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Chemical modification of carboxyl groups in porcine pepsin Ma, Ching-Yung 1979

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CHEMICAL MODIFICATION OF CARBOXYL GROUPS IN PORCINE PEPSIN by CHING YUNG|MA B.Sc,  University of Hong Kong, 1970  M.Sc, University of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF FOOD SCIENCE  We accept t h i s thesis as conforming to the required standard.  THE UNIVERSITY OF BRITISH COLUMBIA ©Febuary, 1979  In  presenting  an  advanced  the I  Library  further  for  degree shall  agree  scholarly  by  his  of  this  thesis  Date  at  University  the  make  that  it  for  partial  freely  may  be  It  Wesbrook  Food  gain  of  British  Canada  1W5  Ap-rii  9U  t  1070  Columbia  for  extensive by  shall  of  the  requirements  B r i t i s h Columbia,  the  understood  Science  Place  of  granted  is  financial  fulfilment  available  permission for  purposes  nf  University  Vancouver, V6T  in  permission.  Department  2075  thesis  representatives.  written  The  this  copying Head  that  not  reference  be  of  of my  I  agree  and this  for that  study. thesis  Department.or  copying  or  publication  allowed  without  my  11  ABSTRACT  Carboxyl groups i n porcine pepsin were chemically modified "by the carbodiimide reaction using l-ethyl-3-(3-dimethylaminopropyl) esters as nucleophiles.  waterrsoluble  carbodiimide and amino acid  The modification resulted i n pro-  found changes i n the a c t i v i t i e s , s p e c i f i c i t y and.some physicochemical properties of the enzyme.  These include* (1) s i g n i -  f i c a n t decrease i n milk c l o t t i n g a c t i v i t y without changes i n p r o t e o l y t i c a c t i v i t y against hemoglobin;  (2) decrease i n  peptidase a c t i v i t y against N-acetyl-L-phenylalanyl-diiodoL-tyrosine;  (3)  increase i n c l o t t i n g a c t i v i t y against  casein but decrease i n c l o t t i n g a c t i v i t y against casein mixture;  (k)  X-  K-  s h i f t -in p r o t e o l y t i c pH p r o f i l e with  pH optimum increased from 2.0 to about 3*5;  (5.) decrease i n  r e l a t i v e electrophoretic mobility and a s l i g h t decrease i n i s o e l e c t r i c point; in k  c a i ;  ;  and  (6) increase i n K  m  without much change  (7) increase i n s t a b i l i t y at pH above  6.0.  Results suggest that the drop i n milk c l o t t i n g was  due to a change i n the charge d i s t r i b u t i o n  activity  on the enzyme  a f f e c t i n g enzyme-micelle i n t e r a c t i o n . The presence of dipeptide substrates i n t e r f e r e d  with  the carboxyl modification suggestive of the proximity of the modified groups to the enzyme active s i t e .  iii  The m o d i f i e d enzyme r e m a i n e d r e a c t i v e fic  i n a c t i v a t o r s but at rates  to  site-speci-  slower than the n a t i v e  The m o d i f i c a t i o n was n o t s p e c i f i c ,  causing s i m i l a r  enzyme.  changes  i n p e p s i n o g e n and c h y m o s i n . The m o d i f i e d a n d n a t i v e p e p s i n s h a d s i m i l a r lytic  p r o p e r t i e s and p r o d u c e d comparable r a t e s  and c u r d t e n s i o n development i n pH s t a b i l i t y s u g g e s t e d better making .  on c u r d l e d m i l k .  of The  caseinosyneresis increase  t h a t t h e m o d i f i e d enzyme may be  c a l f rennet substitute than native pepsin f o r  a  cheese-  TABLE OF CONTENTS PAGE INTRODUCTION  1  LITERATURE REVIEW Chemical M o d i f i c a t i o n of P r o t e i n s M o d i f i c a t i o n of Carboxyl  5  Groups  1.  Esterification  2.  Carbodiimide reaction  9 10  Pepsin  13  ' Chemical M o d i f i c a t i o n of Pepsin  19  MATERIALS AND METHODS Biochemieals  and S p e c i a l Chemicals  22  Methods 1.  M o d i f i c a t i o n of c a r b o x y l groups  2.  Determination of m i l k c l o t t i n g a c t i v i t y  3...  Determination of p r o t e o l y t i c  4.  Determination of peptidase a c t i v i t y  5.  Assay  6.  Determination of r a t e of casein h y d r o l y s i s  30  7.  Agarose g e l  31  8.  Determination of i s o e l e c t r i c  9.  Determination of K  24 ....  