n o r other unknown contaminating substances which have a s i m i l a r e f f e c t . The  d i f f e r e n c e i n s p e c i f i c a c t i v i t y between the A and B  peaks appears t o vary w i t h i n d i v i d u a l p r e p a r a t i o n s . l i v e chicken  preparation  d i d u s u a l l y y i e l d B p r o t e i n higher i n  s p e c i f i c a c t i v i t y than t h a t from c h i c k e n s o b t a i n e d b u t c h e r shops.  the A ( f i g . 2 ) .  Since  from l o c a l  The B p r o t e i n f r o m the l a t t e r source o f t e n  ed enzyme w i t h s p e c i f i c a c t i v i t y c o n s i d e r a b l y  of a l i v e  However, the  Figure  chicken  yield-  lower than t h a t o f  3 shows the i n i t i a l DEAE chromatography  preparationi  s p e c i f i c a c t i v i t y i s c a l c u l a t e d as the r a t e o f conver-  sion of substrate  per u n i t o f enzyme ( u s u a l l y p e r OD^QQ  o  r  P  Q r  mg), I t i s an i n d i c a t i o n o f the e f f i c i e n c y o f the c a t a l y t i c p r o cess.  I f the p r o t e i n i s completely homogeneous throughout an  e l u t e d peak, i t i s expected t h a t a constant s p e c i f i c would be o b s e r v e d i  activity  The chromatographic s t u d i e s on TIM showed  changes i n s p e c i f i c a c t i v i t y which i n d i c a t e d that the p r o t e i n i s not  completely homogeneo\is. Contamination o f the TIM peaks by p r o t e i n  l u t e d w i t h i n the same s a l t  concentration  (which can be e-  range as t r i o s e phosphate  isomerase) c o u l d be a f a c t o r i n the observed s p e c i f i c  activity  30. FIGURE 3: I n i t i a l DEAE Sephadex A50 Chromatography of , a L i v e Chicken P r e p a r a t i o n  -Absorbance at - 2 8 0 nm  Insert!  S p e c i f i c A c t i v i t y and P r o t e i n P r o f i l e s of TIM f r a c t i o n s  Specific Activity units/mc) -7000 -6000 -5000 -4000 -3000 -2000 -1000 120  0  i  20  40  60 80 100 120 140 FRACTION NUMBERS (-18ml)  160  130  31.  profile.  I f the contaminating p r o t e i n ( s ) i s i n h i b i t o r y  (or ac-  t i v a t i n g ) , minute amounts c o u l d produce s u b s t a n t i a l changes i n TIM's a b i l i t y t o c a t a l y z e the l s o m e r i z a t l o n o f g l y c e r a l d e h y d e 3-phosphate and dihydroxyacetone phosphate specific activity  (and hence i n the  profile).  Contaminating p r o t e l n ( s ) which has no e f f e c t upon the enzymatic c a p a b i l i t i e s of TIM would have to be p r e s e n t i n r a t h e r l a r g e amounts to produce the p r o f i l e changes observed.  Evidence  to be p r e s e n t e d i n the next s e c t i o n i n d i c a t e s t h a t the p r o t e i n i s homogeneous as to m o l e c u l a r weight. the  T h i s would tend to r u l e out  p o s s i b i l i t y o f s u b s t a n t i a l l y contaminated p r o t e i n b e i n g e-  l u t e d from the column.  The o n l y o t h e r p o s s i b i l i t y i s t h a t there  i s an i n h e r e n t m o l e c u l a r or c o n f o r m a t i o n a l d i f f e r e n c e i n the ose phosphate  isomerase a t v a r i o u s p o i n t s i n the e l u t e d TIM,peaks.  The p o s s i b i l i t y o f Isozymes ( p r o t e i n s o f s l i g h t l y d i f f e r e n t ino  tri-  a c i d sequence  and/or  am-  conformation which c a t a l y z e the same r e -  a c t i o n ) w i t h d i f f e r i n g t r i o s e phosphate  isomerase a c t i v i t y  capa-  b i l i t i e s i s a p o s s i b i l i t y to be d i s c u s s e d i n the p r o t e i n charact e r i z a t i o n s e c t i o n to f o l l o w . zyme forms o f TIM  The v a r i a t i o n found i n m u l t l e n -  ( l e . number of charges, shape, p r o t e i n aggre-  g a t i o n , e t c . ) c o u l d be the b a s i s f o r a chromatographic of  the v a r i o u s Isozymes.  The presence of Isozymes In the chicken  b r e a s t muscle f i t s w e l l w i t h the observed p r o t e i n and a c t i v i t y p r o f i l e s o f the DEAE chromatography  throughout the v a r i o u s chromatographies:  specific  experiments.  The c h a r a c t e r i s t i c s p e c i f i c a c t i v i t y p r o f i l e was  for  separation  observed  The double peak observed  the A p r o t e i n and s i n g l e one f o r the B might seem to i n d i c a t e  32. two enzyme forms i n the A w i t h o n l y one l n the B.  However, as  w i l l be shown l n the c h a r a c t e r i z a t i o n work to f o l l o w , t h i s does not  seem t o be the case, l n f a c t , there  the c o n t r a r y .  i s strong  evidence to  The p h y s i c a l b a s i s f o r the double a c t i v i t y peak  of the A p r o t e i n i s obscure a t t h i s p o i n t s i n c e i n the c h a r a c t e r i z a t i o n methods used, no s i m i l a r s e p a r a t i o n The  f r e q u e n t l y incomplete s e p a r a t i o n  occurred.  of the A and B TIM  peaks, as w e l l as the observed s p e c i f i c a c t i v i t y p r o f i l e ,  indi-  cated t h a t f u r t h e r p u r i f i c a t i o n and i s o l a t i o n o f the p o s s i b l e molecular forms o f the enzyme should be attempted.  To t h i s end,  separate rechromatography experiments were performed, The  first  experiment u t i l i z i n g DEAE Sephadex A 5 ° r e s i n r e -  s u l t e d i n a s i n g l e peak b e i n g The  peak came o f f very  e l u t e d f o r the A p r o t e i n ( f i g . 4 A )  e a r l y l n the g r a d i e n t  (but n o t w i t h the  v o i d volume) and showed the c h a r a c t e r i s t i c s p e c i f i c a c t i v i t y  pro-  f i l e f o r the A p r o t e i n . The e a r l y e l u t i o n of the A p r o t e i n I n d i cated  t h a t the rechromatography of the B p r o t e i n on the DEAE  Sephadex A 5 0 should and  shallower  be attempted u s i n g a lower s a l t  gradient  ( i n order  more s t r o n g l y to the column).  to a l l o w  concentration  the p r o t e i n t o adhere  However, an e l u t i o n of B p r o t e i n  e a r l y i n the g r a d i e n t , was observed ( f i g . 4 B ) .  The s p e c i f i c a c -  t i v i t y p r o f i l e of the rechromatographad B m a t e r i a l was s i m i l a r to that obtained The at  i n the i n i t i a l  chromatography.  attempts to rechromatograph t r i o s e phosphate isomerase  s a l t concentrations  l n the same range (about 75 M) as t h a t of m  the i n i t i a l Sephadex-DEAE chromatography seemed to be inadequate. The  adherence o f the p u r i f i e d enzyme to the a n i o n exchange c o l -  F i g u r e 4*  Rechromatography o f TIM F r a c t i o n s on DEAE Sephadex A 5 0 (A)  P r o t e i n from Peak A  (B)  P r o t e i n from Peak B  FRACTION NUMBER (-18ML)  34.  umn a p p e a r e d  t o be d i f f e r e n t  f r o m t h a t o f t h e Impure p r o t e i n .  The i n t e r a c t i o n s , w h i c h T I M h a s w i t h t h e o t h e r p r o t e i n s 90% ( N H ^ ) S 0 ^ s a t u r a t i o n c u t ,  could well affect  2  s t r e n g t h w i t h w h i c h TIM i s a b l e Therefore,  w i t h the  to adhere  considerable  column.  changes  90 a n d 50»70mM T r i s t o 5mM T r i s w i t h 0-35mM N a C l ) o f b i n e d peak A and B rechromatography  (fig;  peak d i d n o t  in a *step-like* illustrates  to the  the  recom-  the  The p r o t e i n p r o f i l e o b t a i n e d  the problems attached  s p e c i f i c a c t i v i t y p r o f i l e of  rather  (fig.  to t h i s k i n d of r e s i n .  the rechromatography  How-  first  i n a smoothly s y m m e t r i c a l peak but  manner.  that  column.  the i n t e r r u p t i o n s i n the g r a d i e n t ,  come o f f  ( f r o m 60-  5)» i t was f o u n d  t h e p r o t e i n was a d h e r i n g much more s t r o n g l y e v e r , because, o f  the  t h e manner a n d  to the  gradient  In  5)  The  of pooled A  a n d B ( d o u b l e p e a k f o r A a n d s i n g l e p e a k f o r B ) a l l o w e d one identify  w i t h some c e r t a i n t y ,  the A and B p r o t e i n s .  i n g o f f r a c t i o n s by the i n d i v i d u a l a r r o w s o f A and B peaks i s  shown I n f i g u r e  5*  t h e A p r o t e i n was l o w e r i n s p e c i f i c i n the p r o f i l e . served  The c o m b i n -  the i n d i v i d u a l  The s e c o n d a c t i v i t y p e a k a c t i v i t y than the f i r s t  T h i s was u n u s u a l i n t e r m s o f  the p r o f i l e s  i n t h e i n i t i a l DEAE c h r o m a t o g r a p h y b u t h a s b e e n  i n other rechromatographic  experiments  (eg!  figure  The n e x t r e s i n t o b e t r i e d , i n a n a t t e m p t  s h r i n k a g e p r o b l e m , was c e l l u l o s e DE52 ( f i g .  however,  has a l o w e r  of  peak ob-  observed  7B).  to get  Sephadex  capacity;  to  A g o o d s e p a r a t i o n was  over  the  6) w h i c h , observed  of r e c o m b l n e d A a n d B p e a k s f r o m t h e f i r s t DEAE S e p h a d e x A 5 0 chromatography a l t h o u g h the s p e c i f i c  a c t i v i t y p r o f i l e d i d not  show i t s u s u a l d e f i n i t i o n ( a l t h o u g h i t was g e n e r a l l y  the  same).  FIGURE 5»  Rechromatography of Peak A and Peak B TIM F r a c t i o n s DEAE Sephadex A50  Absorbance at 280 nm  Specific Activity Units/mg AAA  6000 -5000 •WOO r  3000  rEOOO -1000  c n A r T i m i  t i i i M D r o  I~.AO~.I\  FIGURE 6t  Rechromatography of Peak A and Peak B TIM F r a c t i o n s on DE52 C e l l u l o s e  37  L a s t l y , an agarose based i o n exchange was employed of  i n the rechromatography s t e p .  peak A f r a c t i o n s  (fig.  r e s i n , B i o g e l A DEAE, The rechromatography  7A) r e s u l t e d i n e l u t i o n of a s i n g l e  p r o t e i n peak ( w e l l i n t o the g r a d i e n t ) w i t h the c h a r a c t e r i s t i c double peak s p e c i f i c a c t i v i t y p r o f i l e . cal  The s l i g h t l y  assymmetri-  nature of the e l u t e d peak i s a f e a t u r e o f the agarose based  DEAE r e s i n u s e d .  The f a c t t h a t t h i s r e s i n i s capable o f as good  a s e p a r a t i o n o f the A and B p r o t e i n peaks as t h a t shown i n the DEAE Sephadex A50 and c e l l u l o s e DE52 chromatographies o f recombined A and B ( f i g . 5, f i g .  6 ) , i s i l l u s t r a t e d i n f i g u r e 7B.  A  good s e p a r a t i o n o f r e s i d u a l A p r o t e i n and the B p r o t e i n was observed.  The B peak showed a s i n g l e s p e c i f i c a c t i v i t y peak  w h i l e the A p r o t e i n had two (the second b e i n g p r e s e n t as a w e l l d e f i n e d shoulder o f the f i r s t ) ;  The second s p e c i f i c  peak was lower than t h e f i r s t a s was d i s c u s s e d In  activity  earlier.  c o n c l u s i o n , i t might be mentioned t h a t the B i o g e l A DEAE  r e s i n appears t o be the b e s t c h o i c e f o r the a n i o n exchanger to be used, i n the rechromatography s t e p .  I t combines  no shrinkage o f the r e s i n , and the a b i l i t y  high  capacity,  to be used a t a f a i r -  l y low pH, w i t h as good a s e p a r a t i o n o f TIM as has ever been observed c h r o m a t o g r a p h i c a l l y .  FIGURE 7*  Rechromatography of "TIM F r a c t i o n s on B i o g e l A DEAE (A)  Peak A  (B)  Peak B  FRACTION NUMBERS H8nril)  39. CHAPTER I I I CHARACTERIZATION OF PEAK A AND PEAK B 3.1  Methods A.  SDS G e l E l e c t r o p h o r e s i s  A s l i g h t l y m o d i f i e d procedure of F a i r b a n k s e t a l (57) was used.  The procedure was c a r r i e d out i n a v e r t i c a l  electrophor-  e s i s apparatus which c o n t a i n e d 800 mis o f b u f f e r s o l u t i o n i n each e l e c t r o d e compartment and no more than 12 g e l tubes ( f i g . 8 ) . The g e l s c o n t a i n e d 1% SDS and 5 a c r y l a m i d e . To f a c i l i t a t e the p r e p a r a t i o n o f b u f f e r s , g e l s , e t c . , conc e n t r a t e d s t o c k s o l u t i o n s were f i r s t made up and subsequently used to prepare the s o l u t i o n s which were used i n the e l e c t r o p h o r e s i s experiment.  These a r e l i s t e d i n T a b l e I I .  Gels were made by combining l n a s m a l l vaccum f i l t e r  flask,  1.4 mis c o n c e n t r a t e d a c r y l a m i d e and NN*-Methyleneblsacrylamlde (Ac  b i s ) (see Table I I ) , 1.0 ml 10X b u f f e r and 5.6 mis water.  The s o l u t i o n was degassed f o r a p p r o x i m a t e l y 15 minutes' and then to  i t was added 0.5 ml 20$ SDS, 1 ml o f ammonium p e r s u l f a t e :f  (15 mg/ml), and 0.5 ml o f 0.5% TEMED diamlne),  (N,N,N*,N*-tetramethylene-  T h i s mixture was used to f i l l  four.0.5X11  cm pyrex  tubes ( p r e v i o u s l y cleaned i n a c o n c e n t r a t e d HCl b a t h , coated w i t h p h o t o f l o s o l u t i o n , and l e f t the  tube.  to d r y ) to w i t h i n 1 cm o f the top o f  The top o f each g e l was c a r e f u l l y covered w i t h an  o v e r l a y s o l u t i o n which p r e v e n t s d r y i n g out o f the g e l .  They  were then l e f t  complete  polymerization.  to stand f o r a t l e a s t 12 hours t o ensure  40.  Figure. 8t  Electrophoresis  Si  Apparatus  Cross-sectional s i d e view  -kuffer  +  4  gel  <$  tube  buffer  41.  TABLE l i t  Stock s o l u t i o n s and b u f f e r s f o r SDS g e l e l e c t r o p h o r esis  A.  Stock 1)  solutions  Concentrated A c B i s 40 gms a c r y l a m i d e 1.5 S N,N -methylenebisacrylamlde EgO to 100 mis m  2)  I  10X B u f f e r 0.4 M T r i s 0.2 M sodium a c e t a t e 0.02 M EDTA pH = 7»4 w i t h a c e t i c a c i d  3) B.  20$ SDS  (W/W)  Electrophoresis buffer  (per l i t r e )  100 mis o f 10X b u f f e r 50 mis 20$ SDS H 0 to 1 l i t r e o  C.  Denaturing  Solution  2 gms SDS 10 gms sucrose 74.5 mgs EDTA 2 mgs p y r o n i n y 0.242 gm T r i s E.,0 t o 100 mis, pH 8 w i t h HCl D.  Overlay  solution 0.1$ SDS 0.15$ Ammonium p e r s u l p h a t e 0.05$ TEMED  42.  The p r o t e i n s were prepared as f o l l o x * s . the d e n a t u r i n g s o l u t i o n (Table I I ) was tol,  A s m a l l amount of  made 80mM i n d i t h i o t h r e i - -  combined w i t h an equal volume of p r o t e i n s o l u t i o n and  ed f o r 15-20  minutes a t 37°G to completely denature  The molecular weight denatured a t 60 G. p l e was  standards were prepared i n the same way Upon c o o l i n g , up to 100 j*ls  now  but  of p r o t e i n sam-  c o n t a i n e d i n the e l e c t r o p h o r e s i s  and covered w i t h b u f f e r s o l u t i o n ) .  bands.  the p r o t e i n .  c a r e f u l l y a p p l i e d w i t h a m i c r o p i p e t to the top of the  g e l (which was  t e i n was  heat-  apparatus  A maximum of lOOy^g of p r o -  l o a d e d onto each g e l which avoided e x c e s s i v e l y  broad  Samples were run In d u p l i c a t e .  E l e c t r o p h o r e s i s was  c a r r i e d out a t , a c u r r e n t of 5 mamp/gel  and r e q u i r e d approximately 3»5 hours under these c o n d i t i o n s . all  cases, g e l s were prerun f o r one  t i o n which ensured the removal The m o l e c u l a r weight  markers were denatured  trypsin  i n separate t e s t  t e s t tube and a p p l i e d  The marker p r o t e i n s were run i n g e l s  from the sample p r o t e i n s . standards!  hour p r i o r to sample a p p l i c a -  of excess ammonium p e r s u l f a t e .  tubes, then combined together i n one the g e l t o g e t h e r .  bovine serum albumin  (68,600),  ovalbumin  on  separate  The f o l l o w i n g p r o t e i n s were used  (23,800), and myoglobin  In  as  (45,000),  (16,900).  A f t e r removal of the g e l s from the tubes, the p o s i t i o n of the t r a c k i n g dye was dipped i n I n d i a i n k .  marked by n o t c h i n g the g e l s w i t h a needle The g e l s were then t r a n s f e r r e d to stoppered  g l a s s tubes and a g i t a t e d s e q u e n t i a l l y w i t h the f o l l o w i n g p r o t e i n s t a i n i n g and d e s t a i n i n g s o l u t i o n ! 1)  25$ i s o p r o p a n o l ,  (57)  10$ a c e t i c a c i d , 0.025$ Coomassie  43  blue 2)  (overnight)  10$ i s o p r o p a n o l , 10$ a o e t l o a c i d , 0.025$ coomass i e b l u e (6-9 hours)  3)  10$ a c e t i c a c i d , 0.0013$ coomassie b l u e  4)  10$ a c e t i c a c i d  (overnight)  A t t h i s p o i n t , the p r o t e i n s were apparent The  (overnight)  as dark b l u e bands.  subunit molecular weight o f TIM was estimated by f i r s t  plot-  t i n g l o g molecular weight a g a i n s t the R^. v a l u e s measured r e l a t i v e to the t r a c k i n g dye.  The p o i n t s f o r the standard p r o t e i n s f e l l  on a s t r a i g h t l i n e from which any unknown s u b u n i t weight c o u l d be o b t a i n e d by i n t e r p o l a t i o n , B.  molecular  ( f i g . 9)  D i s c Gels  The apparatus  was as f o r SDS g e l e l e c t r o p h o r e s i s . A modi-  f i e d method o f D i e t z and Lubrano was used ( 5 8 ) .  