activity  g l  electrophoresis and k  c  a  27 28  of p e p s i n w i t h x - c a s e i n and < * - c a s e i n  ffl  25  point  29  32 33  t  10.  Assay  of pepsinogen  11.  D e t e r m i n a t i o n o f p e p s i n s t a b i l i t y t o pH . . . .  34  12.  Determination of thermal s t a b i l i t y  35  13.  Determination of curd tension  37  14.  Determination of r a t e  38  34  of syneresis  of pepsin  RESULTS AND DISCUSSION Choice  o f Enzyme S o u r c e  40  Choice  of Carbodiimides  and N u c l e o p h i l e s  Effect  of Nucleophile Concentration  on t h e  Extent  of Carboxyl M o d i f i c a t i o n Activity  o f N a t i v e and C a r b o x y l M o d i f i e d  1.  Milk clotting activity  2.  Proteolytic  3.  Milk clotting:proteolytic  4.  Peptidase a c t i v i t y  Effect  Pepsins  activity activity ratio  ...  o f C a r b o x y l M o d i f i c a t i o n on pH P r o f i l e s  1 . M i l k c l o t t i n g pH p r o f i l e 2.  Proteolytic  pH p r o f i l e  3. p H - A c t i v i t y curves f o r Electrophoretic Modified Pepsins Isoelectric  the h y d r o l y s i s  o f APDT  M o b i l i t y o f N a t i v e and C a r b o x y l on A g a r o s e G e l  P o i n t of N a t i v e and C a r b o x y l  Modified  Pepsins Carboxyl M o d i f i c a t i o n of Pepsin  By  Other Amino  Acid Methyl.Esters Caseinolytic  Properties  of Native  and.Carboxyl  Modified.Pepsins 1.  Rate of  2.  Electrophoretic  patterns  3.  Electrophoretic  patterns of milk curds  Coagulation  casein hydrolysis of hydrolysed  of K - C a s e i n and o < - C a s e i n by g l  casein  Native  and C a r b o x y l M o d i f i e d P e p s i n s Specificity Effect  of Carboxyl M o d i f i c a t i o n  o f C a r b o x y l M o d i f i c a t i o n on K  of Pepsin Effect  of Synthetic  fication  ffl  a n d K'  ^  .<>»<. Dipeptides  on C a r b o x y l  Modi-  vi PAGE Response o f N a t i v e and C a r b o x y l M o d i f i e d Pepsins 91  to I n h i b i t o r s S t a b i l i t y o f N a t i v e and C a r b o x y l M o d i f i e d Pepsins near n e u t r a l pH  9^  •  Thermal S t a b i l i t y o f N a t i v e and C a r b o x y l M o d i f i e d Pepsins  E f f e c t o f C a r b o x y l M o d i f i c a t i o n on Curd and Rate o f S y n e r e s i s  103  ... Tension  112  GENERAL DISCUSSION  117  CONCLUSIONS  131  LITERATURE CITED  133  vii  L I S T OF TABLES TABLE 1  PAGE Some p h y s i c a l p r o p e r t i e s  of porcine  pepsin  and p e p s i n o g e n 2  Adjustment  o f pH o f m i l k u l t r a f i l t r a t e  stability test 3  17 for  of pepsin  36  Carboxyl m o d i f i c a t i o n of pepsin w i t h  differ-  ent n u c l e o p h i l e s 4  Effect  43  o f c a r b o x y l m o d i f i c a t i o n on p e p s i n  activities 5  47  Carboxyl m o d i f i c a t i o n of pepsin  by  differ-  ent amino a c i d m e t h y l e s t e r s 6  Effect  of  activity  67  c a r b o x y l m o d i f i c a t i o n on c l o t t i n g of pepsin  t o X - c a s e i n and  -K-^gj"  casein mixture 7  Comparision of a c t i v i t i e s pepsin to  8  Effect  different  of carboxyl  Kinetics  modified  substrates  78  o f c a r b o x y l m o d i f i c a t i o n on  of pepsin, 9  76  activities  p e p s i n o g e n and chymosin  of the h y d r o l y s i s  of  phenylalanyl-L-diiodotyrosine  81  N-acetyl-Lby n a t i v e  and  carboxyl modified pepsins 10  Effect  of  of pepsin  dipeptides  on c a r b o x y l  86 modification 89  viii  TABLE 11  PAGE Response of native and carboxyl modified pepsins to i n h i b i t o r s  12  E f f e c t of carboxyl modification of pepsin on Q^  0  13  values (milk c l o t t i n g )  107  E f f e c t of carboxyl modification of pepsin on Q values (proteolytic)  Ill  Tension of curds produced by pepsin, carboxyl modified pepsin and chymosin  113  1Q  14  93  IxL I S T OF FIGURES FIGURE 1  PAGE The r e a c t i o n o f p r o t e i n c a r b o x y l  group,  w i t h w a t e r - s o l u b l e c a r b o d i i m i d e and n u c l e o phile 2  12  Dependence o f c h a r g e on n u c l e o p h i l e s modification  3  Effect  on t h e  o f p r o t e i n c a r b o x y l group,  of n u c l e o p h i l e  14  c o n c e n t r a t i o n on t h e  extent of carboxyl m o d i f i c a t i o n of pepsin 4  Effect  of carboxyl m o d i f i c a t i o n of  ...  