The f o l l o w i n g  stock s o l u t i o n s were made up to f a c i l i t a t e p r e p a r a t i o n o f the gels.  S o l u t i o n s A and B were s t o r e d i n the c o l d , i n the dark f o r  up to one month. A.  S o l u t i o n C was f r e s h l y made each  36.3 gms T r i s 0i23 mis TEMED A d d i t i o n HCl t o pH 8.5 H 0 t o 100 mis 2  B.  6 gms a c r y l a m i d e 160 mgs  methylene-bls-acrylamide  H 0 t o 20 mis 2  C.  140 mgs ammonium p e r s u l f a t e H 0 to 100 mis o  time.  45  Gel Mixture t Pour 7.5$ g e l s were made by m i x i n g 1.0 ml A, 2.0 mis B, 1.0 mis water, and 4.0 mis C.  The s o l u t i o n was poured i n t o  0.5X10 cm tubes which had been p r e v i o u s l y coated w i t h solution.  Photoflo  The g e l s were c a r e f u l l y o v e r l a i d w i t h about 50y-/ls o f  B^jO and then l e f t t o p o l y m e r i z e a t l e a s t 8 hours or o v e r n i g h t . D i a l y z e d p r o t e i n was made 20$ i n sucrose and a couple o f milligrams fer  o f bromophenol b l u e was added.  Electrophoresis buf-  f o r the cathode and anode r e s e r v o i r s was made by a 1 J 9 d i l -  u t i o n o f a s t o c k 10X b u f f e r which had been p r e v i o u s l y made: IOX  b u f f e r - 6 gms T r i s 28.8  gms g l y c i n e  to pH 8.3 H 0 to 1 l i t e r 2  The  bottom r e s e r v o i r was f i l l e d  gels placed  i n t o the a p p a r a t u s .  w i t h 800 mis o f b u f f e r and the The v a r i o u s p r o t e i n s o l u t i o n s  to be run were c a r e f u l l y p i p e t t e d onto t h e top o f the g e l s a l l y about  20jig  p r o t e i n i n a volume l e s s than lOOyxls were l a i d  on each g e l ) and then t h e tubes were f i l l e d w i t h b u f f e r . top r e s e r v o i r was f i l l e d w i t h b u f f e r , the e l e c t r o d e s and 4°C.  (usu-  the e l e c t r o p h o r e s i s begun.  The  connected  The experiment was performed a t  The p r o t e i n was concentrated  down Into a t h i n l a y e r on top  of the g e l a t 1 mamp/tube and then the v o l t a g e was i n c r e a s e d to g i v e 2 mamp/tube u n t i l the end o f the experiment. A f t e r e l e c t r o p h o r e s i s , the t r a c k i n g dye was notched w i t h I n d i a i n k and the p r o t e i n was f i x e d by a g i t a t i o n o f the g e l s f o r 30 minutes i n stoppered tubes c o n t a i n i n g  10$ TCA ( t r i c h l o r o a c e t i c  46.  acid).  S t a i n i n g of  t h e p r o t e i n was a c c o m p l i s h e d  12.5$ TCA w i t h 0 i 0 5 $ C o o m a s s i e color in  i n t e n s i t y was d e v e l o p e d  10$ T C A .  Blue for  1 houri  by a g i t a t i o n Finally,  o v e r a 48 h o u r p e r i o d b y  The g e l s w e r e s c a n n e d a t  In.  the  agitation  550nm u s i n g a G i l s o n  gel  scanner. C.  Isoelectric  Column I s o e l e c t r i c  Focusing Focusingt  A diagram of  the  110 m l c a p a c i t y  L K B 8101  column used i n t h e s e experiments  is  descriptions  procedure  of  the  eral references The v a l v e the at  (18 a n d 1 6 ) u n t i l  The f i r s t  used as  12:0 gms s u c r o s e  I t was pumped i n t h r o u g h n i p p l e up t h e b o t t o m o f  i n g 3/4  of  sucrose  dissolved  the  the Ampholytes in it)  ^ of  the Ampholytes  were  prepared.  was  equilibrated  cathode  2  A dense s o l u t i o n ,  as w e l l as a l i g h t  and the  solution  2 i 5-4  6-8  3.5-5  7-10  4-6  8-8.5  5-7  9-11  contain-  containing  f r o m LKB i n c l u d e t p H 5-8  pump a n d  ( a n d 28  d i a l y z e d p r o t e i n d i l u t e d to  avallabe  solution  a n d 14.0 m i s H 0 ) .  d i l u t e d t o 42 m i s w i t h w a t e r  The r a n g e o f A m p h o l y t e s p H 3.5-10  the  sev-  through  (1) w i t h a p e r i s t a l t i c  column.  Detailed  followst  passed  the apparatus  s o l u t i o n t o be a d d e d was diamine,  10.  c a n be f o u n d i n  12 was o p e n e d a n d c o o l i n g w a t e r  ( 0 . 4 mis ethylene  filled  shown i n f i g u r e  (59>60) w i t h t h e p r o c e d u r e  compartments 4°C.  experimental  electrofocusing  60 m i s  gms  FIGURE 10i  A S k e t c h of the I s o e l e c t r i c F o c u s s i n g  Column  F i g . lOt E l e c t r o f o c u s s i n g column of 110 ml c a p a c i t y . The outer c o o l i n g j a c k e t (18) has an i n l e t a t 14, and an o u t l e t a t 5» From the outer j a c k e t the water flows through a tube i n t o the c e n t r a l c o o l i n g j a c k e t at 4 and l e a v e s the column at 3» Two p l a t i n u m e l e c t r o d e s are u s e d . One e l e c t r o d e 13» i s l n c o n t a c t w i t h the p l u g 7? i s In the upper p a r t of the column. The gas formed a t t h i s e l e c t r o d e escapes a t 2. The other i s wound on a T e f l o n bar l l , and gas escapes a t 1. Before d r a i n i n g the column the c e n t r a l tube i s c l o s e d by l i f t i n g the p l u g 12 which has a rubber gasket on the upper s u r f a c e and s e a l s a t 15. I s o e l e c t r i c f o c u s s i n g takes p l a c e i n compartment 16, which i s f i l l e d through n i p p l e 2. At ' the bottom of the column t h e r e i s a p l u g l b , w i t h an a t t a c h ment f o r a c a p i l l l a r y tube to enable the column to be fractionated.  48 Ampholytes were used i n quantities to y i e l d either a 1$ (2.5 mis of 40$) or a 5$ (12:5 mis of 40$) concentration.  In  addition to these ampnolytes, a small quantity of pH 3.5-10 range ampholytes was added to the 1$ (0:2 ml of 40$) and the 5$ (1.0 ml of 40$) columns i n order to 'protect' the ends of the pH gradient from the cathode and anode solutions.  From 2-20 mgs  of protein may be i n each protein zone depending upon the range of ampholytes used and the differences i n the p i ' s of the proteins.  For the experiments descrioed here, about 5 mgs of pro-  tein were added; The dense and l i g h t solutions were added to the column by means of a LKB density gradient mixer through nipple 2 with the a i d of the p e r i s t a l t i c pump.  The f i n a l solution to be added was  the anode solution which contained 1 0 0 j j l s concentrated HgSO^ and 9.9 mis HgO.  The experiment was started with a voltage y i e l d i n g  about 2 watts of power.  ;  The focusing of the c a r r i e r ampholytes and proteins was accompanied by a decrease i n the current passing through the s o l ution.  The current was checked p e r i o d i c a l l y and the voltage i n -  creased (power always kept a t or under 2 watts) u n t i l i t had decreased to a constant value (at constant voltage).  At t h i s point,  most of the c a r r i e r ampholytes should be focused at or near their i s o e l e c t r i c points.  To ensure complete focusing of the  slower t r a v e l l i n g protein, the experiment was continued f o r a further 12 hours;  The e n t i r e focusing experiment takes from 24-  72 hours depending upon the pH range and concentration used. More concentrated and narrower range ampholyte solutions require  ^9.  the  longer focusing times. Upon completion of the p r o c e d u r e , the power i s turned o f f ,  and v a l v e 12 i s c l o s e d to p r e v e n t the c e n t r a l e l e c t r o d e from mixing w i t h the e f f l u e n t ; (20),  solution  The clamp on the c a p i l l a r y  tube  i s opened and the column pumped out a t a f l o w r a t e o f a-  bout 1 ml/minutei  The f r a c t i o n s were c o l l e c t e d on a G i l s o n  frac-  t i o n a t o r and t h e i r pH determined a t 4°0, the temperature of the experiment. t i a l l y , was  P r o t e i n , i f added  to the ampholyte  procedure  ini-  then measured by i t s absorbance a t 280nm and TIM  was  assayed f o r u s i n g the G3P/c(-glycerolphosphatedehydrogenase p r o cedure . The f i r s t pH 7-10  i s o e l e c t r i c f o c u s i n g experiment performed used  and pH 5-8  range ampholytes  t o t a l range of pH 5-10 1%,  (added i n 1 j l r a t i o w i t h a  o b t a i n e d ) a t a f i n a l c o n c e n t r a t i o n of  R e s o l u t i o n of the Peak A and of the Peak B p r o t e i n was i n -  adequate  so 5% runs of pH 7-10  or pH 5-8  (without p r o t e i n ) were  performed and the tubes c o n t a i n i n g c a r r i e r ampholytes 7-8 range were p o o l e d .  i n the pE  An a p p r o p r i a t e amount of the p o o l e d  p h o l y t e s (about 22 mis u s u a l l y ) was  then used In an  f o c u s i n g experiment which i n c l u d e d p r o t e i n .  am-  electro-  The amount o f su-  crose added t o the dense s o l u t i o n was decreased to compensate for  the sucrose p r e s e n t i n the p o o l e d ampholyte  mixture.  best r e s o l u t i o n r e s u l t e d when no more than 3 absorbance of  Peak A o r Peak B was  The units  used;  Polyacrylamide g e l i s o e l e c t r i c  focusings  The apparatus f o r r u n n i n g t h i s experiment was that used f o r d i s c or SDS  gel electrophoresis.  The  the same as polyacryl-  50 .  amide matrix o f the g e l s e r v e d as a s u b s t i t u t e f o r the sucrose g r a d i e n t of the column.  The g e l s were e i t h e r  or c h e m i c a l l y p o l y m e r i z e d .  P h o t o p o l y m e r l z a t l o n was the p r e -  f e r r e d method i n regards t o t l m e j  p r o t e i n seldom needed to be  c o n c e n t r a t e d b e f o r e use and the experiment t e r the p o u r i n g o f the g e l s . only when the average than 7.0-7.5 ( 6 1 ) .  photopolymerized  was s t a r t e d 1 hour a f -  However, the procedure  i s suitable  pH o f the ampholyte range i s no g r e a t e r  T h e r e f o r e the pH 5-8 or 3»5-10 ranges were  s u i t a b l e f o r use b u t not the pH 7-10 range.  The main disadvan-  tage o f the c h e m i c a l l y p o l y m e r i z e d g e l s i s the p r o d u c t i o n o f a r t i f a c t s due to the presence  of p e r s u l f a t e .  T h i s has been comment-  ed on by s e v e r a l authors .(63,6.2,63) Electrofocusing i n gels i s fast, requires r e l a t i v e l y  small  amounts o f p r o t e i n and uses much l e s s ampholytes than the column technique., The disadvantage  i s t h a t i t i s l i m i t e d i n determin-  i n g an a c c u r a t e p i f o r a p r o t e i n s i n c e the g e l i s s h o r t . dards  Stan-  ( i e . d u p l i c a t e g e l s without p r o t e i n ) a r e co-run w i t h the  gels containing p r o t e i n . determined  The pH p r o f i l e o f the standard g e l s Is  by s l i c i n g the g e l r o d , soaking the p i e c e s i n water  i n order to e l u t e the ampholytes, and d e t e r m i n a t i o n o f the pH of the e l u e n t .  The p r o t e i n i n the o t h e r g e l s i s s t a i n e d , t h e i r  p o s i t i o n measured, and the pH a t t h a t p o i n t determined p a r i s o n to the p l o t  by com-  ( o f pH v s . d i s t a n c e ) o f the standard g e l .  T h e r e f o r e , the accuracy o f the p i depends upon an i n d i r e c t d e t e r mination o f the pH a t t h a t p o i n t where the p r o t e i n i s focused and upon the accuracy o f s l i c i n g . The method used f o r the p r e p a r a t i o n o f the photopolymerized  51. g e l s i s g i v e n l n Table I I I .  Upon p o l y m e r i z a t i o n , the g e l s were  p l a c e d i n the e l e c t r o p h o r e s i s apparatus w i t h the anode (bottom) r e s e r v o i r c o n t a i n i n g 800 mis o f 0.2$ s u l f u r i c a c i d and the c a t h ode  ( t o p ) r e s e r v o i r c o n t a i n i n g 800 mis 0:4$ d i e t h a n o l a m l n e .  experiment  was r u n a t 4°C and an i n i t i a l  The  c u r r e n t o f 1 mamp/tube.  T h i s was maintained by i n c r e a s i n g the v o l t a g e u n t i l 350 v o l t s had been r e a c h e d !  The p r o t e i n and ampholytes were c o n s i d e r e d f o -  cused when a constant amperage ( u s u a l l y 0.5-1 mamp) a t 350 v o l t s was observed f o r 1-2 hours took on average  ( t h e IEF e x p e r i m e n t a l r u n n i n g  5 to 7 h o u r s ) .  were s l i c e d a t 5 mm  time  A t t h i s p o i n t , the r e f e r e n c e g e l s  i n t e r v a l s and p l a c e d l n i n d i v i d u a l  test  tubes w i t h 1 ml o f degassed water and then they were capped.  The  ampholytes were a l l o w e d t o e l u t e from the g e l a t 4°C, the tempe r a t u r e o f the experiment. determined  The pH o f the g e l s l i c e s c o u l d be  a f t e r a p p r o x i m a t e l y one h o u r i  I f the e l u t i o n was a l -  lowed to proceed o v e r n i g h t , n i t r o g e n gas was bubbled  i n t o the  tubes and then the tube was f i r m l y s e a l e d w i t h P a r a f i l m . The g e l s to be s t a i n e d  (Vesterburg*s quick s t a i n i n g  method)  (64) were marked w i t h I n d i a i n k a t the anode end and p l a c e d l n stoppered tubes; a)  The s t a i n i n g procedure  includedt  i n c u b a t i o n of the g e l s i n a 60°C water b a t h ( i n a fume hood) l n a s t a i n i n g s o l u t i o n ( o f methanol 75 mis, d i s t i l l e d water 186 mis, t r i c h l o r o a c e t i c a c i d 30 gms, s u l f o s a l i c y l i c a c i d 9 gms, and Coomassie B l u e , for  b)  0.1$)  15 minutes  r e p l a c i n g o f the s t a i n i n g s o l u t i o n w i t h d e s t a l n l n g solution  (of e t h a n o l 250 mis, water 650 mis, g l a c i a l  a c e t i c a c i d 80 mis) and a g i t a t i o n o f the tubes  TABLE I I I :  Isoelectric focusing  solutions f o r gels  P h o t o p o l y m e r i z a t i o n — Stock s o l u t i o n s were kept a t 4°C i n t h e dark f o r about one month. A*  Catalyst 1.0 ml TEMED 14 mgs r i b o f l a v i n H 0 t o 100 mis o  B.  Acrylamide 30 gms a c r y l a m i d e 0.8 gm m e t h y l e n e - b i s - a c r y l a m i d e H 0 t o 100 mis 2  Gel  Mixture  Mix 3 i 0 mis o f B and 0.3 ml ampholytes (40$). For each s e t o f d u p l i c a t e g e l s , take 1.1 ml o f the above'and add t o I t 2.75 mis H2O ( c o n t a i n i n g d i a l l z e d sample i f t h e g e l s a r e t o i n c l u d e p r o t e i n ) . The mixture i s poured i n t o the 0.5X10 cm tubes ( t o approximately 8 cms h e i g h t ) and exposed t o b r i g h t l i g h t f o r a p p r o x i m a t e l y 1 hour to complete polymeri z a t i o n . The tubes had been p r e v i o u s l y coated w i t h Photoflo s o l u t i o n . Chemical p o l y m e r i z a t i o n — Stock s o l u t i o n s were kept a t 4°C i n t h e dark f o r about one month. The c a t a l y s t s o l u t i o n was made f r e s h each time; A;  Acrylamide 3.05 gms a c r y l a m i d e H 0 t o 10 mis 2  B.  Bis-acryl 100 mgs m e t h y l e n e - b i s - a c r y l a m i d e E 0 t o 10 mis o  C;  Catalyst 150 mgs ammonium H O t o 10 mis  persulfate  G e l Mixture Mix 1125 mis A, 1.25 mis B, 3i30 mis H 0, and 2.0 mis p o o l e d ampholyte mlxe. Add t o t h i s s o l u t i o n 25juCLs TSMED a n d 200 / U s o f C. The f o u r g e l s ( o f a p p r o x i m a t e l y 8 cm h e i g h t ) a r e poured Immediately i n t o t h e 0:5X10 cm tubes which had been p r e v i o u s l y coated w i t h P h o t o f l o s o l u t i o n , and a l l o w e d t o p o l y m e r i z e f o r a t l e a s t 8 hours before use; 2  5*. The d e s t a i n i n g s o l u t i o n was f o r the f i r s t  2 hours and  u s u a l l y r e p l a c e d every 30 minutes  then l e f t In a f r e s h s o l u t i o n  n i g h t to complete the d e s t a i n i n g p r o c e s s . a t 550nm when d e s t a i n i n g was The pH range 5-8  was  over-  The g e l s were scanned  complete.  found  to be Inadequate f o r r e s o l u -  t i o n of the p r o t e i n bands, s i n c e the pl»s of the p r o t e i n s l n Peak A and B appear to be v e r y s i m i l a r . i s o e l e c t r i c f o c u s i n g experiment  was  T h e r e f o r e , a 5% column  performed  i n the u s u a l  way  but w i t h a p o o l i n g of those ampholytes i n the pH 7.3-7•9 f r a c tions. t h a t 0.5  The  c o n c e n t r a t i o n of the pooled ampholytes was  ml was  r e q u i r e d f o r each 1$ g e l .  t h i s range of ampholytes was the pH 7-10  Polymerization l n  found to occur w i t h l e s s ease than  range ampholytes (and not a t a l l by  photopolymeriz-  a t i o n ) so t h a t 50$ more p e r s u l f a t e than the u s u a l 1$ ( i e . a 1.5$ ation.  c a t a l y s t used) was  The presence  such  solution  r e q u i r e d to complete p o l y m e r i z -  of sucrose from the pooled ampholytes d i d  not i n t e r f e r e a d v e r s e l y w i t h the experiment.  I t has been  served ( 66) t h a t sucrose s t a b i l i z e s the pH g r a d i e n t and  ob-  enables  the p r o t e i n s to be s u c c e s s f u l l y focused i n a lower p e r c e n t  acryl-  amide g e l , w i t h a lower v o l t a g e and l o n g e r f o c u s i n g time.  Be-  cause of the p o l y m e r i z a t i o n problems e x p e r i e n c e d , a lower  per-  cent a c r y l a m i d e g e l was d e s c r i b e d i n Table I I I .  not used but the g e l s were prepared  as  S i n c e TIM i s not e x c e s s i v e l y l a r g e or  a s s y m m e t r i c a l l y shaped, i t e x p e r i e n c e s no problems i n p a s s i n g through the pores o f the h i g h e r p e r c e n t a c r y l a m i d e g e l s . the main advantages of the 3«5$ g e l s d e s c r i b e d by Doerr  One  of  and  Chramback (66) would be t h a t l a r g e or a s s y m m e t r i c a l l y shaped p r o -  55  t e i n s such as immunoglobulins c o u l d be Upon p o l y m e r i z a t i o n , phoresis  focused!  the g e l s were p l a c e d  In the e l e c t r o -  apparatus w i t h the anode (bottom) b u f f e r i n p l a c e .  presence of sucrose i n the g e l s makes them l e s s r i d g i d and  The less  a b l e to adhere to the s i d e s of the w a l l of the g l a s s tubes when placed  in a vertical position:  y s i s tubing,  Therefore,  s m a l l p i e c e s of  h e l d i n p l a c e by rubber bands around the bottom of  the tube, prevented the g e l s from s l i p p i n g out reservoir. lOOyiils) was pipet.  dial-  P r o t e i n ( i n a 25$  lower  sucrose s o l u t i o n i n a volume under  c a r e f u l l y l a i d on  Next, 100yAls  i n t o the  the top o f the g e l w i t h a micro-  of 20% sucrose was  l a i d on top of the  pro-  t e i n , f o l l o w e d by  lOOyuls o f 10$ s u c r o s e :  The  was  added to f i l l  the r e s t of the tube and  then the top r e s e r v o i r  was  c a r e f u l l y f i l l e d with b u f f e r .  The  cathode b u f f e r  b u f f e r s , experimental run-  n i n g c o n d i t i o n s , temperature, e l u t i o n o f ampholytes, and ing  of p r o t e i n were as d e s c r i b e d  method. the 5-7  The  g e l s were a l l o w e d  f o r the  photo-polymerization  to focus f o r 13 hours r a t h e r  hours used f o r the photopolymerized g e l s .  pH range of the ampholytes used i n the c h e m i c a l l y g e l s take l o n g e r were scanned a t  upon completion of  D.  Amino A c i d  The  amino a c i d d a t a was  was  than  narrower  polymerized The  gels  destaining.  Analysis obtained  d r o l y s e s of approximately 0 . 0 5 y U m o l e s Tryptophan  The  to t r a v e l to t h e i r i s o e l e c t r i c p o i n t . 550nm  stain-  from d u p l i c a t e 24 hour 2X  chromatographed Peak A.  assayed f o r u s i n g Wltkop's procedure  a c t i o n of N-bromosuccinimide (NBS)  hy-  (66) of r e -  w i t h the i n d o l e r i n g of t r y p t o -  56..  phane to y i e l d o x i n d o l e . The assay procedure was c a r r i e d out i n 8M u r e a ( a d j u s t e d to pH 4 a t 22°C w i t h a c e t i c a c i d ) t o ensure all  tryptophan  d i t i o n s of 5  residues. o  f  complete  The t i t r a t i o n proceeds "by stepwise ad-  10mM NBS t o 2.0 mis of p r o t e i n  ( O D 8 o » 5 - 2 ) contained i n a 3«0 ml c u v e t t e . 2  t i t r a t i o n of  =1  ceeded w i t h a decrease i n the absorbance  solution  The t i t r a t i o n  a t 280nm.  pro-  5 y i l addi-  t i o n s continued u n t i l a minimum a b s o r b t l o n was reached with c o r r e c t i o n s f o r volume i n c r e a s e b e i n g made. at  The absorbance  280nm may be r e l a t e d t o the OD o f tryptophan  decrease  i n the p r o t e i n  sample by the e m p i r i c a l f a c t o r of 1.31 which a l l o w s f o r the oxi d a t i o n product o f o x l n d o l e .  The E _  Q  f o r tryptophan  i s 5500M *  3.2  Results A.  Subunit M o l e c u l a r Weight  An SDS was  g e l e l e c t r o p h o r e s i s molecular weight d e t e r m i n a t i o n  performed u s i n g p r o t e i n from the two a c t i v e TIM peaks of the  I n i t i a l DEAE Sephadex A50  chromatograph.  The f o l l o w i n g  were r u n ( l n a d d i t i o n t o the molecular weight  gels  markers)i  A.  20 jxes  of peak A  B.  20y*gs o f Peak B  C.  20^Ugs of peak A p l u s 20yjigs of Peak B  P r o t e i n was  taken from the c e n t e r f r a c t i o n s of the two  TIM. peaks.  I n a l l cases, s i n g l e bands were observed upon com-  p l e t i o n of the e l e c t r o p h o r e s i s experiment c o n t a i n i n g marker p r o t e i n s ) . 24,500 was  active  (with e x c e p t i o n o f g e l s  A s u b u n i t m o l e c u l a r weight of  observed f o r Peak A, 24,700 f o r Peak B, and 24,500  when center f r a c t i o n s of peak A and B were co-run i n the same g e l . The presence o f s i n g l e bands was geneity.  an i n d i c a t i o n of p r o t e i n homo-  I t would appear t h a t the TIM a c t i v e f r a c t i o n s from  both peaks have the same, or n e a r l y the same m o l e c u l a r weight. The v a l u e s o f 49,000-49,400 o b t a i n e d f o r the d l m e r l c molecular weight are w i t h i n a r e a s o n a b l e d e v i a t i o n from the molecular weight of 48,500 which M c V i t t l e c a l c u l a t e d from p a r t i a l  s p e c i f i c volume  and s e d i m e n t a t i o n e q u i l i b r i u m measurements ( 1 1 ) .  SDS g e l mol-  e c u l a r weight d e t e r m i n a t i o n s are c o n s i d e r e d a c c u r a t e to w i t h i n 5-10$  of the t r u e m o l e c u l a r weight. SDS  g e l e l e c t r o p h o r e s i s performed on twice  chromatographed  m a t e r i a l gave r e s u l t s which were w i t h i n 5$ of the subunit molecul a r weight determined f o r the once chromatographed  protein.  58. These second values were 23,300 f o r Peak A, and gel.  23»000 f o r when Peak A and Once a g a i n ,  found to be The  tein  Peak B were co-run i n the same  s i n g l e bands were o b t a i n e d .  homogeneous by SDS  c o n c l u s i o n which may  phoresis  23,100 f o r Peak B,  protein  be drawn from the SDS  gel electro-  of the TIM  c h r o m a t o g r a p h i c . p r o t e i n and  active  i s not based on molecular weight d i f f e r e n c e s .  t e i n ) was  (with the e x c e p t i o n  homogeneous by SDS  B.  Disc Gel  The  r e s u l t s of the pH  t e i n s , were obtained  chicken  only one  The  modified  pro-  gel electrophoresis.  8.5  disc gel electrophoresis  The  R^, v a l u e s ,  reported  experi-  f o r the  electrophoretic  band f o r d i s c g e l e l e c t r o p h o r e s i s 8.5»  hut  pro-  gels.  samples (4$ g r e a t e s t d i f f e r e n c e ) .  at pH  g e l e l e c t r o p h o r e s i s had component.  of the FEM  s i g n i f i c a n t d i f f e r e n c e i n the  muscle TIM  Even the IX chrom  from measurements taken from the  m o b i l i t y of the v a r i o u s (20) r e p o r t e d  follows)  Electrophoresis  ment i s shown i n f i g u r e 11.  There i s l i t t l e  pro  activity  p r o f i l e s as w e l l as the i s o e l e c t r i c f o c u s i n g data which  atographed m a t e r i a l  was  gel electrophoresis.  r e s u l t s i s that the h e t e r o g e n e i t y  (as observed i n the  The  he a l s o i n d i c a t e d t h a t  Scope of starch  demonstrated the presence of a minor  minor component c o u l d correspond to the peak B  p r o t e i n which has been shown i n t h i s t h e s i s to be  separable  chro  atographically. The  r e s u l t s shown here, support Scope's o b s e r v a t i o n  band by d i s c g e l e l e c t r o p h o r e s i s . from the two one  chromatographic (TIM  p r o t e i n zone.  The  mixing of the  of  one  protein  a c t i v e ) peaks s t i l l r e s u l t s i n  Even the peak B p r o t e i n which was  one  year  FIGURE lit  D i s c G e l E l e c t r o p h o r e s i s Using F r e s h P r o t e i n from Peak A (A) and. peak B (B>, and O l d P r o t e i n from Peak B ( B ) v  60 o l d was homogeneous  (by t h i s method).  The homogeneity of TIM  by t h i s method and the very minor contaminent observed i n the starch g e l electrophoresis erature  t h a t chicken  has encouraged t h e b e l i e f i n the l i t -  muscle TIM i s r e l a t i v e l y f r e e from Isozymes.  However, the i s o e l e c t r i c f o c u s i n g  d a t a t o be d e s c r i b e d i n  the f o l l o w i n g s e c t i o n as w e l l as the chromatographic separat i o n observed, i n d i c a t e s the presence o f isozymes.  The p r o t e i n  i n peak B comprises almost 8% of the p r o t e i n found t o possess t r i o s e phosphate isomerase a c t i v i t y . The  f a i r l y l a r g e degree of isozymlc  contamination could a f -  f e c t the v a l i d i t y of c r y s t a l l o g r a p h i c and amino a c i d sequence data which has been r e c e n t l y p u b l i s h e d  ( 1 8 ) . I t appears ijrob-  a b l e that both the x-ray c r y s t a l s t r u c t u r e a t 2.5A r e s o l u t i o n and  the amino a c i d sequence were performed on p r o t e i n which was  not pure.  The i m p l i c a t i o n of t h i s ( i n terms of the  o f the p u b l i s h e d  r e s u l t s ) c o u l d be Important i f the  s t r u c t u r a l d i f f e r e n c e s are s i g n i f i c a n t .  correctness isozymic  I n the I n t e r p r e t a t i o n  of the e l e c t r o n d e n s i t y map, t h e r e were 23 s i d e chains which were a poor f i t w i t h the map i n r e g i o n s s i t y was r e l a t i v e l y  strong  where the e l e c t r o n den-  (including 6 p a i r s of equivalent  res-  idues from the two s u b u n i t s ) and t h e r e was one s h o r t s e c t i o n of polypeptide  chain, r e s i d u e s  not be f o l l o w e d  168-176 i n one subunit,  e a s i l y i n the e l e c t r o n d e n s i t y map.  which  could  These prob-  lems were p a r t i a l l y r e s o l v e d by the a u t h o r s o f the c r y s t a l s t r u c t u r e by c o n s i d e r i n g  t h a t they were l o o k i n g a t two independ-  ent Images of e s s e n t i a l l y i d e n t i c a l s t r u c t u r e subunits).  (ie. 2 identical  However, i f there was s u b s t a n t i a l Isozymic  Impurity  61. present i n the p r o t e i n c r y s t a l s used, the assumption would no l o n g e r v a l i d and. the p o s s i b i l i t y of. i n c o r r e c t of amino a c i d s  C.  he  assignments  exists.  I s o e l e c t r i c Focusing;  I s o e l e c t r i c focusing  (IEF) i s a s e n s i t i v e method of sep-  a r a t i n g ampholytesj e s p e c i a l l y p r o t e i n s , a c c o r d i n g to t h e i r electric point.  iso-  T h e r e f o r e , the IEF t e c h n i q u e , which i s char-  a c t e r i z e d by very h i g h a n a l y t i c a l r e s o l u t i o n and by  simplicity  of apparatus and method, can be used f o r both p r e p a r a t i v e sepa r a t i o n s o f p r o t e i n s as w e l l as i s o e l e c t r i c p o i n t a c t e r i z a t i o n of p r o t e i n s . i n p r o t e i n s may  As l i t t l e as a 0.02  be observed.  ( p i ) char-  p i difference-  As w i l l be demonstrated i n t h i s  s e c t i o n , i t i s p o s s i b l e to observe s e p a r a t i o n s which cannot be seen by d i s c g e l e l e c t r o p h o r e s i s . . Probably the IEF method which has the most p o t e n t i a l f o r s e n s i t i v i t y and a c c u r a c y i s the column method, as d i s c u s s e d i n the methods s e c t i o n .  I t has a l l o w e d , because o f i t s high r e -  s o l u t i o n and r e p r o d u c i b i l i t y of p i v a l u e , a c h a r a c t e r i z a t i o n of the c h r o m a t o g r a p h i c a l l y s e p a r a t e Peaks A and B o f TIM. The f i r s t  experiment t o be r e p o r t e d here i s the column  Iso-  e l e c t r i c f o c u s i n g o f 6 absorbance u n i t s of rechromatographed peak A u s i n g the narrow range ampholytes prepared from a 5$ ampholyte  column r u n .  (pH 7-8) which had been The r e s u l t s shown In  f i g u r e 12 i n d i c a t e the presence of a major p r o t e i n and. a major s p e c i f i c a c t i v i t y peak w i t h a p i o f 7*64.  The A y 2  0  p l o t showed  a shoulder w i t h pl=7»57 as w e l l as a minor peak e t 7*^7  which  ml  effluent  63.  might have some s i g n i f i c a n c e when compared  to other runs, which  contain Peak B protein, which w i l l "be described s h o r t l y .  There  was also a protein with p l = 7 « 7 3 which has some TIM a c t i v i t y and one with a pl=5»87 which has none.  The l a t t e r was probably en-  zyme which had been aggregated by the pH conditions of the experiment. In the electrofocusing of 4 . 3 absorbance units of rechromatographed Peak. B(see figure 1 3 ) the minor peak a t pH 7*72 r e appeared as d i d the nonactive 280nm absorbing peak a t pH 5 » 9 2 . However the major features were a protein peak at  7*57  and a  well defined shoulder at 7.44. Resolution of the 7 . 5 7 and 7.44 p i peaks could probably be improved by a narrower gradient or less p r o t e i n .  The s p e c i f i c a c t i v i t y p r o f i l e showed two peaks  which appear to correspond to the proteins with i s o e l e c t r i c points of 7'57 and 7.44.  The presence of the two small peaks  at pH 7 . 5 7 and 7.47 i n the protein p r o f i l e of the i s o e l e c t r i c focusing of Peak A (fig.12) can now be ascribed (with some degree of certainty) to the presence of Peak B contaminant. Better r e s o l u t i o n of the two proteins i n Peak B i s v i s i b l e i n figure 14 which i l l u s t r a t e s a column IEF experiment of r e chromatographed Peak A and Peak B.  Three major protein peaks  were eluted from the IEF column with p i ' s of 7.66, 7.55 and 7.46.  Once again there i s a minor amount of protein possessing  some TIM a c t i v i t y at about pH 7 * 7 .  In addition, there was a  small peak of some a c t i v i t y at pH 7 * 3 8 . than Peak B was used i n t h i s experiment.  Less Peak A protein The s p e c i f i c  activity  p r o f i l e showed three peaks but they only roughly corresponded  ml  effluent  65.  66  to the t h r e e major peaks v i s i b l e by determining the absorbance of f r a c t i o n s at 280nm. The r e s u l t s of the column IEF i n d i c a t e d the presence- of three p r o t e i n s w i t h t r i o s e phosphate Isomerase a c t i v i t y !  the  f i r s t w i t h a p i of about 7«65 ( s e p a r a b l e c h r o m a t o g r a p h l c a l l y Into Peak A) and the second and  t h i r d w i t h p i ' s of 7.56  and  7.45  r e s p e c t i v e l y i s o l a t e d c h r o m a t o g r a p h l c a l l y together i n Peak B. There i s approximately  0.1  pH u n i t s d i f f e r e n c e between p r o t e i n / I  (pl=7.65) and p r o t e i n I I (pl=7»56) and between p r o t e i n I I and p r o t e i n I I I (pl=7.48). The  s e p a r a t i o n of the t h r e e p r o t e i n s was  s t r a t e d i n the g e l IEF experiment  which was  f u r t h e r demon-  performed  u s i n g the  narrow range ampholytes, s p e c i a l l y prepared by $% column IEF runs.  The r e s u l t s i l l u s t r a t e d In the g e l scans of f i g u r e  15  i n d i c a t e s the f o c u s i n g of twice chromatographed Peak A i n t o a / s i n g l e component w i t h pi-7.64, twice chromatographed peak B i n t o three components of p i ' s equal to 7.66,  7.56  and  7.49,  and  fin-  a l l y the f o c u s i n g of Peak A and p e a k B i n t o three components with p i ' s of 7.64,  7.56,7.^-6:  The photograph of the g e l s ( f i g .  16)  demonstrates the c l a r i t y of the s e p a r a t i o n of the three p r o t e i n s . Minute t r a c e s of bands other than the major t h r e e p r o t e i n s are s l i g h t l y v i s i b l e i n the photograph but are not v i s i b l e at a l l l n the g e l scans which i n d i c a t e s t h a t contamination to any  significant  The presence  extent. of the t h r e e t r i o s e phosphate isomerase  t i v e p r o t e i n s as observed by the IEF experiments, s t r o n g l y t h r e e TIM  i s not p r e s e n t  isozymes.  suggests  acvery  68 FIGURE 1 6 1  Sample Gels o f G e l I s o e l e c t r i c F o c u s s i n g o f Peak A and Peak B  A  B  r  A +B  (-)  pi =7.64 pi =756 pi =747  (Hh)  6.9  The presence  o f more than one enzyme form f o r chicken  b r e a s t muscle TIM has an e v o l u t i o n a r y b a s i s .  Gracy  e t a l has  </ has p u b l i s h e d r e s u l t s g i v i n g the e l e c t r o p h o r e t i c m o l u l i t y from s t a r c h g e l e l e c t r o p h o r e s i s o f t r l o s e phosphate isomerases various t i s s u e s . ent.  from  He a l s o i n d i c a t e d t h e number of isozymes p r e s -  A l l v e r t e b r a t e s r e p o r t e d possess t h r e e isozymes.  I t was  necessary t o go t o the e v o l u t i o n a r y l e v e l of a crab t o observe two  isozymes and o f a b e e t l e to observe  one.  I t would t h e r e -  f o r e be i n c o n s i s t e n t I f only one molecular form o f TIM was  found  i n chicken b r e a s t muscle. On the b a s i s of the i s o z y m i c forms o f other v e r t e b r a t e t r i o s e phosphate isomerase  ( e g : human and r a b b i t ) , as w e l l as  the p i r e s u l t s , i t i s p o s s i b l e to t e n t a t i v e l y suggest  t h e basis  of the chicken isozymes to be a r e s u l t o f two p r o t e i n .chains ai andp .  