pepsin  on m i l k , . c l o t t i n g . a c t i v i t y 5  Effect  6  Effect  46  of carboxyl m o d i f i c a t i o n of  on p r o t e o l y t i c  Effect  pepsin  activity  49  of c a r b o x y l m o d i f i c a t i o n of  on m i l k c l o t t i n g * p r o t e o l y t i c 7  pepsin  activity ratio..  of carboxyl m o d i f i c a t i o n of  53  M i l k c l o t t i n g pH p r o f i l e s  of n a t i v e  and  carboxyl modified pepsins 9  P r o t e o l y t i c pH p r o f i l e s  51  pepsin  on p e p t i d a s e a c t i v i t y 8  44  of c r y s t a l l i n e  55 pepsin,  c a r b o x y l m o d i f i e d c r y s t a l l i n e p e p s i n and chymosin 10  Proteolytic  56 pH p r o f i l e s  of crude n a t i v e  carboxyl modified pepsins  and 58  X  FIGURE 11  12  13  PAGE pH-Activity curves f o r the action of native and carboxyl modified pepsins on N-acetylL-phenylalanyl-diiodo-L-tyrosine  60  E f f e c t of carboxyl modification of pepsin on r e l a t i v e electrophoretic mobility  63a  Determination of i s o e l e c t r i c points of native and carboxyl modified pepsins  14 15  16  Rate of casein hydrolysis by native and carboxyl modified pepsins at pH 5*3 •  70  Rate of casein hydrolysis by native and carboxyl modified pepsins at pH 6.5  71  Lineweaver-Burk plots of the hydrolysis of Nacetyl-L-phenylalanyl-diiodo-L-tyrosine by native and carboxyl modified pepsins at pH 2.0  17  o 18  19  65  84  Lineweaver-Burk plots of the hydrolysis of N-acetyl-L-phenylalanyl-diiodo-L-tyrosine by native and carboxyl modified pepsins at pH 4.5  85  Inactivation of native and carboxyl modified pepsins by EPNP  92  S t a b i l i t y of c r y s t a l l i n e native and carboxyl modified pepsins i n 0.05 M phosphate buffer, pH 6.5  95  xi  FIGURE 20  PAGE S t a b i l i t y of crude native and carboxyl modified pepsins i n 0.05 M phosphate buffer, pH 6.5  21  97  S t a b i l i t y of c r y s t a l l i n e native and carboxyl modified pepsins i n milk u l t r a f i l t r a t e under cheese-making conditions  22  98  S t a b i l i t y of crude native and carboxyl modified pepsins i n milk u l t r a f i l t r a t e under  23;  24  cheese-making conditions  99  Thermal p r o f i l e s (milk c l o t t i n g ) of c r y s t a l l i n e native and carboxyl modified pepsins  104  Thermal p r o f i l e s (milk c l o t t i n g ) of crude native and carboxyl modified pepsins  25  105  Thermal p r o f i l e s (proteolytic) of c r y s t a l l i n e native and carboxyl modified pepsins  26  108  Thermal p r o f i l e s (proteolytic) of crude native and carboxyl modified pepsins  27  109  Syneresis of curd by c r y s t a l l i n e pepsin, carboxyl modified c r y s t a l l i n e pepsin and mosin  28  chy•  115  Syneresis of curd by crude native and carboxyl modified pepsins  116  xii  L I S T OF PLATES PLATE I  II  PAGE Agarose  gel electrophoretic  patterns  native  and c a r b o x y l m o d i f i e d p e p s i n s  Agarose  gel electrophoretic.patterns  whole  c a s e i n h y d r o l y s e d by n a t i v e and  of 6l of car-  boxyl modified pepsins III  Agarose  72  gel electrophoretic  curds produced by n a t i v e f i e d pepsins  patterns  of  milk  and c a r b o x y l m o d i 74  X l l l  ACKNOWLEDGEMENTS  I w o u l d l i k e t o e x p r e s s my g r a t i t u d e supervisor,  Dr. S.  Nakai, for his  and u n f a i l i n g guidance preparation of t h i s  throughout  t o my a c a d e m i c  enthusiastic this  study,  support and i n  thesis.  I would l i k e t o thank M r s . V . Skura f o r her a s s i s t a n c e i n the amino a c i d Finally, help, be  the  technical  analysis.  I w i s h t o t h a n k my w i f e ,  Stephanie,  for  her  u n d e r s t a n d i n g and l o v e w i t h o u t w h i c h t h i s work c o u l d n o t  finished.  1  INTRODUCTION  Chemical m o d i f i c a t i o n of p r o t e i n s by s p e c i f i c agents i s a u s e f u l technique i n the study of the  re-  physico-  c h e m i c a l b a s i s and mechanisms o f p r o t e i n f u n c t i o n i n t h e b i o l o g i c a l systems industry,  (Means a n d F e e n e y ,  I n the  1971).  food  c h e m i c a l m o d i f i c a t i o n i s p l a y i n g an i n c r e a s i n g l y  important r o l e .  Many f o o d p r o t e i n s , p a r t i c u l a r l y t h o s e  non-conventional sources, functional properties.  have been m o d i f i e d t o improve  t o a l t e r the f u n c t i o n a l i t y of food proteins 1977).  their  A l k a l i t r e a t m e n t and m o d i f i c a t i o n w i t h  v a r i o u s a c y l a t i n g a g e n t s a r e t h e two m o s t w i d e l y u s e d  Ryan,  from  A l a r g e number o f p a t e n t s  methods  ( K i n s e l l a , 1976i  have a p p e a r e d ,  i n most p u b l i s h e d c a s e s , improvements i n f u n c t i o n a l  and  properties  were o b t a i n e d making i t p o s s i b l e t o extend and r e p l a c e t i n g food proteins w i t h novel proteins i n processed f o r t h e f a b r i c a t i o n o f new  f o o d and  foods.  C h e m i c a l m o d i f i c a t i o n o f enzymes i s u s u a l l y to elucidate  t h e a c t i o n mechanism and a c t i v e - s i t e  o f the b i o l o g i c a l c a t a l y s t s . is  exis-  undertaken residues  I m m o b i l i z a t i o n o f enzymes,  one f o r m o f c h e m i c a l m o d i f i c a t i o n , h a s b e e n  which  extensively  u t i l i z e d i n b i o - m e d i c a l r e s e a r c h as w e l l as i n t h e f o o d i n dustry.  The f e a s i b i l i t y o f m o d i f y i n g f o o d - r e l a t e d  conform to p a r t i c u l a r p r o c e s s i n g requirements  or to  enzymes avoid  to  2  undesirable characteristics it  has not been e x p l o r e d ,  although  should h o l d great promises i n enhancing the a p p l i c a b i l i t y  o f many enzymes  i n food  manufacture.  The a i m o f t h e p r e s e n t whether the performance  investigation i s to  of a food-related  proved by chemical m o d i f i c a t i o n . an a c i d protease  enzyme c a n be i m -  P o r c i n e p e p s i n ( E . C . 3f4,23»l)»  f o u n d i n t h e stomach mucosa o f p i g ,  chosen f o r the f o l l o w i n g reasonst most w i d e l y a c c e p t e d a substitute  determine  is  (1) P o r c i n e p e p s i n i s  milk coagulant  the  from an a n i m a l source  f o r c a l f rennet i n cheese-making,  and has  as  been  u s e d a s 50*50 m i x t u r e s w i t h c a l f r e n n e t i n t h e c h e e s e i n d u s t r y ( P h e l a n , 1973; 1976).  B o t t a z z i e t a l . , 1976;  Carbone and E m a l d i ,  (2) The u s e o f s w i n e p e p s i n a l o n e i s g e n e r a l l y  s i d e r e d u n s u i t a b l e f o r cheese-making  (Sardinas,  has been p a r t l y a t t r i b u t e d t o slower p r o t e o l y s i s  stable  1974).  i n cheeses  F o l t m a n n , 1966;  Dumas, 1971), s u g g e s t i n g extent  rennet  P o r c i n e p e p s i n i s known t o b e  t h a n c h y m o s i n ( m a j o r enzyme i n c a l f r e n n e t )  ( H e r r i o t t , 1955;  6.0  This  1972).  made w i t h s w i n e p e p s i n t h a n i n t h o s e made w i t h c a l f (Green and F o s t e r ,  con-  less  a t pH a b o v e  A n t o n i n i and Ribadeau  t h a t i t may be i n a c t i v a t e d t o a  than rennet d u r i n g the cheese-making  process.  greater  (3)  P o r c i n e p e p s i n h a s b e e n t h e m o s t t h o r o u g h l y s t u d i e d among a l l acid proteases. et  The p r i m a r y s t r u c t u r e was d e t e r m i n e d  a l . , 1973), a n d i t s  well  characterized.  (Tang  p r o p e r t i e s a n d modes o f a c t i o n w e r e  3  The p r i m a r y o b j e c t i v e selectively  the r e a c t i v e  i n pepsin with specific the a c t i v i t y , the  enzyme.  groups  residues change  i f beneficial,  P r e l i m i n a r y experiments  i n pepsin.  tryptophan,  The e n z y m a t i c  coagulation  had been  tyrosine  and  investigated.  phase and a  still  the present  unclear  pro-  secondary  The m e c h a n i s m b y w h i c h m i c e l l e s  gate t o form curd i s  found  o f m i l k i s a complex  cess i n v o l v i n g a primary p r o t e o l y t i c phase.  the  C a r b o x y l m o d i f i c a t i o n was  t o be most p r o m i s i n g a n d was f u r t h e r  of  should en-  u t i l i z a t i o n of porcine p e p s i n i n  of cheese.  