The presence  of 2 d i s t i n c t  p o s s i b i l i t y of 3 Isozymes: subunits has been observed ( 6»24).  rip,  c h a i n s ^ ands would g i v e t h e and p  Heterogeneity of  i n other t r l o s e phosphate Isomerases  The r e l a t i v e s t a b i l i t i e s o f the t h r e e forms as w e l l  as the a c t u a l r e l a t i v e q u a n t i t i e s o f  and ^ chains would de-  termine the r a t i o s o f cK^ enzyme t o  and to f^*  with the data  a v a i l a b l e a t t h i s p o i n t a r a t i o o f about 80:6:1 f o r d i^pi^ n  may  be c a l c u l a t e d i f the major enzyme form p r e s e n t i n Peak A (pl= ?.64) i s a s s i g n e d the  d e s i g n a t i o n and the two miner isozymes  i n the peak B a r e assigned. <k $ (pl=?.57) andy6'  2  (pl=?.49) d e s i g n a -  tions respectively. The  two p r o t e i n chains c o u l d have some g e n e t i c o r i g i n  70  ( i e . two separate genes) or I t i s p o s s i b l e t h a t they a r i s e from some e p i g e n e t i c process ( l e . p o s t t r a n s l a t i o n a l changes i n the a c t u a l p r o t e i n I t s e l f which may occur i n the ' i n v i v o * s i t u a t i o n ) . The f i r s t p o s s i b i l i t y would r e s u l t from the presence  of two d i s -  t i n c t genes ( p o s s i b l y a r i s i n g from mutation a t one or more s i t e s ) which code f o r p r o t e i n chains o f d i f f e r i n g amino a c i d sequence. The second p o s s i b i l i t y o f e p i g e n e t i c p r o c e s s e s would  involve  some s o r t o f chemical m o d i f i c a t i o n o f the p r o t e i n o c c u r r i n g l n the c e l l i t s e l f .  A w e l l known example o f t h i s i s the f o r m a t i o n  of p r o t e o l y t i c enzymes from pro-enzymes. The presence o f a f o u r t h enzyme form which has no ' i n v i v o ' b a s i s was observed when another g e l i s o e l e c t r i c f o c u s i n g iment was performed  which I n v o l v e d the f o c u s i n g o f f r e s h twice  chromatographed Peak A w i t h o l d e r ographed Peak B ( f i g . 17)« at  exper-  (about 1 y e a r ) twice chromat-  The B p r o t e i n used,  l e a s t 50$ o f i t s i n i t i a l s p e c i f i c a c t i v i t y .  still  possessed  The f o c u s i n g of  the A p r o t e i n was s i m i l a r t o that o b t a i n e d i n other IEF e x p e r i ments ( f i g . 15) w i t h a pl=7«66 found f o r the major p r o t e i n zone. However t h e B p r o t e i n was now found to c o n t a i n 4 p r o t e i n s o f different p i *  7»66, 7*62 (not observed p r e v i o u s l y w i t h f r e s h B  p r o t e i n ) , 7»58 and 7«50. parison  Three peaks were accounted  f o r by com-  to the g e l IEF runs of f r e s h A and f r e s h B p r o t e i n t  p r o t e i n I (pl=7.66), p r o t e i n I I (pI=7-58) and p r o t e i n I I I ( p l = 7.50)•  The presence o f a peak w i t h pl=7.66 l n the B p r o t e i n  probably i n d i c a t e s contamination w i t h A p r o t e i n . The p r o t e i n w i t h pl=7»62 (from Peak B p r o t e i n ) was a new occurrence.  There was no t r a c e o f i t when f r e s h B p r o t e i n was  FIGURE 1?s  Gel I s o e l a c t r l c Focussing of Fresh Protein Peak A and Old P r o t e i n from peak B  " pH| £.t.  T.C.  7.6.  1 7.4, ! 1  .i  A  550nm  •7J  ^Onm 7.6C  V D I S T A N C E (cm)  from  72 used  ( f i g . 15).  While the pI--7«62 p r o t e i n has taken over  as  the dominant p r o t e i n i n the B peak, the p r o t e i n w i t h pl=7»58 has d i m i n i s h e d i n q u a n t i t y r e l a t i v e to the other B peak proteins.  I t would seem l i k e l y t h a t , w i t h age,  there i s a change  i n the pIn.-7.58 p r o t e i n to g i v e an a l t e r e d p r o t e i n w i t h pl=7»62. The appearance of the pl=7»62 p r o t e i n a f t e r a c o n s i d e r a b l e l e n g t h of time and not i n the o r i g i n a l f r e s h p r e p a r a t i o n of B, makes i t c l e a r t h a t the 7-62  form has no g e n e t i c o r i g i n but  r a t h e r a r i s e s from a m o d i f i c a t i o n of e x i s t i n g p r o t e i n .  Mod-  i f i c a t i o n o f r e a c t i v e c a r b o x y l , amino, or hydroxyl groups i n the ' i n v i t r o ' as w e l l as ' i n v i v o ' s i t u a t i o n may concomittant  change i n p i .  occur w i t h a  Some of the most l i k e l y  would be l o s s of NH3 from asparaglne or glutamine SH o x i d a t i o n (of c y s t e i n e ) to -SOH,  changes  as w e l l as  -S0 H, or -SO^E. 2  Replace-  ment of even a s i n g l e c a r b o x y l group l n hemoglobin has been known to cause d i s t i n c t  changes i n i t s m o b i l i t y (67).  a l s o the p o s s i b i l i t y t h a t w i t h time, the conformation  There i s of the p r o -  t e i n has changed, w i t h a r e s u l t a n t a l t e r a t i o n i n the exposure of charged amino a c i d s i d e chains and hence a change i n i t s movement i n an e l e c t r i c f i e l d and i n i t s p i . D.  Amino A c i d A n a l y s i s  The amino a c i d data f o r 1 subunit i s g i v e n below, along w i t h the p u b l i s h e d v a l u e s  (17)•  TABLE IV:  Amino a c i d  Amino A c i d A n a l y s i s o f peak A TIM  2X chromatographed  L i t e r a t u r e value  TIM-Peak A cys  4.01  4.0  arg  7.40  7.5  meth  1.98  2.0  tyr  3.80  3.9  his  7-32  7.6  asp  16;52  20.0  val  17.29  24.4  ileu  14.42  16.6  lev  16.00  17.0  phe  7.00  7.8  pro  7.95  8.9  lys  21.28  23.1  ala  25.18  28.2  gly  23.90  27.0  glu  24.90  25.8  ser  10.94  13.5  thr  10:26  10.2  trp  5.02  5.0  TOTAL (nearest whole i n t e g e r ) 225  253  74. A molecular  weight o f 4 8 , 0 6 4 was c a l c u l a t e d f o r the Peak A,  (^2 isozyme) t r i o s e phosphate isomerase.  This i s s i g n i f i c a n t l y  lower than the p u b l i s h e d l i t e r a t u r e value o f from the above ( l i t e r a t u r e ) amino a c i d d a t a .  5^*^00  obtained  There a r e s i g n i f -  i c a n t d i f f e r e n c e s i n a s p a r t i c a c i d and v a l i n e amino a c i d numbers with much s m a l l e r d e v i a t i o n s apparant i n i s o l e u c l n e , l e u c i n e , p h e n y l a l a n i n e , p r o l i n e , l y s i n e , a l a n i n e , g l y c i n e , g l u t a m i c and serine residues.  The l e u c i n e , p h e n y l a l a n i n e , p r o l i n e , and g l u -  tamic a c i d r e s i d u e s d e v i a t e by o n l y 1 r e s i d u e which c o u l d be w i t h i n the e r r o r o f the experiments. Amino a c i d d a t a from the other two isozymes w i l l be necessary b e f o r e any assumptions can be made concerning a c i d d i f f e r e n c e s between the t h r e e p r o t e i n forms.  the amino  75CHAPTER IV PROTEIN MODIFICATIONS 4.1  Introduction P r o t e i n m o d i f i c a t i o n i s a s t r a t e g y used by  the  biological  p r o t e i n chemist to probe the s t r u c t u r e of a p r o t e i n .  In  the  case of enzymes, the e f f e c t of m o d i f i c a t i o n upon the a c t i v e and  hence the c a t a l y t i c c a p a b i l i t i e s of the system, can  valuable  information  example, i n the  concerning  I n t e r e s t has been focused l y e s s e n t i a l glutamic tail  give  the enzymatic mechanism.  case of t r i o s e phosphate isomerase, on the m o d i f i c a t i o n of an  acid residue.  site,  For  considerable enzymatical-  T h i s i s covered i n some de-  i n the i n t r o d u c t i o n to t h i s t h e s i s . P o s s i b l e s i t e s of m o d i f i c a t i o n i n any  protein include  s u l f h y d r y l group of c y s t e i n e , the i m i d a z o l e hydroxyl  group of  the  histidine,  group of s e r i n e , the f-amlno group of l y s i n e , the u»-  c a r b o x y l group of a s p a r t l c and  glutamic  a c i d s and  the  phenolic  group of t y r o s i n e . The due  s u l f h y d r y l group o f c y s t e i n e has  to i t s high n u c l e o p h l l i c and  attracted attention  redox r e a c t i v i t y and  i t y to enter i n t o c h a r a c t e r i s t i c and  its abil-  selective reactions.  s p e c i f i c i t y of the m o d i f i c a t i o n i s Important f o r both the y t i c a l determination  of numbers of s u l f h y d r y l r e s i d u e s  as w e l l as a s t r u c t u r a l probe of the p r o t e i n . fortunate  Therefore  The anal-  present i t is  t h a t the p r o t e i n s u l f h y d r y l group of c y s t e i n e has  been  found to be very r e a c t i v e to many r e a g e n t s . The  high n u c l e o p h i l l c l t y of mercaptide i o n s i s g i v e n by  the  76.  p a r t i c u l a r e l e c t r o n s t r u c t u r e of the s u l f u r atom w i t h i t s high polarlzablllty.  The t h i o l a t e a n i o n i s c o n s i d e r e d  the s t r o n g e s t b i o l o g i c a l n u c l e o p h l l e s ;  to he one o f  i n a d d i t i o n to the po-._  l a r i z a b l l i t y o f the s u l f u r e l e c t r o n s , t h e r e a r e empty d - o r b i t a l s , p e r m i t t i n g d - o r b i t a l o v e r l a p and thereby i n c r e a s i n g n u c l e o phlliclty  .  F o r these reasons, c y s t e i n e i s a good choice  for*  m o d i f i c a t i o n of p r o t e i n s . The  SH group o f c y s t e i n e takes p a r t i n most r e a c t i o n s i n  the form o f the mercaptide anion (RS~).  I t was c a l c u l a t e d by  Eenesch and Benesch ( 68 ) t h a t a t pH 7«^> ( p h y s i o l o g i c a l pH) 6% of the SH's of f r e e c y s t e i n e were i o n i z e d .  In the a c t i v e  s i t e s of enzymes, i t has been found t h a t t h e pK o f c y s t e i n e may vary from 7 t o 9»  Examples Include  the a c t i v e s i t e SH o f  phosphoenolpyruvate carboxykinase w i t h a pH o f 7»3 (69 ) and f i c i n which has been found to i n c l u d e a c y s t e i n e r e s i d u e w i t h pK of 8.55 ( 70 ) i n i t s a c t i v e s i t e .  Microscopic  which cause t h i s v a r i a t i o n i n c l u d e p r o x i m i t y (pK TT 0  d e c r e a s e s ) and n e g a t i v e  charges (pK„  TT  environments  t o p o s i t i v e charges increases).  Ioniz-  a t i o n of sulfhydr.yls a r e p a r t i c u l a r l l y depressed when the c y s t e i n e i s i n a hydrophobic microenvironment, b u r i e d w i t h i n the protein. The  The p K s i n t h i s case a r e commonly found to be above 9« f  p r o t e i n chemist"s i n t e r e s t i n c y s t e i n e i s a r e s u l t o f  the s i g n i f i c a n c e of the SH group f o r s p e c i f i c f u n c t i o n s o f a number o f enzymes, hormones and other b i o l o g i c a l l y a c t i v e p r o t e i n s which p l a y a c e n t r a l r o l e i n the normal course o f many p h y s i o l o g i c a l p r o c e s s e s ^ a s w e l l as t h e i r e x c e p t i o n a l  reactivity.  77  As a b i o l o g i c a l l y a c t i v e f u n c t i o n a l group, the s u l f h y d r y l has been known to be r e s p o n s i b l e  f o r noncovalent b i n d i n g  s t r a t e s and c o f a c t o r s , d i r e c t c o v a l e n t  of sub-  p a r t i c i p a t i o n i n the c a t -  a l y t i c a c t and maintenance of the n a t i v e  catalytically  active  conformation o f an enzyme. With the b i o l o g i c a l Importance of c y s t e i n e  i n mind as w e l l  as the p o t e n t i a l r o l e o f chemical s u l f h y d r y l m o d i f i c a t i o n s i n e l u c i d a t i n g p r o t e i n s t r u c t u r e and f u n c t i o n ,  i t i s w e l l to note  the high r e a c t i v i t y and d i v e r s i t y o f chemicals r e a c t i o n s d i s t i n g u i s h e s the SH group*  alkylatlon, acylation,  which  oxidation,  t h i o l - d i s u l f i d e exchange, r e a c t i o n s w i t h s u l f e n y l h a l i d e s , and the f o r m a t i o n o f mercaptides, hemimercaptols and m e r c a p t o l s . The  f o l l o w i n g i s a short  listing  o f some o f the r e a c t i o n  classes  along w i t h examples o f r e a g e n t s . Reaction 1.  Class  Transition. Metals  2.  Oxidation  3-  4.  Reagent Coll, Cull,  Nill, Hgll  Comment mercury complexes among the most s t a b l e  2°2  i n absence o f metals, f a i r l y s p e c i f i c f o r c y s t e i n e and methionine  Nucleophilic Addition  N-ethyl malelmide  vary from r e v e r s i b l e condens a t i o n s w i t h aldehydes and ketones to f a i r l y i r r e v e r s i b l e r e a c t i o n s w i t h malelmide  Displacement  a)  highly s p e c i f i c , r e s u l t i n g i n s t a b l e products  H  haloacetol hates b) DTNB phosiD  78«  The  a l k y l a t i n g l a b e l s i n c l u d e some of the more r e a c t i v e r e -  agents a l t h o u g h the problem does e x i s t of the r e s u l t i n g t h i o l product being drolyzlng.  quite unstable  However, one  have c o n s i d e r a b l e with c y s t e i n e .  i n the aqueous environment and  c l a s s of l a b e l s , maleimides, u s u a l l y  success i n forming a s t a b l e c o v a l e n t  The  hy-  reaction i t s e l f  linkage  i n v o l v e s a d d i t i o n of the  sulf-  h y d r y l to an a c t i v a t e d double bond:  E-S  +  T h i s M i c h a e l - t y p e conjugate a d d i t i o n i s I r r e v e r s i b l e and r a p i d l y i n a l k a l i n e media. pH i n c r e a s e s reactive An  goes  There i s an enhancement of r a t e  s i n c e the mercaptide a n i o n of c y s t e i n e i s the most  species. important competing r e a c t i o n c o u l d be  £-amino groups of l y s i n e or the i m i d a z o l e a malelmlde v i a an analogous mechanism.  the a d d i t i o n of  pH  the  group of h i s t i d i n e to The  s p e c i f i c i t y of  reagent f o r c y s t e i n e i s m a i n t a i n e d by keeping the pH a t or low  as  the be-  7«0 a t which p o i n t the r e a c t i o n of £-amino groups (or i m i -  d a z o l e ) i s i n s i g n i f i c a n t i n the time p e r i o d r e q u i r e d f o r  titra-  t i o n of the SH groups which i s u s u a l l y under an hour.  At pH  the r a t e of r e a c t i o n f o r simple t h i o l s i s on the order  of  times f a s t e r than f o r simple amines ( 71 ).  maleimides  Therefore,  can be h i g h l y s p e c i f i c reagents a l t h o u g h t h e r e sibility  that the malelmlde may  7«0,  1000  i s always the pos-  r e a c t w i t h some r e s i d u e  other  than c y s t e i n e which possesses i n c r e a s e d r e a c t i v i t y as a r e s u l t of i t s p a r t i c u l a r environment l n the  protein.  79 •  A w i d e l y used malelmide i s N - e t h y l malelmide (NEM) i s employed as a s u l f h y d r y l r e a g e n t .  NEM  which  absorbs s t r o n g l y a t  -1-1 around 300nm (£=620M  cm  ) which a l l o w s one  to f o l l o w the r e -  a c t i o n of the l a b e l by o b s e r v i n g  the decrease i n absorbance a t  300nm as the r e a c t i o n p r o c e e d s .  T h i s reagent  s u i t a b l e f o r chemical  was  found to  be  m o d i f i c a t i o n of t r i o s e phosphate isomer-  ase, the r e s u l t s of which are r e p o r t e d i n t h i s s e c t i o n . A f l u o r i n e c o n t a i n i n g analogue of NEM D.G.  was  synthesized  by  C l a r k f o r t h i s work: |[^0  —»ir^N-CE^CF^  + HgN-CHg-CF^  +  H0 2  0 The r e s u l t i n g t r l f l u r o - N - e t h y l malelmide (FEM)  has a broad max-  imal absorbance c e n t e r e d a t about 280nm (£=390). ^  makes d i r e c t s p e c t r o p h o t o m e t r i c  t l o n by FEM  difficult  protein absorbtion  o b s e r v a t i o n of SH  (and hence problems w i t h b l a n k i n g ) . spectrophotometric  have a p o s s i b i l i t y of about 10$  error.  F i g u r e 18 i l l u s t r a t e s the u l t r a v i o l e t The  modifica-  s i n c e there i s a s t r o n g i n t e r f e r e n c e w i t h  values o b t a i n a b l e by the d i r e c t  and FEM.  T h i s 280nm  The  observation  s p e c t r a of both  e l e c t r o n withdrawing e f f e c t of the f l u o r i n e atoms  appears to a c t i v a t e the mallelmlde  double bond even more s t r o n g -  l y r e s u l t i n g i n a more f a c i l e a d d i t i o n to c y s t e i n e than the drogen analogue, The  FEM  NEM  hy-  NEM.  m o d i f i c a t i o n s reported, here have a s p e c i a l  i n l i g h t of the reagent's  p o t e n t i a l as a NMR  label.  interest  FIGURE IVt  U l t r a v i o l e t S p e c t r a of NEM  -i  250  and  FEM  ———i  300  \  ( \M)  ~r~  350  81.  M o d i f i c a t i o n o f t r l o s e phosphate isoraerase was r i e d out u s i n g S H - d i s u l p h i d e i n t e r c h a n g e  also  car-  methods based on  a c t i o n s w i t h d i s u l f i d e s which a r e c o n s i d e r e d  re-  t o be among the  most s p e c i f i c r e a g e n t s f o r p r o t e i n SH groups,  A  particularily  e f f e c t i v e reagent which has been used i n t h i s work i s Ellman's (72)  reagent, 5 , 5 - d i t h i o b i s f  (2-nitrobenzroic a c i d ) which r e a c t s  w i t h t h i o l s as f o l l o w s : E-S~  + N0 -^^S_S-^~JJ-N0 2  ""00C  2  —E-S»S-^~^-N0  COO"  2  + ~S-<^^-NG  COO"  (DTNB)  COO"  (E-TNB)  (TNB")  The  strongly colored thionitrobenzoate  may  be quantated by i t s absorbance a t 4l2nm (£=1.36X10 M  a t pH  2  a n i o n which i s l i b e r a t e d 4 —1 —1 cm  8.0). However, I t must be r e a l i z e d t h a t w i t h some p r o t e i n s  ( 73 , 7 4 , 75 )» two r e a c t i o n s are p o s s i b l e a f t e r some SH*s have reacted: (1) the normal i n t e r m o l e c u l a r r e a c t i o n o f the f i r s t SH*s (as above) (2)  an i n t r a m o l e c u l a r  r e a c t i o n o f SH w i t h the  d i s u l f i d e product which r e s u l t s from the t i o n o f p r o t e i n -SH  w i t h DTNB. P  S  mixed reac_^ •NO,  82.  Whether or not the I n t r a m o l e c u l a r r e a c t i o n succeeds ing  i n compet-  s u c c e s s f u l l y w i t h the I n i t i a l l n t e r m o l e c u l a r r e a c t i o n , the  end r e s u l t i s the same*  r e l e a s e o f one e q u i v a l e n t o f n i t r o t h l -  opholate a n i o n (TNB") f o r each SH group t h a t r e a c t s . The absorbance o f the r e a c t i o n products a t 4l2nm i s h i g h l y dependent upon pH.  Consequently,  there i s a l i m i t  t o the range  of pH's which may be used w i t h DTNB ( u s u a l l y pH 7.5-8 o n l y ) . In a d d i t i o n , t h e r e i s a problem w i t h a u t o x i d a t l o n o f the n i t r o phenolate a n i o n as w e l l as d i f f i c u l t i e s l n u s i n g t h e reagent w i t h c o l o r e d p r o t e i n s ( e g . heme c o n t a i n i n g P r o t e i n s ) which absorb s t r o n g l y a t 412nm. Some o f the d i f f i c u l t i e s may be g o t t e n around by f o l l o w i n g Butterworth*s  p r o c e d u r a l changes ( 7 6 j * the p r o t e i n i s f i r s t  m o d i f i e d w i t h DTNB, then I s o l a t e d and f i n a l l y r e a c t e d w i t h d l thiothreitol  (DTT).  The DTT v e r y r a p i d l y l i b e r a t e s TNB~ from  the p r o t e i n and a l l o w s d e t e r m i n a t i o n o f t h e number o f SH's by o b s e r v i n g the Increase i n absorbance a t 4l2nm.  T h i s method may  not be used f o r those p r o t e i n s which a r e a b l e t o undergo the i n tramolecular, d i s u l f i d e f o r m a t i o n and r e s u l t a n t e l i m i n a t i o n o f TNB".  However, when t h i s method I s used,  i t i s p o s s i b l e t o ob-  t a i n v a l u e s as c o r r e c t as by t h e method I n i t i a l l y d e s c r i b e d a l though i t i s not p o s s i b l e t o observe  the k i n e t i c s o f the mod-  i f i c a t i o n o f the p r o t e i n w i t h DTNB. Another method o f l i b e r a t i n g TNB~ i s by displacement cyanide j (CN") E-S-(TNB)  ^ E-S-CN  +  TNB"  with  83*  T h i s r e a c t i o n a l s o has the advantage o f a l l o w i n g the p r o t e i n to become l a b e l l e d w i t h C  1 3  v i a C N~ or C ^ v i a C^N". 13  13 C  N  The  1  l4 p r o t e i n c o u l d be used In CMR s t u d i e s while  t e i n would make t h e m o d i f i e d  the C  N pro-  protein radioactive.  A second aromatic d i s u l f i d e was used i n the m o d i f i c a t i o n s t u d i e s t o be d e s c r i b e d .  There a r e two analogues o f the d i t h i -  o d i p y r i d i n e d i s u l f i d e d e s c r i b e d by G r a s s e t t i and Murray ( 7 7 ) possiblei  (1) The  (2)  4,4»dithiopyridlne(2), i s the most s e n s i t i v e reagent s i n c e upon  r e a c t i o n w i t h a SH group i t r e l e a s e s a 4 - t h i o p y r i d o n e  which has  as e x t i n c t i o n c o e f f i c i e n t o f 19,800 a t 324nm which i s c o n s i d e r a b l y l a r g e r than the maximum molor e x t i n c t i o n c o e f f l c l e n t , o f 7060 a t 3^3 f o r the 2 - t h i o p y r i d o n e .  The 4,4* reagent was used  exclusively:  (4-PD3) The  (4-TP)  p y r i d i n e d i s u l f i d e s have the advantage o f being a b l e to be  used over a much wider pH range than DTNB.  It i s particularily  u s e f u l f o r t h e lower pH's, where DTNB cannot be employed, s i n c e the d i m i n i s h i n g l e v e l o f E-S~ i s compensated f o r by the i n c r e a s i n g r e a c t i v i t y o f the r e a g e n t i t i e s of the p y r i d i n e r i n g  The e l e c t r o n withdrawing p r o p e r -  (and hence the r e a c t i v i t y o f the d i -  s u l f i d e ) becomes s t r o n g e r w i t h p r o t o n a t i o n  o f the n i t r o g e n .  •84* 4.2  Methods A.  (a)  M o d i f i c a t i o n w i t h Malelmld.es  N-ethylmaleimlde (NEM)  1  Approximately 20 mgs o f twice chromatographed t r i o s e phosphate i s o m 3 r a s e from Peak A i n a lOOmM pH 6.5 phosphate b u f f e r was g e n t l y  s t i r r e d overnight.in  the c o l d w i t h 25mM d i t h i o t h r e i -  tol  (DTT) t o ensure complete r e d u c t i o n  The  p r o t e i n was d e s a l t e d by p a s s i n g  of cysteine  residues.  i t down a Sephadex G25 c o l -  umn ( 1 . 5 X 2 0 c m ) e q u i l i b r a t e d and e l u t e d w i t h the lOOmM pH 6 . 5 phosphate b u f f e r i  Approximately 2 ml f r a c t i o n s were c o l l e c t e d  on a G l l s o n m i c r o f r a c t i o n a t o r  and the absorbance a t 280nm f o r  each f r a c t i o n was determined;  A f r a c t i o n with A230 equal to  8.02 was s e l e c t e d f o r t h e m o d i f i c a t i o n and k i n e t i c s t u d y . A Z e i s s PMQ I I v i s l b l e - U V spectrophotometer was s e t up f o r constant temperature runs a t 20°C.  The r e a c t i o n was i n i t i a t e d  by p l a c i n g 1 ml o f p r o t e i n i n t o a 3 ml cuvette  containing  of a N - e t h y l malelmide s o l u t i o n i n t h e pH 6 . 5 b u f f e r . elmide: s o l u t i o n was o f such a c o n c e n t r a t i o n  The mal-  as t o g i v e 20X molar  excess o f malelmide over p r o t e i n ; f i n a l c o n c e n t r a t i o n s 4l.8yA.M I n p r o t e i n and 850y^M i n NEM.  2 mis  were  A r e a c t i o n blank c o n s i s t -  ed o f 2 mis o f the NEM s o l u t i o n and 1 ml c f b u f f e r .  The m o d i f i -  c a t i o n o f t h e p r o t e i n was monitored by f o l l o w i n g the change i n absorbance a t  300nm.  as a f u n c t i o n o f time.  ( f i g . 21)  A decrease  was observed u n t i l a c o n s t a n t v a l u e was o b t a i n e d i n l e s s than 1 hour. (b)  T r l f l u o r o - N - e t h y l malelmide (FEM) Peak A TIM was p r e p a r e d f o r chemical m o d i f i c a t i o n  as was  85 • the p r o t e i n In the NEM pH 6.5  lOOmM phosphate b u f f e r was  action conditions. modification, fer  experiments.  When pH  6.0  the p r o t e i n was  However, e i t h e r pH  t i o n was  carried  b u f f e r was  to be used i n  s t i r r e d overnight  i n pH  i f i e d p r o t e i n was  c o l d w i t h 20X  ance a t 280nm over 1.0 The  vette containing  buf-  modifica-  2.0  c o n s i s t e d of 2.0  70 mgs  excess FEM.  enzyme  The  mod-  column, e l u t e d w i t h  those f r a c t i o n s w i t h an  absorb-  i n i t i a t e d by adding 1 ml  about 1 absorbance u n i t p r o t e i n to a 3 ml  lOOmM phosphate pH  ml o f the reagent s t o c k  8.0,  mis  lOmM EDTA and  The  solution  5.25mM DTNB.  of s t o c k reagent and  lOOmM phosphate b u f f e r . the i n c r e a s e  7.0  were kept f o r s u l f h y d r y l d e t e r m i n a t i o n  DTNB r e a c t i o n was  enzyme c o n t a i n i n g  6.0),  on a Sephadex G25  desalted  lOOmM phosphate b u f f e r and  with DTNB.  the  out.  a l l o w e d to r e a c t In the  pH 6.0  re-  In a l l o t h e r  reduced a t the same pH as the  In the f i r s t s e r i e s of experiments (pH was  or  used f o r the m o d i f i c a t i o n  so as to ensure the s t a b i l i t y of the p r o t e i n .  cases, the p r o t e i n was  6.0  r e a c t i o n was  1.0  of cu-  containing  The  blank  ml of pH  followed  J  by  8.0 observing  i n absorbance a t 4l2nm u n t i l a c o n s t a n t v a l u e  was  observed a f t e r 2 hours. In the same e x p e r i m e n t a l s e r i e s , an a l i q u o t of the modified reacted  p r o t e i n was  s t i r r e d In DTT  overnight  then  w i t h DTNB.  A s i m i l a r s e r i e s of experiments was FEM  (25mM) and  FEM  m o d i f i c a t i o n a t pH  i f i e d p r o t e i n and performed a t pH  6.5•  performed but w i t h  Both the r e d u c t i o n  of the unmod-  i t s d e s a l t i n g on the Sephadex G25  6.5.  The  p r o t e i n was  modified  the  column were  under 20X  molar  86  excess reagent  c o n d i t i o n s and then t h e m o d i f i e d p r o t e i n was de-  s a l t e d on a pH 8.0 lOOmM phosphate conlumn.  The DTNB  a t i o n proceeded as b e f o r e b u t made use o f a reagent  determin-  stock s o l -  a t i o n c o n t a i n i n g 1% sodium d o d e c y l 3 u l f a t e (SDS) i n a d d i t i o n t o the lOOmM phosphate pH 8.0, lOmM EDTA and  5'25mK  DTNB.  An attempt was made t o f o l l o w the k i n e t i c s o f the FEM modi f i c a t i o n by o b s a r v i n g t h e decrease  i n the absorbance a t 280nm  as the p r o t e i n r e a c t s w i t h t r i f l u o r o m a l e i m i d e a t pH 6.5. was  allowed  Thus,  to react with  complete r e a c t i o n t a k i n g p l a c e i n under 30 s e c .  Thus the modi-  f i c a t i o n was too r a p i d (and would n e c e s s i t a t e use o f a s t o p flow spectrophotometer) t o f o l l o w u s i n g normal techniques.  spectrophotometry  However, i t d i d a l l o w a d i r e c t d e t e r m i n a t i o n  of the  number o f SH*s which had r e a c t e d w i t h FEM. The was  s p e c i f i c a c t i v i t y o f the NEM and FEM m o d i f i e d p r o t e i n  determined u s i n g the TIM assay procedure d e s c r i b e d  earlier.  P r o t e i n which had been m o d i f i e d by NEM as above a t pH 6.5 was d e s a l t e d on a Sephadex G25 column which was e l u t e d w i t h lOOmM pH  7.5 t r i e t h a n o l a m i n e b u f f e r , the a p p r o p r i a t e d i l u t i o n was made,  and the a c t i v i t y was determined.  The same procedure f o l l o w e d i  the FEM m o d i f i c a t i o n experiment.  A c t i v i t y measurements were  a l s o performed on a c o n t r o l o f unmodified DTT  reduction.  Peak A p r o t e i n a f t e r  87  B. (a)  M o d i f i c a t i o n s with  Disulfides  5 , 5 * - d i t h i o b i s ( 2 - n l t r o b e n z o i c a c i d ) (DTNB) Twice chromatographed Peak A p r o t e i n was reduced  i n the c o l d w i t h 25mM d i t h l o e r y t h r e t o l  overnight  (DTE) o r DTT i n pH 7.0  lOOmM phosphate, and then d e s a l t e d on a Sephadex G25 column e l u t ed with lOOmM phosphate pH 8 . 0 b u f f e r .  Eluted fractions  (~2mls)  of A28O g r e a t e r than 0 . 8 were used f o r t h e SH d e t e r m i n a t i o n . The r e a c t i o n was i n i t i a t e d by a d d i t i o n o f 1 . 0 ml enzyme s o l u t i o n to 2 . 0 mis o f s t o c k reagent  (lOOmM phosphate pH 8 . 0 , lOmM EDTA,  5.25mM DTNB) i n a 3 ml c u v e t t e .  The r e a c t i o n c e l l was blanked  a g a i n s t a c u v e t t e c o n t a i n i n g 1 . 0 ml b u f f e r and 2 . 0 mis r e a g e n t . The r e a c t i o n was f o l l o w e d v i a an i n c r e a s e i n t h e absorbance a t 4l2nm u n t i l a constant value was o b t a i n e d a f t e r 2 hours.  approximately  Upon completion o f t i t r a t i o n o f the 'exposed* SH's, a  s m a l l q u a n t i t y (^10 mgs) o f s o l i d SDS was added t o the r e a c t i o n cell.  W i t h i n one minute, t h e p r o t e i n was completely  denatured,  thus f a c i l i t a t i n g t h e r e a c t i o n o f the *burled» SH*s.  The f i n a l  absorbance a t 412 was determined tein sulfhydryl.  giving a value f o r t o t a l  pro-  The above procedure was r e p e a t e d f o r twice  chromatographed Peak B p r o t e i n . Experiments  i n v o l v i n g the displacement  o f TNB  were  performed  on twice chromatographed Peak A p r o t e i n which had been m o d i f i e d w i t h DTNB as b e f o r e except  t h a t r e a c t i o n was c a r r i e d out i n a  37°C water bath I n s t e a d o f room temperature the r e a c t i o n time:  i n o r d e r t o decrease  The m o d i f i e d p r o t e i n was d e s a l t e d on a Seph-  adex G25 column ( 1 . 5 X 2 0 cm) e l u t e d w i t h a pH 7*5 t r i e t h a n o l a m i n e buffer.  Approximately  4 mis o f A 8 o 2  = 0 , 1 8 1  P r o t e i n sample was  88.  obtained. cuvette TNB~  One h a l f o f the p r o t e i n  ( i e . 2 mis)  was p l a c e d  and 1 mg o f d l t h i o e r y t h r e l t o l was added.  in a  L i b e r a t i o n of  (as observed by an I n c r e a s e i n absorbance a t 4l2nm) was  almost i n s t a n t a n e o u s .  The second h a l f o f the p r o t e i n  ( i e . 2mls) was a l s o p l a c e d (1 mg) was added.  i n a cuvette  and a c r y s t a l o f KCN  The r e a c t i o n c e l l h o l d e r  meter was thermostated a t 20°C.  sample  i n the spectrophoto-  L i b e r a t i o n o f TNB~ was a g a i n  observed by f o l l o w i n g the i n c r e a s e  i n absorbance a t 4l2nm.  The  dispacement by CN*" was slower than t h a t by DTE and data f o r a k i n e t i c a n a l y s i s was c o l l e c t e d , (b)  4,4* d i t h i o p y r i d i n e The  modifications  o f TIM w i t h the second d i s u l f i d e , 4,4*-  d i t h i o p y r i d i n e were performed i n a s i m i l a r manner t o the DTNB r e a c t i o n except t h a t pH 6.5 was chosen f o r t h e r e a c t i o n tion.  As b e f o r e ,  t h e p r o t e i n was reduced o v e r n i g h t  i n 25mM DTE o r DTT i n p r e p a r a t i o n  f o r the chemical  condi-  ( i n the c o l d ) modifications.  D e s a l t i n g o f the reduced, unmodified p r o t e i n was performed on a Sephadex G25 column (1.5X20 cm) e l u t e d w i t h pH 6.5 lOOmM phosphate b u f f e r .  Fractions  containing  employed i n the experiment.  protein with  were  &2Q0~^*®  The r e a c t i o n was i n i t i a t e d by p l a c -  i n g 5 0 j u l s o f s t o c k reagent (26 mgs o f 4 , 4 " d i t h i o p y r i d i n e d e l i v e r e d v i a a Lang-Levy p i p e t i n t o a 3 ml c u v e t t e 1.95 The  p e r ml)  containing  mis o f pH 6 i 5 lOOmM phosphate b u f f e r and 1.0 ml enzyme. m o d i f i c a t i o n was observed by f o l l o w i n g the i n c r e a s e  i n ab-  sorbance a t 324nm u n t i l a c o n s t a n t v a l u e was o b t a i n e d , u s u a l l y ..less t h a t 2 hours.  A d e t e r m i n a t i o n o f t o t a l s u l f h y d r a l s was  89.  accomplished  by adding a s m a l l amount o f s o l i d SDS a t t h i s p o i n t  and o b s e r v i n g the i n c r e a s e i n A  ,. 324  A 4 , 4 ' d i t h i o p y r i d i n e t i t r a t i o n a t pH 6.5 was a l s o performed on p r o t e i n which had been m o d i f i e d by DTNB a t pH 8.0. m o d i f i e d Peak A p r o t e i n was reduced  The un-  i n t h e u s u a l way, d e s a l t e d  a t pH 8.0, r e a c t e d with DTNB ( i n the c o l d f o r s e v e r a l h o u r s ) , d e s a l t e d a t pH 6 i 5 and f i n a l l y allowed dine d i s u l f i d e .  Upon completion  t o r e a c t w i t h the p y r i -  o f the t i t r a t i o n o f r e a c t i v e  groups, SDS was added and an a d d i t i o n a l i n c r e a s e i n t h e absorbance a t 32^nm was  observed.  A s e r i e s o f s p e c i f i c a c t i v i t y measurements o f the v a r i o u s m o d i f i e d p r o t e i n s was performed.  In a l l cases, t h e p r o t e i n s  were m o d i f i e d under the c o n d i t i o n s d e s c r i b e d p r e v i o u s l y .  The p r o -  t e i n samples i n c l u d e d j (1)  peak A TIM m o d i f i e d w i t h DTNB  (2)  DTNB m o d i f i e d p r o t e i n which had been a l l o w e d t o r e a c t  with CN" (3)  DTNB m o d i f i e d p r o t e i n which had been a l l o w e d  with DTE (4)  ' c o n t r o l ' sample o f unmodified  protein  to r e a c t  90 .  4.3  Results A.  (a)  Maleimides  N-ethylmaleimida F i g u r e 19 i l l u s t r a t e s the pseudo f i r s t  t r i o s e phosphate isomerase  o r d e r m o d i f i c a t i o n of  w i t h N-ethylmalelmide.  Data p o i n t s  were p l o t t e d to the t h i r d h a l f l i f e and a l e a s t squares  technique  was  A pseudo  employed to fi:.t the b e s t s t r a i g h t l i n e to the d a t a . _*a  first  order r a t e constant o f 2.39X10  J  -1  sec  was  calculated  (20 C ) . A v a l u e of 2.05 dryls modified.  was  c a l c u l a t e d f o r the number of s u l f h y -  A t the pH of the experiment (6.5) i t i s improb-  a b l e t h a t any r e s i d u e s o t h e r than c y s t e i n e was S i n c e TIM one  being  i s a dimer, the near i n t e g r a l v a l u e of 2 suggests  c y s t e i n e per monomer i s exposed to a t t a c k by NEM  protein i s i n a native statei  However, i t s h o u l d be  t h a t there i s a p o s s i b i l i t y t h a t the conformation is  modified. that  when the realized  of the p r o t e i n  such t h a t 2 c y s t e i n e r e s i d u e s are exposed on one monomer but  none on the o t h e r .  