c a r b o x y l groups  aggre-  ( E r n s t r o m , 1974).  It  study can c o n t r i b u t e t o the  d a t i o n o f t h i s mechanism and h e l p t o d e f i n e  the  between m i l k c l o t t i n g and g e n e r a l p r o t e o l y s i s , similar  modify  c h e m i c a l r e a g e n t s s o as t o  These a l t e r a t i o n s ,  i s hoped t h a t  to  o f t h e amino a c i d  c a r r i e d out t o modify a r g i n i n e ,  aggregation  is  s t a b i l i t y and p h y s i c o c h e m i c a l p r o p e r t i e s  hance t h e p o t e n t i a l manufacture  of the p r o j e c t  eluci-  relationship two  closely  processes. In the present  project,  with limited quantities  p o r c i n e p e p s i n was  of reagents to y i e l d  m o d i f i c a t i o n of the c a r b o x y l groups.  modified  limited  Extensive  derivati-  z a t i o n of c a r b o x y l groups  is  since  a m i n o a c i d s w e r e shown t o  t h e two a c t i v e - s i t e  aspartic  acid  (Bayliss  detrimental to pepsin  activity  e t a l . , 1969$ Chen a n d T a n g ,  be 1972).  4  The a c t i v i t i e s ious substrates  of the modified pepsin against  including synthetic  dipeptides,  var-  hemoglobin,  r e c o n s t i t u t e d s k i m m i l k , whole c a s e i n and /c- and o e - c a s e i n s , g l  were measured.  Some p h y s i c a l p r o p e r t i e s  m o d i f i e d enzyme s u c h as pH p r o f i l e s mobility,  and p r o p e r t i e s  pepsin i n cheese-making and r a t e  of syneresis  and  of the  carboxyl  electrophoretic  p e r t i n e n t to the performance s u c h a s pH s t a b i l i t y ,  were s t u d i e d .  curd tension  Some c h e m i c a l  ties  o f t h e m o d i f i e d enzyme s u c h a s t h e r e s p o n s e  tors  and k i n e t i c s were a l s o  studied.  The  of  to  properinhibi-  characteristics  of the m o d i f i c a t i o n , i n c l u d i n g the s p e c i f i c i t y of the a c t i o n and t h e e f f e c t were  investigated.  of substrates  re-  on t h e m o d i f i c a t i o n ,  5  LITERATURE REVIEW  Chemical M o d i f i c a t i o n of In its involves  broadest  Proteins  s e n s e , any t r a n s f o r m a t i o n  the formation or rupture  be r e g a r d e d  which  of a covalent  b o n d may  as c h e m i c a l m o d i f i c a t i o n o f a p r o t e i n .  This  w o u l d i n c l u d e p r o c e s s e s s u c h as p r o t o n t r a n s f e r ,  metal  chelation,  hydrogen  bonding ever, the  enzyme-substrate  (Cohen,  i n t e r a c t i o n and even  I n a more r e s t r i c t e d  1970).  chemical modification i s  intentional alteration  it  involves  r e a g e n t s o f some r e a c t i v e molecule  s u c h as c h a r g e d  hydroxyi,  amide and t h i o l  referred  of p r o t e i n s t r u c t u r e  f o r m a t i o n by c h e m i c a l agents Essentially,  generally  sense,  the d e r i v a t i z a t i o n by s i d e - c h a i n groups  to  or  (Means a n d F e e n e y ,  to the pharmaceutical,  tries.  The t r e a t m e n t  groups,  d y e i n g and c l o t h i n g  of animal hides  use as i n the t a n n i n g o f l e a t h e r  hyde,  is  or h a i r s  one o f t h e  chemical modification.  was r e c e n t l y  This  i m p r o v e d by t h e use  a c r o s s - l i n k i n g reagent of p r o t e i n s .  several  modifications  performance  of  for  Formaldehyde  history indushuman  oldest  pro-  ancient glutaralde-  Similarly,  were used t o g i v e w o o l f i b r e s  for clothing.  protein  residues.  related  procedure  con-  specific  Chemical m o d i f i c a t i o n of p r o t e i n s has a l o n g  cesses u l t i l i z i n g  as  1971).  