In view of the f a c t t h a t r e c e n t x-ray work  on c h i c k e n muscle TIM demonstrated a symmetrical  molecule  (18)  t h i s appears u n l i k e l y . The k i n e t i c a n a l y s i s demonstrates t h a t both SH's be r e a c t i n g w i t h NEM  a t the same r a t e .  appear to  T h i s i s i n c o n t r a s t to  r e s u l t s which w i l l be r e p o r t e d i n the l a s t chapter w i t h of  disulfide modification.  experiments,  With the d a t a a v a i l a b l e from  i t cannot be d e c i d e d w i t h any  t e i n e s are b e i n g m o d i f i e d by  NEM.  kinetics these  c e r t a i n t y which cys-  91.  FIGURE 19:  Pseudo F i r s t Order A n a l y s i s Modification  of the  o f TIM w i t h NEM  I n s e r t : I n c r e a s e i n the Absorbance R e a c t i o n Proceeds  -I  4  r12  1  8 TIME  (MIN)  as the  92* (b)  T r i f l u o r o N-ethylmaleimide (FEM) Peak A t r i o s e phosphate isomerase, when m o d i f i e d  a t pK 6.0  or 6.5 by FEM, was found'to a l s o be a b l e t o r e a c t w i t h DTNB. I t w i l l be shown i n l a t e r segments o f t h i s s e c t i o n t h a t b o t h FEM and  DTNB a r e a b l e t o modify 2 c y s t e i n e r e s i d u e s  o f TIM.  f o r e , i f one assumes t h a t the same r e s i d u e s a r e being  There-  modified  by a FEM m o d i f i c a t i o n o r a DTNB m o d i f i c a t i o n , i t i s necessary t o account f o r t h e type o f r e a c t i o n o c c u r r i n g I f DTNB i s shown t o r e a c t w i t h p r o t e i n which i s a l r e a d y  modified  case p r o t e i n which had been m o d i f i e d had  by FEM.  In t h i s  by FEM a t pH 6.0,  and hence  2 r e s i d u e s r e a c t was observed t o have 1.10 r e s i d u e s  exposed  to r e a c t i o n w i t h DTNB under nondenaturing c o n d i t i o n s and a t o t a l of 7.28 exposed t o r e a c t i o n imder d e n a t u r i n g  conditions.  d i f f e r e n c e , 6.28, probably denotes the number o f r e s i d u e s  The which  are b u r i e d and n o t exposed t o r e a c t i o n w i t h e i t h e r DTNB or FEM. T h i s value obtained  i s s i m i l a r to the value  o f 6 'hidden' SH's u s u a l l y  by DTNB t i t r a t i o n s alone (see d i s u l f i d e  modification  segment o f t h i s s e c t i o n ) . When the FEM m o d i f i e d  p r o t e i n was a l l o w e d to s t i r  overnight  In DTT and was then assayed w i t h DTNB, s i m i l a r r e s u l t s were obtained The  :  t  1.01 r e s i d u e s r e a c t e d under nondenaturing  d i f f e r e n c e i n t h i s case i s 5«67 which i s s t i l l  to the i n t e g r a l value  o f 6 u s u a l l y obtained  hidden SH's by DTNB.  I f the FEM modified  conditions. quite  close  f o r a t i t r a t i o n of  p r o t e i n i s denatured  by SDS, 5*77 r e s i d u e s were found t o r e a c t w i t h DTNB, vihich a g a i n denotes a value  c l o s e t o 6 f o r the number o f hidden  residues.  If  i t i s a g a i n assumed, t h a t FEM and DTNB a t t a c k the same  r e s i d u e , a mechanism f o r displacement be g i v e n .  Under normal circumstances,  are c o n s i d e r e d  o f t h e FEM by DTNB must maleimide m o d i f i c a t i o n s  t o be i r r e v e r s i b l e .  However, a mechanism such as the f o l l o w 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  i n t h i s p a r t i c u l a r m o d i f i c a t i o n o f TIM*  The g e n e r a l base (Bs) c o u l d w e l l be a b a s i c amino a c i d c h a i n i n the v i c i n i t y o f t h e FEM m o d i f i e d set  up t o accept a proton  side  s i t e which i s u n i q u e l y  from t h e malelmide.  The b a s i c pH (8.0) o f the DTNB m o d i f i c a t i o n makes the poss i b i l i t y of b a s i c residues being  i n t h e unprotonated form more  likely. When the p r o t e i n which had been m o d i f i e d by FEM was allowed to  r e a c t w i t h DTNB i n nondenaturing s o l u t i o n f i r s t and then i n  denaturing  s o l u t i o n , t h e t o t a l number o f e q u i v a l e n t s o f DTNB  averaged out t o about 7 (one r e s i d u e r e a c t s i n s o l u t i o n without SDS, the remaining the m o d i f i e d  s i x r e a c t when SDS i s added).  However, when  (FEM) p r o t e i n was denatured b e f o r e a d d i t i o n o f DTNB,  . o n l y 5-77 r e s i d u e s were found t o r e a c t . a maleimide displacement  T h i s g i v e s support t o  which i s a s s i s t e d by a s t r a t e g i c a l l y  p l a c e d b a s i c amino a c i d s i d e c h a i n . mide appears t o n o t be a b l e t o occure  T h e displacement  c f malei-  i f t h e p r o t e i n i s denatured.  94'.  The  f a c t t h a t only one e q u i v a l e n t o f maleimide i s l i b e r a t e d  suggests t h a t there i s a nonequivalence o f the two m o d i f i e d which i s due t o some c o n f o r m a t i o n a l  difference.  sites  I f the d i s p l a c e -  ment i s indeed dependent upon some b a s i c amino a c i d s i d e c h a i n , even s m a l l d i f f e r e n c e s c o u l d a f f e c t i t s a b i l i t y  t o c a t a l y z e the  reaction. The  FEM m o d i f i c a t i o n o f TIM has -a p r a c t i c a l a p p l i c a t i o n i n  f l u o r i n e NMR o f the p r o t e i n .  F i g u r e 20 g i v e s the s p e c t r a ob-  t a i n e d from 200 mgs o f m o d i f i e d TIM a l o n g w i t h a r e f e r e n c e and a model compound ( a l l a t pH 6.5). The r e f e r e n c e i n t h i s case i s t r l f l u r o a c e t l c a c i d (TFA) which has been a r b i t r a r i l y  set at zero.  The model compound,  which was formed by combining equimolar p o r t i o n s o f N - a c e t y l t e i n e and FEM (pH 6.5), was found to absorb 1192 h e r t z from TFA.  cys-  downfield  However the p r o t e i n sample gave a s i g n a l a t 375 h e r t z  u p f i e l d from TFA which i s a t o t a l o f 1567 h e r t z from the model compound.  A l l s p e c t r a were performed, a t 100MHz on the XL100. I t  was necessary  t o accumulate 100 t r a n s i e n t s f o r the model compound.  (.02 hours) and 100,000 (5.57 hours) f o r the p r o t e i n sample. The  l a r g e chemical  s h i f t d i f f e r e n c e between the model com-  pound and the FEM l a b e l l e d p r o t e i n i s i n d i c a t i v e o f both the sens i t i v i t y o f the f l u o r i n e nucleus  to changes i n environment and  a l s o the c o n s i d e r a b l e a l t e r a t i o n o f the chemical the FEM l a b e l .  environment of  The s i n g l e p r e l i m i n a r y experiment i l l u s t r a t e d i n  f i g u r e 22 can o n l y g i v e l i m i t e d Information  but i t does i n d i c a t e  the f e a s i b i l i t y o f f u r t h e r i n v e s t i g a t i o n w i t h t h i s  system.  "  FIGURE 20»  F NMR  of. FEM M o d i f i e d  T r l o s e Phosphate Isomerase  0 HERTZ  ^  96.  Further  studies at various  the absence of  substrate  cerning  the  nucleus  as  w e l l as  binding  of  substrate.  pH"s  should  and give  b o t h l n the valuable  i o n i z a t i o n of groups i n the  information  vicinity  changes l n c o n f o r m a t i o n  p r e s e n c e and  of the  in  con-  fluorine  o f p r o t e i n upon  the  97 .  (c)  S p e c i f i c A c t i v i t y Measurements on M o d i f i e d TIM A s i m i l a r k i n e t i c a n a l y s i s as t h a t done f o r t h e NEM modi-  f i c a t i o n o f TIM c o u l d not be performed f o r FEM (without flow  stop  t e c h n i q u e s ) because o f t h e r a p i d i t y o f t h e m o d i f i c a t i o n r e -  action.  As d i s c u s s e d  i n the i n t r o d u c t i o n t o t h i s s e c t i o n , t h i s  Is p r o b a b l y due t o a c t i v a t i o n o f the double bond due t o the e l e c t r o n withdrawing p r o p e r t i e s o f the-CF^ group. there i s the p o s s i b i l i t y t h a t the f l u o r o a n a l o g u e r e a d i l y 'reach'  In a d d i t i o n , i s a b l e t o more  t h e r e a c t i o n s i t e s i n t h e p r o t e i n than t h e NEM  i s , due t o the changed e l e c t r o n i c c h a r a c t e r  o f FEM.  However, t h e experiment which was performed a l l o w e d ect determination extent The  t h a t I.96 SH's had been m o d i f i e d .  a dir-  Thus, the  o f m o d i f i c a t i o n i s t h e same as t h a t o c c u r r i n g w i t h NEM.  two exposed SH's found i n the FEM t i t r a t i o n a r e e s p e c i a l l y  i n t e r e s t i n g i n l i g h t o f the a b i l i t y o f DTNB t o t i t r a t e one SE equivalent  p e r mole o f FEM m o d i f i e d  protein.  be a nonequivalence o f the two FEM m o d i f i e d t i o n by DTNB.  There appears t o groups t o m o d i f i c a -  As w i l l be demonstrated i n the k i n e t i c s s e c t i o n  to f o l l o w , there i s a l s o a nonequivalence o f t h e unmodified p r o t e i n t o DTNB m o d i f i c a t i o n . cysteine residues  The nonequivalence of t h e two  c o u l d be p r e - e x i s t i n g i n the n a t i v e u n m o d i f i e d  p r o t e i n but a l s o might be induced by t h e m o d i f i c a t i o n o f e i t h e r one  or both o f the SH's. S i m i l a r i t i e s between t h e NEM and FEM m o d i f i e d  proteins are  demonstrated by t h e d a t a on t h e s p e c i f i c a c t i v i t y o f the modif i e d s p e c i e s , as summarized belowt  98" Protein  Specific A c t i v i t y (units)  % Activity  100.0  0  #SH  Modified  1.  Control  5233  2.  NEM  modified  1204  23.0  2.0  3.  FEM  modified  1307  25.0  2.0  The  s i m i l a r i t y of the  s p e c i f i c a c t i v i t i e s o f the  modified  p r o t e i n s suggests t h a t the e f f e c t o f the r e s p e c t i v e maleimides on the t r l o s e phosphate isomerase molecule i s a l s o s i m i l a r . Even though the r a t e s of m o d i f i c a t i o n of the two  maleimides  were q u i t e d i f f e r e n t , i t i s l i k e l y t h a t the a c t u a l m o d i f i c a t i o n ( i e . the s i t e ) i s the same. ure  A t t h i s p o i n t , i t would be  premat-  to assume t h a t s i n c e the s p e c i f i c a c t i v i t y i s decreased  by  the m o d i f i c a t i o n j t h a t the s i t e of m o d i f i c a t i o n i s a t or near the a c t i v e s i t e .  However, the r e c e n t  structure places  the c y s t e i n e r e s i d u e  a c t i v e s i t e o f the enzyme.  A t the  2.5A  r e s o l u t i o n x-ray  126 In the  catalytic  same time, i t i s p o s s i b l e  t h a t m o d i f i c a t i o n remote from the a c t i v e s i t e c o u l d induce a conformational  change t h a t would a l t e r T I M s c a t a l y t i c c  ability.  Thus, i f a change i n conformation d i d r e s u l t i n a c t u a l changes i n the s t r u c t u r e of the a c t i v e s i t e which made c a t a l y s i s more d i f f i c u l t , a decrease i n the s p e c i f i c a c t i v i t y would be  observed.  T h i s p o s s i b i l i t y has a s p e c i a l s i g n i f i c a n c e upon examination of the t r l o s e phosphate Isomerases prepared f o r the 2.5A  resol-  u t i o n x-ray s t r u c t u r e which i n d i c a t e d t h a t the most r e a c t i v e c y s t e i n e was  a t a s i t e remote from the c a t a l y t i c c e n t e r .  It  was  99 • found t h a t the SH o f r e s i d u e 21? was  the most r e a c t i v e to mercury  compounds such as 2 - c h l o r o B i e r c u r i - 4 - n I t r o p h e n o l . s m a l l e r and more hydrophobic  However, a  m e r c u r i a l , ethyl-mercury  phosphate,  r e a c t e d w i t h a l e s s r e a d i l y a c c e s s i b l e p a i r of s u l p h y d r y l groups at  r e s i d u e 41. B. In  t h a t TIM to  Disulfides a d d i t i o n t o the aforementioned  m a l e i i s l d e s , i t was  reacted r e a d i l y with d i s u l f i d e s .  This i s i n contrast  r e a c t i o n w i t h 3 - h r o m o - l , l , l - t r i f l u r o p r o p a n o n e or  a c i d which d i d not occur to any Goetze,  BSc  t h e s i s , UBC,  found  n o t i c e a b l e extent  brompacetic  (Andrew  M.  1973).  However, when DTNB i s a l l o w e d to r e a c t w i t h Peak A TIM,  the  r e s u l t s I n d i c a t e t h a t t h e r e are 2 SH groups which a r e exposed for  ready m o d i f i c a t i o n by the d i s u l f i d e : upon d e n a t u r a t i o n 6 more  are a b l e to r e a c t to g i v e a t o t a l o f 8 c y s t e i n e s p r e s e n t . agrees w e l l w i t h the 8 c y s t e i n e r e s i d u e s per dimer found amino a c i d a n a l y s i s .  Repeated experiments  This l n the  u s u a l l y gave r e s u l t s  which v a r i e d o n l y one or two p e r c e n t from the Integer v a l u e s of 2.0  and  8.0  f o r the r e s p e c t i v e 'exposed' and  The  r e s u l t s f o r Peak B p r o t e i n were s i m i l a r w i t h 1.7  titrated initially upon a d d i t i o n o f The  by DTNB, and an average  cyanide displacement  o f TNB" The  a n a l y s i s w i t h d a t a g i v e n to the t h i r d f i g u r e 21..  o f 8.0  titration. SH's  being  being obtained  SDS.  protein occurred quite r e a d i l y .  in  t o t a l SH  A l e a s t squares  from the DTNB m o d i f i e d pseudo f i r s t half l i f e  treatment  order  kinetic  is illustrated  y i e l d e d the best  straight  FIGURE 21»  Pseudo F i r s t Order A n a l y s i s o f the Cyanld Displacement o f TNB~ from the DTNB M o d i f i e d peak A P r o t e i n Insert i  I n c r e a s e i n the Absorbance the R e a c t i o n Proceeds  I  ,  .  ,  6 T ~i 1  2  f TIME  :  (MIN)  d 3  as  r—  12 (MIN) :  r 4  IOI. l i n e f o r the d a t a and the s l o p e thus c a l c u l a t e d gave a v a l u e of —3  —1  1 1 . 1 6 x 1 0 " - ' sec  f o r the pseudo f i r s t o r d e r r a t e constant f o r  the displacement  o f TMB by CN" a t pH 7 * 5 and 2 0 ° C .  I t was observed of TNB" (average  t h a t CN" i s a b l o t o d i s p l a c e 0 . 8 5 e q u i v a l e n t s  of 3 runs).  be a nonequivalence  I n the a b i l i t y o f m o d i f i e d c y s t e i n e r e s i d u e s  to r e a c t w i t h r e a g e n t s . due  Thus, once a g a i n t h e r e appears t o  The same e x p l a n a t i o n s o f nonequivalence  t o c o n f o r m a t i o n a l d i f f e r e n c e may be invoked here a l s o .  schematic  A  summary o f the r e a c t i o n s i n c l u d e s s DTNB E  > E(TNB) pH 8  The nonequivalence  ?  CN" —-» E(TNB)CN pE 7 . 5  o f the DTNB m o d i f i e d p r o t e i n was not ob-  served upon a c t i o n o f d i t h l o e r y t h r e i t o l on t h e E ( T N B ) the enzyme.  form o f  2  Upon a d d i t i o n o f DTE 2 . 0 8 e q u i v a l e n t s o f TNB~ p e r  mole o f TIM were l i b e r a t e d .  The d i f f e r e n c e between the r e s u l t s  of the CN" and DTE experiments might be e x p l a i n e d by d i f f e r e n t a b i l i t i e s o f the reagents  t o e n t e r the s i t e o f DTNB m o d i f i c a t i o n ,  on the enzyme. R e s u l t s from t h e s p e c i f i c a c t i v i t y measurements performed upon the v a r i o u s m o d i f i e d p r o t e i n s a r e summarized belows Specific Activity Specific Activity Protein (units) {%) ' (1)  Control  8368  100  (2)  E(TNB)  2059  24.6  (3)  E(TNB)CN  2410  28,8  (4)  E(TNB)  8477  101.3  2  2  + DTE.  .  102  The  t o t a l a c t i v i t y o f the enzyme was r e g a i n e d upon a d d i t i o n  of DTE. TNB"  T h i s a l o n g w i t h t h e o b s e r v a t i o n o f 2.08 e q u i v a l e n t s o f  l i b e r a t e d i n d i c a t e s t h a t the TIM molecule has resumed a na-  t i v e unmodified  state.  I t i s i n t e r e s t i n g t o note t h a t the s p e c i f i c a c t i v i t y o f the E(TNB)  2  form o f the enzyme (24.6$) i s e s s e n t i a l l y i d e n t i c a l t o  t h a t o f the malelmlde m o d i f i e d s p e c i e s ( 2 5 . 0 $ ) .  T h i s l e n d s sup-  p o r t to our e a r l i e r assumption t h a t DTNB and maleimides ( l e . NEM and FEM) modify the same s i t e s on TIM.  The s p e c i f i c a c t i v i t y o f  the E(TNB)CN form o f TIM (28.8$) i s a l s o very  similar.  The r e a c t i o n o f TIM w i t h the second d i s u l f i d e idine  (4-PDS) f o l l o w e d a l o n g the same l i n e s as DTNB to y i e l d  ues o f 2.0? exposed SH t i t r a t e d under nondenaturing and  4,4-dithlopyr-  8.02 t o t a l SH's t i t r a t e d under d e n a t u r i n g  a t o t a l o f 5.95 SH's 'hidden*.  val-  conditions  conditions to y i e l d  The s p e c i f i c i t y o f DTNB appears  to apply a l s o t o 4-PDS when t i t r a t i n g TIM.  However, t h e g r e a t e r  working range o f pH c o u l d make I t more u s e f u l f o r f u r t h e r m o d i f i c a t i o n s t u d i e s o f TIM ( o r indeed any other c y s t e i n e c o n t a i n i n g protein). A r a t h e r I n t e r e s t i n g example o f the nonequivalence  o f the  DTNB m o d i f i e d s i t e s o f TIM was observed  when 4-PD3 was allowed  to r e a c t w i t h the E ( T N B )  The experiment  2  enzyme form.  illustrates  t h a t 1.01 e q u i v a l e n t s o f 4-PDS were a b l e t o r e a c t w i t h the enzyme under non-denaturing  contitlons.  