i n the  a n i o n i c and c a t i o n i c  how-  superior  had been used  to  6  modify b a c t e r i a l  toxins,  c i t i n g a t o x i c response t o produce  r e n d e r i n g them i n c a p a b l e but s t i l l  retaining its  an immunological response  of  eli-  ability  when i n j e c t e d  into  an a n i m a l . With the a v a i l a b i l i t y  o f new, s p e c i f i c  r e a g e n t s a n d more s o p h i s t i c a t e d  analytical  chemical  techniques,  c h e m i c a l m o d i f i c a t i o n h a s become one o f t h e most tools  of p r o t e i n chemists f o r the study  functions Stark,  of b i o l o g i c a l l y - a c t i v e Means a n d F e e n e y ,  1970j  G l a z e r e t a l . . , 1976). used t o i n v e s t i g a t e chains  proteins  residues  of proteins  in  (Cohen,  Knowles,  1971;  and  1968}  1974;  are  o f i n d i v i d u a l amino  i n r e l a t i o n to the p h y s i c a l ,  properties  of structures  Chemical modifications  the roles  powerful  routinely acid  c h e m i c a l and b i o l o g i c a l  and t o determine  the  active-site  enzymes.  P r o t e i n m o d i f i c a t i o n has wide a p p l i c a t i o n i n other areas of biochemical research X-ray crystallography  i n i t s a b i l i t y to interact obtained  (Singer, structure  with antigen,  H a b e r , 1968).  information can  involved i n the bonding  Determination of the  of a protein at high resolution requires  preparation  o f h e a v y atom d e r i v a t i v e s  w i t h the parent  crystal.  By  and d e t e r m i n a t i o n o f changes  on t h e f u n c t i o n a l groups  1965;  immunochemistry,  and p u r i f i c a t i o n o f p r o t e i n s .  m o d i f i c a t i o n of an antibody  be  including  which are  H e a v y atoms may be  crystal the  isomorphous  incorporated  7  by s e l e c t i v e chemical modification which makes important contributions to X-ray crystallography (Benisek and Richards,  1968).  Some proteins form reversible complexes  with other substances and have properties which can be used to separate proteins by conventional methods.  One  example i s the separation of a group of c l o s e l y r e l a t e d antifreeze glycoproteins from blood serum of Antarctic fishes by complexing with borate (Vandenheede, 1972).  In the food industry, chemical modification i s mainly used to improve functional properties of proteins. A l k a l i treatments have been used i n the s o l u b i l i z a t i o n of protein-containing materials i n preparation f o r extrusion processing  (Van Beek et a l . . 1974).  Hydrolysates  of some  proteins have improved f l a v o r c h a r a c t e r i s t i c s , better emulsifying property and improved foaming a b i l i t y  (Richard-  son, 1977). The other chemical approach to modify the functiona l i t y of proteins which has received much attention i s the use of various acylating agents.  Egg white modified  with 3»3-dimethyl-glutaric anhydride shows increased heat s t a b i l i t y while the foaming capacity i s not s i g n i f i c a n t l y affected (Gandhi et a l . . 1968).  Vegetable proteins such  as soy proteins have been modified with acetic and succinic anhydrides to be used i n coffee whitener  formulations  8  ( M e l n y c h y n and S t a p l e y , decreased flavor  viscosity  and odor.  isolates,  1973).  and i s o e l e c t r i c  p o i n t as w e l l a s  or t e a .  N - S u c c i n y l a t e d egg y o l k  and s a l a d d r e s s i n g  Other a c y l a t e d  proteins  protein  made f o r u s e i n may-  (Evans and I r o n s ,  i n c l u d i n g whey,  and i n i c e cream m i x ( E v a n s ,  fibrillar viscous  proteins  added  proteins  1971a: b ) .  casein,  serum and  g e l a t i n are a l s o used f o r s t a b i l i z i n g o i l - i n - w a t e r sions  have mild  o r p r e c i p i t a t e when  and o v a l b u m i n have been s u c c e s s f u l l y onnaise  proteins  Unlike conventional vegetable  t h e y do n o t " f e a t h e r "  to hot coffee  The a c y l a t e d  1970a; b ) .  emul-  Fish  myo-  have been s u c c i n y l a t e d and f o u n d t o  aqueous d i s p e r s i o n s w i t h good h e a t s t a b i l i t y ,  high emulsifying capacity, stability  (Groninger,  Chen e t a l . ,  bland flavor  1973;  and i m p r o v e d  G r o n i n g e r and M i l l e r ,  form a  foaming  1975;  1975).  A p a r t from i m p r o v i n g the f u n c t i o n a l p r o p e r t i e s , proteins ative  a r e m o d i f i e d b y c h e m i c a l methods  reactions  to block  and t o improve n u t r i t i o n a l v a l u e .  