When SDS was added ( a f t e r  tit-  r a t i o n of the 1.01 r e s i d u e s ) 5.53 a d d i t i o n a l groups r e a c t e d t o y i e l d a t o t a l o f 6.61 r e a c t i v e groups.  Again  i t appears t h a t  t h e r e i s one o f the two DTNB m o d i f i e d s i t e s which i s a b l e t o r e -  i03 act further.  The 6 'hidden* c y s t e i n e s a r e o n l y a b l e t o r e a c t  when the p r o t e i n I s u n f o l d e d . In T o r c h l n s k i i * s e x c e l l e n t t e x t d e a l i n g w i t h s u l f h y d r y l and d i s u l f i d e groups o f p r o t e i n s , he g i v e s a p o s s i b l e account o f the r e a c t i o n s o c c u r r i n g i n a d i s u l f i d e exchange r e a c t i o n ( ? 8 ) . R'-S-S-R' + R»-S-S-R*  s  2R'-S-S-R*  Three mechanisms f o r the exchange a r e g i v e n :  one f o r n e u t r a l  and b a s i c s o l u t i o n s , one f o r a c i d i c and f i n a l l y a f r e e r a d i c a l mechanism.  A t the pH of the experiment (6.5) t h e r e  p o s s i b i l i t y o f more than one o c c u r r i n g . (a)  should be a  They a r e g i v e n  below:  Basic or n e u t r a l s o l u t i o n  The r e a c t i o n i s c a t a l y z e d by t h i o l s  ( c a t a l y t i c q u a n t i t i e s can  a r i s e by h y d r o l y t i c cleavage o f d i s u l f i d e s ) which c a r r y out n u c l e o p h l l l c a t t a c k on a s u l f u r atom o f the d i s u l f i d e as i n a t h i o l disulfide  (b)  exchange.  (1)  R\S~ + R S-SR*  (2)  R^S" + R«S-SR  » R'S-SR« + R S "  W  W  f  =====  R*S-SR' + R'S~  A c i d Media  The exchange takes p l a c e through a s u l f e n l u m  c a t i o n which i s  formed through a t t a c k o f a p r o t o n on the S-S bond. (1)  R'S-S-R' + H  (2)  R'S + R*S-SR  (3)  R*S  +  +  ==?  +  W  =====  + R'S-SR* =====  R-S-S-R H. R»S-SR*  =====  R'SH + R'S  + R S W  R"S-SR' + R ' S  +  The r e a c t l o n ( s ) would be i n h i b i t e d by t h i o l s : BSH.+ R'S = = * RS-SR' + H +  +  +  +  104. o)  Free r a d i c a l The reaction occurs with p a r t i c i p a t i o n of free r a d i c a l s  which can a r i s e either from high temperatures (>100°C) or u l t r a violet radiation.  Since the reaction i s followed a t 324nm,  there i s the p o s s i b i l i t y of some photolysis occurring.  Radicals  a r i s i n g from thermal cleavage can be ruled out. (1)  R'S-SR  (2)  H ' S ' + . R«-S-SR  (3)  R*S«  1  +  2R»S« R*S-SR'  W  R'S-SR"  R®S-3R'  +  R S* M  • B ' S -  Assuming that one, two or a l l mechanisms are operative to some extent i n the case of E(TNB) reacting with 4-PDS, the f o l 2  lowing scheme can be writtent  E  DTNB PH 8  E (TNB) g  4-PDS pH 6.5  E(TNB) (4-TP)  105,  CHAPTER V • KINETICS OF DISULFIDE MODIFICATION OF 5.1  TIM  Methods Twice chromatographed  Peak A p r o t e i n was prepared f o r k i n e t i c  runs by g e n t l e s t i r r i n g o v e r n i g h t a t 4°C i n 25mM d i t h i o t h r e i t o l or d i t h l o e r t h y r e l t o l .  The p r o t e i n was  d e s a l t e d on a 1 . 5 X 2 0 cm  Sephadex G25 column e l u t e d w i t h 200mM phosphate b u f f e r , pH  6.5  i n the case of ^ j ^ ' d l t h i o p y r i d l n e runs and pH 8.0 f o r d i t h i o bis-(2-nitrobenzoate) k i n e t i c s .  Approximately 2 ml f r a c t i o n s  were c o l l e c t e d by a G l l s o n M i c r o f r a c t i o n a t o r , and t h e i r ance a t 280nm determined on a Z e i s s PMQII U V - V i s i b l e photometer.  absorb-  spectro-  F r a c t i o n s w i t h Aggg^O.S were kept i n i c e u n t i l  ready f o r u s e . The most e x t e n s i v e s e r i e s of k i n e t i c s i n v o l v e d of t r i o s e phosphate  modification  isomerase w i t h ^ j V d l t h l o p y r i d i n e .  Kinetics  of the m o d i f i c a t i o n of TIM were performed a t 37°,31°,2?°,25°,22°, and 15°C.  The k i n e t i c s were f o l l o w e d both l n the presence and  i n the absence  of s u b s t r a t e (G3P) f o r each temperature  The p r o g r e s s of the r e a c t i o n was  cited.  f o l l o w e d by o b s e r v i n g the  lib-  e r a t i o n o f the t h i o p y r l d o n e v i a an i n c r e a s e i n the absorbance a t 324nm.  The runs were done i n e i t h e r d u p l i c a t e or t r i p l i c a t e ;  the c a l c u l a t e d r a t e c o n s t a n t s b e i n g an average of the i n d i v i d u a l v a l u e s o b t a i n e d from each i n d i v i d u a l experiment.  A l l constit-  uents of the r e a c t i o n mixture as w e l l a s the b l a n k were e q u i l i b r a t e d a t the temperature of the experiment.  The  temperature  106.  was h e l d c o n s t a n t throughout t h e experiment by means o f a c i r c u l a t i n g water b a t h which passed water o f a c o n s t a n t temperature through t h e c e l l h o l d e r o f the spectrophotometer. I n d i v i d u a l k i n e t i c runs were i n i t i a t e d by r a p i d d e l i v e r y o f o f 4, V d l t h l o p y r l d i n e , 26 mgs/ml, v i a a Lang-Levy  SOjxls  i n t o a 3 ml c u v e t t e which c o n t a i n e d l ml o f p r o t e i n  sample  ( A g = 1) and 1 . 9 5 mis o f 200mM pH 6 . 5 phosphate b u f f e r . 2  pipet  0  The r e -  a c t i o n mixture was blanked a g a i n s t a 3 ml c u v e t t e c o n t a i n i n g 50 ^utls o f reagent and 2 . 9 5 mis b u f f e r . to  F o r those runs which were  i n c l u d e s u b s t r a t e , l O O y ^ l s o f g l y c e r a l d e h y d e 3-phosphate  (0.57mM) was added t o both r e a c t i o n c e l l and b l a n k i n p l a c e o f 100jO.B  of buffer.  A t t h e completion o f each experiment, a  s m a l l amount (~10 mgs) o f sodium d o d e c y l s u l f a t e  (SDS) was added  to  the r e a c t i o n c e l l and t h e f i n a l A-^gZ* r e s u l t i n g from  of  a l l c y s t e i n e s , both hidden and exposed was determined.  titration  The k i n e t i c experiments i n v o l v i n g m o d i f i c a t i o n o f TIM by DTNB were performed s i m i l a r i l y .  Runs a t 3 5 - 5 ° and 22°C were done,  both i n t h e presence and i n t h e absence o f G3P. the  Preparation of  p r o t e i n was a t pH 8.0 a s d e s c r i b e d p r e v i o u s l y ; r e a g e n t s ,  b u f f e r , and p r o t e i n were p r e - e q u l l i b r a t e d a t t h e temperature o f the  experiment.  The k i n e t i c runs were performed a t c o n s t a n t  temperature. The experiments were i n i t i a t e d by t h e p i p e t t i n g o f 1.0 ml of  protein  (A25Q=  1»0) Into a 3 ml c u v e t t e c o n t a i n i n g 2.0 mis  reagent s t o c k s o l u t i o n c o n s i s t i n g o f lOOmM phosphate pH 8.0, lOmM EDTA, and 5»25mM DTNB.  F o r those k i n e t i c runs which were  107  to  c o n t a i n s u b s t r a t e , l O O y i l o f G3P («57mM) was added along w i t h  1.90 of  mis r e a g e n t , and 1.0 ml p r o t e i n s o l u t i o n .  The m o d i f i c a t i o n  TIM by DTNB was f o l l o w e d by o b s e r v i n g the l i b e r a t i o n o f TNB~  as shown by the i n c r e a s e i n absorbance a t 4l2nm. at  4l2nm was determined  a t v a r i o u s time i n t e r v a l s u n t i l a con-  s t a n t value was o b t a i n e d . was  then determined  Increase i n A ^  l 2  The absorbance  The t o t a l t h i o l content o f the p r o t e i n  upon a d d i t i o n o f s o l i d SDS; w i t h a f u r t h e r  r e s u l t i n g from d e n a t u r a t l o n o f the p r o t e i n and  exposure o f hidden  c y s t e i n e r e s i d u e s to m o d i f i c a t i o n by DTNB.  Data Treatment The pseudo f i r s t lows w i t h " a  1  equal to i n i t i a l c o n c e t r a t l o n o f reactant ( l e . r e -  active t h i o l s ) , • t*  order r a t e equation may be w r i t t e n as f o l -  (a-x) meaning c o n c e n t r a t i o n o f r e a c t a n t a t time  and k b e i n g the pseudo f i r s t  order r a t e c o n s t a n t .  a In  . - kt a-XL  Since  a  i s p r o p o r t i o n a l t o the f i n a l absorbance a t 4l2nm ( f o r  DTNB k i n e t i c s ) o r 324nm ( f o r 4-PDS k i n e t i c s ) the s u b s t i t u t i o n may be made whereby In  . - kt  T h e r e f o r e a p l o t o f In  versus time t h e o r e t i c a l l y g i v e s a Ao~ t A  s t r a i g h t l i n e going through was  p l o t t e d t o the t h i r d  the o r i g i n w i t h s l o p e k.  A l l data  half-life.  However, the pseudo f i r s t  o r d e r p l o t s were not always ob-  served to be l i n e a r s i n c e there were two ( o r more) r e a c t i v e t h i ols  and each SH n o t n e c e s s a r i l y r e a c t i n g a t t h e same r a t e .  When  3.0S. the k i n e t i c p l o t was mine the  slope  bi-phasic,  i t was  necessary to f i r s t  of t h a t l i n e a r p o r t i o n of the  curve, a t l a t e r time  ( t ) where r e a c t i o n of the more r e a c t i v e t h l o l ( s ) was  complete  the s l o p e r e p r e s e n t s the r a t e of r e a c t i o n f o r the slower thiol.  T h i s s l o p e was  l i n e a r p o r t i o n and performed.  then s u b t r a c t e d  In the case of b i - p h a s i c  now  be l i n e a r and  for  the f a s t e r r e a c t i n g A  from the  a r e p l o t of the new  deter-  and  reacting  e a r l i e r non-  values against  time  k i n e t i c s , the r e p l o t  the s l o p e would r e p r e s e n t the r a t e  was should  constant  thiol.  t y p i c a l example of mono-phasic k i n e t i c s i s i l l u s t r a t e d  f i g u r e 22 which shows the pseudo f i r s t thiopyridine  (4-PDS) m o d i f i c a t i o n  of s u b s t r a t e  (G3P).  order p l o t f o r  of TIM  A t y p i c a l bi-phasic  in  4,4"-di-  a t 22°C In the  absence  p l o t , along with i t s  r e p l o t , i s shown i n f i g u r e 23 which demonstrates the 4-PDS modi f i c a t i o n o f TIM  a t 25°C i n the absence of s u b s t r a t e  r e s u l t s of these p l u s the s e c t i o n f o l l o w i n g . was  The  the other temperature runs are g i v e n i n In a l l c a s e s , a l e a s t squares a n a l y s i s  performed on the l i n e a r p l o t s . A u s e f u l quantity  to know i s the  of a r e a c t i o n which i s the  1  half-life*  * half-period'  There i s a simple r e l a t i o n -  s h i p between the h a l f - l i f e of the r e a c t i o n and r a t e constant  or  time i t takes f o r h a l f the o r i g i n a l  substance t o d i s a p p e a r ( i e . r e a c t ) .  the f i r s t  0.693 k  f o r T i n minutes when k i s i n min  h a l f - l i f e , as a u s e f u l concept f o r s e m i - q u a n t i t a t i v e  s i o n s , was  order  • k't  TJL = — * The  (G3P).  calculated for a l l rates  determined.  discus-  .  FIGURE 2 2 :  A T y p i c a l Example o f Monophaslc K i n e t l c s 4-PDS M o d i f i c a t i o n  of TIM a t 22°G i n the  Absence of G l y c e r a l d e h y d e  5  10  3-Phosphate  15 T I M E (MIN )  20  25  no.  "FIGURE 23J  A T y p i c a l Example of E i - p h a s i c K i n e t l c s 4-PDS M o d i f i c a t i o n of TIM a t 25°G i n the Absence of G l y c e r a l d e h y d e 3-Phosphate Insert:  R e p l o t of B i - p h a s l c  Area  .2(h  5  10  20  40 T I M E  ( MIN)  •  60  ill. An important  r e l a t i o n s h i p i n k i n e t i c s which p r o v i d e s much  i n f o r m a t i o n concerning mechanism,, i s one t h a t connects constant of a r e a c t i o n w i t h the temperature! The  law may be expressed k = : A e  s  E  the A r r h e n i u s  Law.  as f o l l o w s $ -Ea/RT  w i t h 'k* the r a t e c o n s t a n t , 'A* the frequency action,  the r a t e  f a c t o r of the r e -  the a c t i v a t i o n energy, *R" the u n i v e r s a l gas con-  f a  s t a n t and *T* as temperature (°Kelvin).  T h e r f o r e a p l o t of  l o g kCsec"" ) v e r s u s 1/T K y i e l d s a s l o p e o f -Ea/2.303R o r -Ea/4.57 1  w i t h i n t e r c e p t A.  T h i s energy o f a c t i v a t i o n r e p r e s e n t s the energy  t h a t the r e a c t a n t s must a c q u i r e i n order t o undergo r e a c t i o n and as such i s one measure of the ease o f r e a c t i o n ;  112.  5.2  Results A.  M o d i f i c a t i o n o f TIM w i t h 4-PDS  The r e s u l t s o b t a i n e d from t h e pseudo f i r s t  order  kinetic  p l o t s o f t h e m o d i f i c a t i o n o f TIM w i t h 4-PDS a r e shown belows TABLE V i  (1) T°C  37  M o d i f i c a t i o n w i t h 4-PDS  In presence  o f G l y c e r a l d e h y d e 3-phosphate , , •. #SH"s' m o d i f i e d *  S p e c i f i c Rate Constant (mln-1)  (min)  = .3950  1.75  2.0  .3259 .1681  2.13 4.12  2.0  = .2804  2.47  2.0  .2413  2.87 11.42  2.5  .1297  5.34 10.88  2.4  = .0637 = .0149  46.51  2.6  k  l  31 k  2  k  l  27 25  k  22  2 = .0607  k  15  k  2  l  T.L  In a l l cases, 8.0 SH's were m o d i f i e d under d e n a t u r i n g c o n d i t i o ] (2)  In absence of G l y c e r a l d e h y d e  3-phosphate  37  .3421  2.03  2.0  31  = .2880  2.41  2.0  .2760 .1432  2.51 4.84  2.0  = • 2731  2.54 18.63  2.0  .0372  = .0788  8.79  2.0  .0289  23.98  2.0  27 25  K  2  k  l 2  k  22 15  k  l  113  From r e s u l t s p r e s e n t e d i n T a b l e to be any  V - 1 , 2 , t h e r e does not seem  s p e c i a l dependence upon temperature or presence or  sence of s u b s t r a t e when o b s e r v i n g i n the k i n e t i c p l o t s .  ab-  mono- or b i - p h a s l c behaviour  However, the presence of b i - p h a s l c behav-  i o r does i n d i c a t e t h a t under c e r t a i n c o n d i t i o n s or a v a i l a b i l i t y of s u b s t r a t e )  there was  (of temperature  a non-equivalence i n the  p o t e n t i a l s i t e s o f m o d i f i c a t i o n such t h a t the r e a c t i o n r a t e s were a b l e to d i f f e r by a f a c t o r of as much as e i g h t absence of s u b s t r a t e ) . concerning  As  previously discussed  p r o t e i n m o d i f i c a t i o n s , there  (eg:  i n the  chapter  i s the p o s s i b i l i t y  r e a c t i o n of a s i n g l e t h i o l Inducing a c o n f o r m a t i o n a l the p r o t e i n such t h a t the second t h i o l i s now  25°C>  of  change In  i n a less favor-  a b l e p o s i t i o n to r e a c t . I t i s evident were m o d i f i e d  from the l i s t i n g of the number of SH's  i n the v a r i o u s  which  experiments t h a t the b i n d i n g of sub-  s t r a t e a t temperatures a t or below 25°C Is a b l e to the t i t r a t i o n of a p o r t i o n of a t h i r d t h i o l .  influence  I t Is known that  the s u b s t r a t e G 3 P or DHAP i s a b l e to change the conformation of TIM  to a s i g n i f i c a n t degree ( 3 8 ) which might account, i n p a r t ,  f o r the exposure of the a d d i t i o n a l t h i o l .  However, an a d d i t i o n a l  temperature dependent mechanism appears to be a t work s i n c e modi f i c a t i o n of more than 2.0 e r a t u r e s above 25°C.  s u l f h y d r y l s does not occur a t temp-  At the same time, the  complete absence of  a t i t r a t i o n of a t h i r d t h i o l i n those experiments which were p e r formed i n the absence of s u b s t r a t e , pendence upon the c o n f o r m a t i o n a l  seems to i n d i c a t e some de-  changes which the b i n d i n g  of  ii4  s u b s t r a t e can Induce. the t h i r d t h i o l  .  The o b s e r v a t i o n t h a t o n l y a p o r t i o n o f  ( 0 . 4 t o 0.6) was t l t r a t a b l e c o u l d i n d i c a t e t h e  e x i s t e n c e o f an e q u i l i b r i u m between some • r e a c t i v e * and "nonreactive*  s t a t e of the t h i r d t h i o l and hence i n i t s p a r t i a l mod-  i f i c a t i o n by 4 , 4 ' d i t h l o p y r i d i n e . The r a t e c o n s t a n t s o f the most r a p i d l y r e a c t i n g t h i o l ( k ) 1  were employed  i n an A r r h e n i u s p l o t o f l o g k v e r s u s 1/T K.  The  r e s u l t s f o r those experiments which c o n t a i n e d G3P i s shown i n f i g u r e 24 and f o r those which l a c k e d G3P i s i l l u s t r a t e d  i n figurs  25.  A very prominent b i - p h a s i c c h a r a c t e r i s t i c i s apparent i n both p l o t s .  F o r those experiments performed w i t h substrate,, the  break comes a t 24.6°C w i t h t h e a c t i v a t i o n energy o f the 4-PDS m o d i f i c a t i o n b e i n g 7 » 2 k c a l / m o l e above t h i s temperature and 5 0 » 0 kcal/mole below i t .  However, t h e second p l o t  (without G3P) ex-  h i b i t e d a break a t 2 5 . 7 ° C w i t h the upper l i m b ( T > 2 5 . 7 ° C ) g i v i n g an a c t i v a t i o n energy of 4 . 4 (T<25.7°C) 3 9 . 9  kcal/mole and t h e lower limb  kcal/mole.  The presence o f t h i s type o f A r r h e n i u s p l o t i s not unknown i n the l i t e r a t u r e .  Copious examples a r e a v a i l a b l e which  t r a t e temperature dependent  illus-  a c t i v a t i o n e n e r g i e s o f the c a t a l y t i c  p r o c e s s o f an enzyme, which i s analogous to the temperature dependent r e a c t i v i t y o f a s p e c i f i c group, t h a t i s t h e t h i o l o f c y s t e i n e , which has been i l l u s t r a t e d l n t h e A r r h e n i u s p l o t s o f f i g u r e 2 4 and 2 5 . Some i n s i g h t i n t o t h e temperature dependent  mechanism i n TIM  U5 .  126, ^FIGURE 2 5 J  A r r h e n l u s P l o t f o r t h e 4-PDS M o d i f i c a t i o n of TIM i n t h a Absence of G l y c e r a l d e h y d e 3-Phosphate  117 • might be gained by l o o k i n g more c l o s e l y a t some of the examples i n the l i t e r a t u r e of temperature  dependent p r o c e s s e s o c c u r l n g i n  enzymes. G l u t a t h i o n e r e d u c t a s e from P. chrysogenum has been shown to o have a c a t a l y t i c a c t i v a t i o n energy of 14.3 kcal/mole between 20 and 30°C and 11.8  kcal/mole between 30°and 40°C (79).  ment of c o n f o r m a t i o n a l change was  Invoked  The  argu-  t o e x p l a i n the r e s u l t s .  Massey et a l (80) a l s o o b t a i n e d n o n l i n e a r A r r h e n i u s p l o t s f o r amino a c i d oxidase which was  found to undergo a temperature  de-  pendent c o n f o r m a t i o n a l change as observed by changes i n the sedi m e n t a t i o n c o n s t a n t , d i f f e r e n c e B p e c t r a , and f l u o r e s c e n c e . c r i t i c a l temperature  f o r these phenomena c o i n c i d e d w i t h the break  i n the A r r h e n i u s p l o t .  An even more r a d i c a l example of a b l -  p h a s i c A r r h e n i u s p l o t a r i s i n g from temperature a t i o n a l change was mutase ( 8 1 ) .  observed i n the CMII isozyme  dependent  was  observed.  The p l o t I l l u s t r a t e d an a c t i v a t i o n energy of 9«i80  However, the l a r g e  changes l n conformation which may  of TIM  No  further  pH, or s u b s t r a t e i n f l u e n c e d c o o p e r a t i v e i n t e r a c t i o n (11X) a c t i v a t i o n energy  ence d i s p l a y e d by the mutase i s an i n d i c a t i o n of the  temperature.  conform-  of chorismate  kcal/mole above 25°C and 99.000 kcal/mole below 25°C. temperature,  The  differ-  significant  take p l a c e as a f u n c t i o n of  The r e a c t i v i t y of the most e a s i l y m o d i f i e d t h i o l  (by 4-PDS) changes by a f a c t o r of about  7 l n the a c t i v a -  t i o n energy i n the presence of s u b s t r a t e and approximately 9 when s u b s t r a t e i s not p r e s e n t .  The m o d i f i c a t i o n r e s u l t s i n the  p r e c e d i n g chapter do suggest t h a t Peak A TIM i s s e n s i t i v e to c o n f o r m a t i o n a l change.  However, an i n t e r e s t i n g c o n t i n u a t i o n of  118  the d i s c o v e r y o f b i - p h a s i c A r r h e n i u s would be  p l o t s f o r the Peak A p r o t e i n ,  i n v e s t i g a t i o n Into whether the phenomenon occurs  other two  isozymes.  i n the  T h i s c o u l d be an i n d i c a t i o n of e s s e n t i a l  d i f f e r e n c e s between the d i f f e r e n t enzyme forms of chicken  muscle  TIM. An  i n t e r e s t i n g example of a break i n an A r r h e n i u s  found i n muscle phosphorylase k i n a s e  plot  was  (82) i n which the phenom-  enon was  e n t i r e l y absent a t 7 ^ g s / m l p r o t e i n but p r e s e n t at  28  jlgs/ml.  The  an  marked d i s c o n t i n u i t y was  centered at l 5 ° C with  a c t i v a t i o n energy of 18.200 kcal/mole a t h i g h e r temperatures 2.600 kcal/mole a t lower temperatures.  T h i s unusual example of  an enzyme w i t h a higher a c t i v a t i o n energy a t the higher e r a t u r e was  and  temp-  e x p l a i n e d by i t s c a t a l y t i c a c t i v i t y b e i n g c o n t r o l l e d  by a combination of s u b s t r a t e b i n d i n g and a s s o c i a t i o n - d i s s o c i a t i o n of the enzyme.  T h i s example has  some i n t e r e s t s i n c e i t has  been r e p o r t e d t h a t TIM's c a t a l y t i c a b i l i t y i s a f f e c t e d by i t s concentration The  (83).  k i n e t i c t i t r a t i o n s of TIM  SH's  i n t h i s t h e s i s were c a r r i e d  out a t p r o t e i n c o n c e n t r a t i o n s of about 0.3 of Poole e t a l (83) c r y s t a l l i n e TIM was  mg/ml.  In the  i s o l a t e d from r a b b i t muscle  d i l u t e d to c o n c e n t r a t i o n s between 1 mg/ml and  5 mgs/ml.  g r e s s i v e i n c r e a s e of enzymatic a c t i v i t y w i t h time was w i t h r a t e of c a t a l y s i s by TIM  study  observed  i n c r e a s i n g up to t w i c e the  value observed immediately a f t e r d i l u t i o n .  Pro-  initial  In a d d i t i o n , i t was  found t h a t the c o n c e n t r a t i o n of m e r c u r i a l i n h i b i t o r s r e q u i r e d to reduce TIM  a c t i v i t y by h a l f , as w e l l as the r a t e o f r e a c t i o n of  t h i o l s to i o d o a c e t a t e , was  dependent upon c o n c e n t r a t i o n of p r o t e i n ^  119  I t has been suggested t h a t the observed e f f e c t s a r e due  to con-  formational  d i f f e r e n c e s of the  enzyme a t d i f f e r e n t p r o t e i n  centrations  which c o u l d be due  to the p r o t e i n s  present i n aggregation.  The  k i n e t i c experiments r e p o r t e d  concentration In t h i s t h e s i s  enough to show these conformation and The  protein  interactions  of p r o t e i n used i n (0;3  mg/ml) are  son^s r e p o r t s  f u r t h e r expanded by  on f o u r e s t e r a s e s  (84).  Hipps and  They found t h a t the  and 14.5  8.5  kcal/mole a t higher temperatures and  kcal/mole a t lower temperatures.  The  Nel-  ester-  characterized  by double s l o p e d A r r h e n i u s p l o t s w i t h a c t i v a t i o n e n e r g i e s  and  high  aggregation e f f e c t s .  a s e s , as p u r i f i e d from the American cockroach were  11.6  the  p o s s i b i l i t y of temperature dependent mechanisms other  than c o n f o r m a t i o n a l change was  tween 6.3  con-  be-  between  data  was  l i n k e d to a s s o c i a t i o n d i s s o c i a t i o n phenomenon ( l e . f o r m a t i o n of enzyme aggregates) which i n d i c a t e d t h a t the ases p r o b a b l y e x i s t as  thermally  cockroach gut  dependent m o l e c u l a r aggregates  w i t h d i f f e r e n t r a t e s o f h y d r o l y t i c a c t i v i t y i n the and  d i s s o c i a t e d forms.  Thus, l o w e r i n g the  i s known to form c o n c e n t r a t i o n  In a d d i t i o n , i t has been w e l l s u b s t a n t i a t e d a s s o c i a t i o n of g l y c o l y t i c p r o t e i n s u n i t s In the  associated  temperature d i s s o c i a t e s  the aggregates i n t o l e s s a c t i v e s u b u n i t s ( 8 4 ) . e a r l i e r TIM  ester-  As  mentioned  dependent aggregates. t h a t t h e r e i s some  ( i n c l u d i n g TIM)  'In v i v o ' s i t u a t i o n (85,86,87,83):  Into  large  Therefore,  the  p o s s i b i l i t y of some s o r t of a s s o c i a t i o n - d i s s o c i a t i o n mechanism f o r the b l p h a s i c A r r h e n i u s p l o t b e h a v i o r of TIM  cannot be  ruled  out. In c o n c l u s i o n ,  It i s difficult  to suggest w i t h any  certainty,  120  the o p e r a t i v e temperature dependent mechanism i n TIM with the data a v a i l a b l e a t t h i s p o i n t ;  However, t h e mechanism i s not  o b v i o u s l y s e n s i t i z e d by s u b s t r a t e b i n d i n g a l t h o u g h  t h e (-) sub-  s t r a t e case does have somewhat lower a c t i v a t i o n e n e r g i e s . break i n the p l o t s i s about i°C a p a r t which i s w i t h i n the experimental  (24.6*0 versus  error.  The  25*7*0)  There i s a g r e a t de-  gree o f d i f f i c u l t y i n drawing a c c u r a t e A r r h e n i u s p l o t s w i t h t h e few  temperatures which were i n v e s t i g a t e d . B.  M o d i f i c a t i o n o f T I M w i t h DTNB  The k i n e t i c s o f the DTNB m o d i f i c a t i o n s o f Peak A p r o t e i n was  i n v e s t i g a t e d a t two temperatures, 22°C and 35»5 C. <>  The r e -  s u l t s a r e shown belowt (1)  (+) s u b s t r a t e S p e c i f i c Rate Constant  (2)  #SH m o d i f i e d  35-5  k-, = .3625 kg = .1885  2.0  22  k j = .0425  2.0  (-) s u b s t r a t e T°  S p e c i f i c Rate Constant  #SH modified  35-5  K = .3555 kg = .2311  2.0  22  k  2.0  1  = .0355  121  The phenomenon o f t i t r a t i o n o f more than 2.0 t h i o l s i s absent i n the DTNB m o d i f i c a t i o n o f TIM. was s t i l l  However, b i p h a s i c b e h a v i o r  observed i n the case o f those runs performed a t 35«5°C  which i n d i c a t e s some s i m i l a r i t y i n enzyme changes o c c u r i n g . However, the b i p h a s i c b e h a v i o r was n o t n e a r l y as marked as t h a t o b t a i n e d w i t h 4-PDS and so, w i t h the time l i m i t a t i o n s which were present f o r t h i s segment of the t h e s i s , t h e m o d i f i c a t i o n s w i t h DTNB were not pursued beyond two temperatures.  However, the  c l o s e chemical r e a c t i v i t y o f DTNB and ^ j V d i t h i o p y r i d l n e the p o t e n t i a l i n t e r e s t o f more extended temperature  indicates  variation.  With the l i m i t e d d a t a a v a i l a b l e , i t would seem i n a p p r o p r i a t e to r e p o r t a c t i v a t i o n e n e r g i e s o f the (+) s u b s t r a t e and (-) subs t r a t e systems.  Only two p o i n t s would be a v a i l a b l e f o r each  A r r h e n i u s p l o t and thus a low degree of c o n f i d e n c e would be p l a c e d i n the v a l u e s o b t a i n e d .  In a d d i t i o n , c a u t i o n must be e x e r c i s e d  i n d e r i v i n g any i n t e r p r e t a t i o n from r e s u l t s o b t a i n e d from such A r r h e n i u s p l o t s s i n c e t h e temperature i n t e r v a l a c r o s s the b r e a k i n g p o i n t  (22-35.5°C) cuts  (about 25°C) observed i n the A r r h e n i u s  p l o t s of 4-PDS m o d i f i c a t i o n .  122 .  CONCLUSIONS In the  c o n c l u s i o n , i t might he advantageous t o b r i e f l y  discuss  s i g n i f i c a n c e o f the r e s u l t s r e p o r t e d i n t h i s t h e s i s i n r e -  l a t i o n t o the e x i s t i n g knowledge  of t # r i o s e phosphate isomerase.  An i n d i c a t i o n o f the p u r i t y o f t h e i s o l a t e d c h i c k e n muscle enzyme i s t h a t I t was found t o possess s p e c i f i c a c t i v i t y up t o 12,000 units/mg which i s a t l e a s t as h i g h o r h i g h e r than the optimum o f 10,000 units/mg which has been r e p o r t e d .  Also, the  chromatographic s e p a r a t i o n o f TIM c l e a r l y demonstrated t h e f i r s t s e p a r a t i o n o f c h i c k e n muscle isozymes.  The f i r s t  e l u t e d peak,  d e s i g n a t e d as "A" was shown t o c o n s i s t o f one e l e c t r o p h o r e t i c moiejby w h i l e t h e second *B* peak was observed t o c o n t a i n two. The presence o f t h e double a c t i v i t y peak i n t h e s p e c i f i c ity  activ-  p r o f i l e o f t h e DEAE-Sephadex chromatography o f Peak A i s  difficult  t o account f o r . The p o s s i b i l i t y o f p r o t e i n aggrega-  t i o n has been d i s c u s s e d I n t h i s t h e s i s and might p r o v i d e some e x p l a n a t i o n f o r t h e observed a c t i v i t y p r o f i l e ;  I t has been w e l l  e s t a b l i s h e d t h a t TIM forms p a r t o f an i n v i v o g l y c o l y t i c aggregate  which c o n s i s t s o f g l y c e r a l d e h y d e 3-phosphate  dehydrogenase,  a l d o l a s e , p y r u v a t e k i n a s e , and l a c t a t e dehydrogenase., (65,86,87) T h e r e f o r e i t would n o t be t o o s u r p r i s i n g i f the i s o l a t e d p r o t e i n aggregated as w e l l .  T h i s p a r t i c u l a r type o f p r o t e i n : p r o t e i n I n -  t e r a c t i o n c o u l d have some e f f e c t upon t h e enzyme's c a t a l y t i c a b i l i t y and hence on i t s s p e c i f i c a c t i v i t y .  Ultracentrifugation  s e d i m e n t a t i o n measurements c o u l d l e a d t o I n f o r m a t i o n concerning the  p o s s i b i l i t y t h a t these aggregates do, i n f a c t form.  123 •  There i s v e r y l i t t l e p o s s i b i l i t y t h a t the observed, t h r e e Isozymes a r e pseudo-isozymes which o r i g i n a t e from v a r i o u s ext e n t s of s u l f h y d r y l o x i d a t i o n as was found f o r r a b b i t phosphoglucose isomerase (90) :  muscle  The c a r e f u l r e d u c t i o n of a l l  p r o t e i n w i t h d i t h i o l t h r e l t o l b e f o r e any e x p e r i m e n t a l manipulat i o n s , have ensured t h a t a l l shown by DTNB a s s a y ;  t h i o l s a r e i n a reduced s t a t e as  However, t h i s does suggest a p o s s i b i l i t y  f o r the double s p e c i f i c a c t i v i t y p r o f i l e observed f o r the Peak A isozyme.  The chromatographies were n o t c a r r i e d out i n the  presence o f a r e d u c i n g agent and i f t h e t h i o l s o f the Peak A isozyme a r e p a r t l c u l a r l l y l a b i l e , isozyme-formation:  i t c o u l d r e s u l t l n pseudo-  These pseudo-isozymes  would n o t be d e t e c t e d  i n the e l e c t r o f o c u s i n g o f the p r o t e i n s i n c e t h e enzyme i s r e duced Immediately before-hand;  The type o f f a c i l e  which t h i o l s a r e p a r t i c u l a r i l y prone t o i s u s u a l l y  oxidation reversible.  The I m p l i c a t i o n s o f the Isozymic s e p a r a t i o n on the s t r u c t u r a l d e t e r m i n a t i o n a l r e a d y performed has a l r e a d y been d i s c u s s e d i n s e c t i o n B of the c h a r a c t e r i z a t i o n chapter.  Extreme  caution  w i l l have to be e x e r c i s e d i n i n t e r p r e t i n g t h e p u b l i s h e d amino a c i d sequence and x - r a y s t r u c t u r e , e x p e c l a l l y i n l i g h t o f the f a c t that t h e r e were s i g n i f i c a n t d i f f e r e n c e s i n many amino a c i d  res-  idues i n the amino a c i d a n a l y s e s r e p o r t e d i n t h i s t h e s i s , f o r the Peak A p r o t e i n :  I n a d d i t i o n , the c a l c u l a t e d m o l e c u l a r weight  was d i f f e r e n t from t h e l i t e r a t u r e v a l u e by a t l e a s t 5»000 gms/mole: The c h e m i c a l m o d i f i c a t i o n s r e p o r t e d were i n d i c a t i v e o f s i g n i f i c a n t temperature induced c o n f o r m a t i o n a l changes.  Information  124.  concerning foundations  t h i o l m o d i f i c a t i o n has,  to d a t e , been s c a n t y .  l a i d 'with these o b s e r v a t i o n s w i l l be important  only f o r g e n e r a l u n d e r s t a n d i n g  .There i s a p r o m i s i n g  w i t h the  f o r the PEM  F NMR  l a r g e chemical  not  of the p r o t e i n , but a l s o f o r such  s p e c t r o s c o p i c techniques,,as-NMR.  and  The  s p e c t r a observed  beginning  m o d i f i e d TIM.  The  s h i f t d i f f e r e n c e of the l a b e l e d p r o t e i n complex  the l a b e l e d model compound g i v e s some i n d i c a t i o n of the  j  sen-  s i t i v i t y w i t h which t h i s technique w i l l be a b l e to d e t e c t changes i n environment and  hence the s u b s t r a t e - i n d u c e d p r o t e i n conforma-  t i o n a l changes; The k i n e t i c s chapter was  a b l e to expand upon the m o d i f i c a t i o n s  performed and l e d to some e x c i t i n g d i s c o v e r i e s of temperature pendent mechanisms;  de-  L i t t l e information i n t h i s area i s a v a i l a b l e  but the lower a c t i v a t i o n energy of the s u b s t r a t e enzyme complex c o r r e l a t e s w i t h some i n f o r m a t i o n a v a i l a b l e i n the l i t e r a t u r e concerning r e a c t i o n of r a b b i t muscle TIM w i t h DTNB.  In one  (16) paper,  K r l e t s c h et a l r e p o r t e d a second order r a t e constant f o r the DTNB m o d i f i c a t i o n i n presence of s u b s t r a t e DHAP which was of  t h a t observed  i n the absence of s u b s t r a t e .  only a  third  Further i n v e s t i g a -  t i o n i n t h i s a r e a shows promise of y i e l d i n g r e s u l t s which a l l o w f r u i t f u l s t u d i e s of the chemical and p h y s i c a l p r o p e r t i e s of chicken muscle t r i o s e phosphate  isomerase.  125 References (1  A. 3. S c h n e i d e r , W.N.  V a l e n t i n e , M. H a t t o r l , and H.L.  N. E n g l . J . Med. 2 £ 2 , 229-235 (2  M.A.  (1965)  Baughan, N.N. V a l e n t i n e , D.E. P a g l i a , P.O.  E.R. Simon, and Q.B. DaMarsh,  Ways,  Blood, 2,2, 2 3 6 - 2 5 8  (3  E.A. Noltman, The Enzymes V I , 2 7 1 - 3 5 4  (4  E.W.  (I968)  U97<0  Lee, J i A . B a r r i s o , M. Pepe, and R. Snyder, Biochem.  B i o p h y s . A c t a , 242, 2 6 l  (1971)  European J . 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