amino groups have been m o d i f i e d by a c y l a t i o n Carpenter,  1970)  proteins  deteriorProtein  (B j a m a s on a n d  and d i m e t h y l a t i o n (Galembeck  block the M a i l l a r d r e a c t i o n .  food  e t a l . , 1977)  The n u t r i t i o n a l q u a l i t y o f  food  c a n be i m p r o v e d b y i n c r e a s i n g t h e d i g e s t i b i l i t y  the proteins,  i n a c t i v a t i n g t o x i c or i n h i b i t o r y  or attaching essential  substances  n u t r i e n t s to the p r o t e i n s ;  and f l a v o r i n g a g e n t s c a n a l s o be a t t a c h e d  of  coloring  to improve  the  to  9 acceptability  of food proteins  M o d i f i c a t i o n of Carboxvl Two r e a c t i o n s c a r b o x y l groups  1.  1977).  Groups  m o s t commonly e m p l o y e d t o m o d i f y  of proteins  with nucleophiles  (Feeney,  are  e s t e r i f i c a t i o n and  mediated by a water-soluble  the  coupling  carbodiiraide.  Esterification P r o t e i n c a r b o x y l g r o u p s may be e s t e r i f i e d  of procedures.  C a r b o x y l groups  c a n be c o n v e r t e d  e s t e r s i n m e t h a n o l c o n t a i n i n g s m a l l amounts o f acid  ( W i l c o x , 1967).  t h a t may r e s u l t esterification shift  can l e a d to s i d e r e a c t i o n s ( W i l c o x , 1967;  procedure  its  c a r b o x y l groups  i n more s t a b l e  ( F r a e n k e l - C o n r a t and O l c o t t , trypsinogen  hydrochloric  C o h e n , 1968).  acyl  The  changes  However,  the  to study the r o l e  p r o t e i n s such as  19451  conditions  s u c h a s N-*0  use w i t h most p r o t e i n s .  has been used s u c c e s s f u l l y  methyl  acid-catalysed  r e a c t i o n i s a l s o accompanied by c o n f o r m a t i o n a l which preclude  to  U n l e s s done u n d e r l i m i t i n g  i n incomplete r e a c t i o n ,  or deamidatibn  b y a number  of  lysozyme  F r i e d e n , 1956), c h y m o -  ( D o s c h e r a n d W i l c o x , 1961)  and b o v i n e  serum  a l b u m i n (Ram a n d M a u r e r , 1959). D i a z o compounds  s u c h as d e r i v a t i v e s  a c i d are w i d e l y used t o e s t e r i f y acetates r e a c t w i t h water  carboxylic  of  diazoacetic  acids.  a n d many s i m p l e i n o r g a n i c  Diazoanions,  10  l i m i t i n g m o d i f i c a t i o n t o t h e more a c c e s s i b l e groups.  carboxyl  M o d i f i c a t i o n o f o n l y 20-30$ o f t h e c a r b o x y l  i s usually possible with a t y p i c a l protein. react  Diazoacetates  o p t i m a l l y w i t h p r o t e i n ; ^ c a r b o x y l g r o u p s n e a r pH 5»  A t l o w e r p H * s , h y d r o l y s i s o f r e a g e n t becomes and l i m i t s  the extent  of modification.  Scheraga,  appreciable  T h i s procedure has  been used t o modify p a n c r e a t i c r i b o n u c l e a s e  (Riehm and  1965)» c h y m o t r y p s i n o g e n A ( D o s c h e r a n d W i l c o x ,  1961) a n d p e p s i n ( R a j a g o p a l a n e t a l . , 1966a? Stein,  group  Lundblad and  1969). Alkylhalides are too unspecific f o r e s t e r i f i c a t i o n  o f p r o t e i n c a r b o x y l groups u n l e s s they a l s o behave as an affinity  l a b e l , as i n t h e e s t e r i f i c a t i o n o f t h e a c t i v e - s i t e  a s p a r t i c a c i d of p e p s i n by p-bromophenacyl bromide and M o r e l l ,  19661  Erlanger e t . a l . ,  (Gross  1966) a n d t h e a c t i v e -  s i t e glutamic a c i d of ribonuclease T i by  iodoacetate  ( T a k a h a s h i e t a l . , 1967)*  Triethyloxonium fluoroborate  has been used t o e s t e r i f y  s p e c i f i c a l l y a s p a r t i c a c i d 52  i n lysozyme  2,.  (Parsons and R a f t e r y ,  1969).  Carbodiimide reaction The m o s t p o p u l a r m e t h o d f o r m o d i f y i n g c a r b o x y l  i n p r o t e i n s i n v o l v e s the use o f w a t e r - s o l u b l e  groups  carbodiimides.  The c a r b o d i i m i d e s . r e a c t w i t h c a r b o x y l g r o u p s a t s l i g h t l y a c i d i c pH t o g i v e a n 0 - a c y l i s o u r e a  ( K h o r a n a , 1953)t a n  11 activated acylurea If  intermediate or react  that  can e i t h e r rearrange  is  an a m i n e , i t w i l l  1.  with  0-  The c a r b o d i i m i d e r e a c t i o n h a s b e e n w i d e l y u s e d  in  to y i e l d the corresponding  condense amide.  the d e t e r m i n a t i o n of the c a r b o x y l group content as w e l l as  of  proteins  i n the study of c a r b o x y l group f u n c t i o n .  mild conditions,  o n l y t h e more a c c e s s i b l e  c a r b o x y l groups r e a c t , excess reagents,  be o b t a i n e d land,  an  w i t h a n u c l e o p h i l e a s shown i n F i g .  the n u c l e o p h i l e  acylisourea  and  to  or  w h i l e i n the presence  nearly quantitative  ( H o a r e a n d K o s h l a n d , 1967;  Under  reactive of  denaturants  substitution  can  Carraway and K o s h -  1972). This procedure  the choice soluble  offers  considerable  flexibility in  o f c a r b o d i i m i d e and n u c l e o p h i l e .  Several  c a r b o d i i m i d e s have been used f o r p r o t e i n  cation .  water-  modifi-  l-Gyclohexyl-3-(2-morpholinyl-4-ethyl)-carbodi-  imide metho-p-toluenesulfonate aminopropyl)  carbodiimide are  and  commercially  l-Benzyl-3-(3-dimethylaminopropyl) frequently  used  imides react be expected carboxyl  l-ethyl-3-(3-dimethylavailable.  carbodiimide i s  ( H o a r e a n d K o s h l a n d , 1966).  also  A l l carbodi-  s i m i l a r l y although the smaller reagents t o be more a c c e s s i b l e  might  to p a r t i a l l y buried  groups.  Different nucleophiles experimental conditions.  c a n be u s e d t o s u i t  The i o n i c c h a r a c t e r  of  particular proteins  12  R N Protein-COOH + C II  R O NH Protein-C-O-C H  N  N  R'  R'  i  rearrange  + R"Nu-H  O OR'  O  Protein - C - N u - R  Protein-C-NHCNHR O RNHCNHR' FIGURE 1. THE REACTION OF PROTEIN CARBOXYL GROUP WITH WATERSOLUBLE CARBODIIMIDE AND NUCLEOPHILE. Nu : NUCLEOPHILE.  13  c a n be v a r i e d c o n s i d e r a b l y d e p e n d i n g on t h e c h a r g e o f t h e nucleophiles  ( F i g . 2).  R a d i o a c t i v e or c o l o r e d amines can  be employed t o f a c i l i t a t e  q u a n t i t a t i o n and i d e n t i f i c a t i o n  ( M a t y a s h e t a l . , 1973). C a r b o d i i m i d e a l s o r e a c t s w i t h b o t h s u l f h y d r y l and tyrosyl-OH groups.  T y r o s i n e c a n be r e g e n e r a t e d  by  ment o f t h e d e r i v a t i v e w i t h h y d r o x y l a m i n e a t pH 7 a n d K o s h l a n d , 1968).  Successful  regeneration  of  treat(Garraway  thiol  g r o u p s h a s n o t b e e n r e p o r t e d due t o e x c e s s i v e s t a b i l i t y o f t h e r e s u l t i n g product  ( C a r r a w a y a n d T r i p l e t ! , 1970).  Pepsin The c a t a l y t i c  a c t i v i t y of pepsin i n the  digestive  f u n c t i o n o f s t o m a c h was f i r s t i d e n t i f i e d b y t h e I t a l i a n researcher I898).  Abbe S p a l a n z a n i in,1783  T h i s was p e r h a p s t h e f i r s t  of enzymatic a c t i v i t y .  ( c i t e d by G i l l e s p i e , scientific  P e p s i n was t h e f i r s t  demonstration enzyme t o b e  named ( b y T . Schwann i n 1825) a n d t h e s e c o n d enzyme t o b e crystallized  ( N o r t h r o p , 1930).  The t e r m p e p s i n i s a p p l i e d t o t h e g a s t r i c formed by p a r t i a l p r o t e o l y s i s pepsinogens.  of t h e i r inactive  The n o m e n c l a t u r e  t h e m u l t i p l i c i t y o f t h e enzyme.  proteinases zymogens,  of p e p s i n i s complicated by However, p e p s i n ( E . C .  3 » 4 , 2 3 1 . ) h a s b e e n r e f e r r e d t o b y most a u t h o r s a n d b y t h e  14  PROTEIN -C  + NH - CH CH SO; 2  V  0"  2  R N  2  - ' C  N R  .  //> PROTEIN - C /^CH^O-j NH  TAURINE  N  u.  fi  -  PROTEIN -G V  fi  RN=C=NR'  + NH -CH C 2  2  O"  N  OCH . 3  GLYCINE METHYL  /fi  ESTER  /fi  +  + NH -CH CH NH 2  2  2  CH C 2  N  PROTEIN - C V  ... /fi  PROTEIN-C  NH  N  /  0CH 3  RN=C=NR' 3  ETHYLENEDIAMINE fi  PROTEIN-C  CH CH NH 2  2  3  NH  FIGURE 2. DEPENDENCE OF CHARGE ON NUCLEOPHILES ON THE MODIFICATION OF PROTEIN CARBOXYL GROUP.  15 Enzyme C o m m i s s i o n a s p o r c i n e p e p s i n A , t h e m a j o r proteinase  from p i g .  Most o f t h e a v a i l a b l e  c h e m i c a l s t r u c t u r e and k i n e t i c p r o p e r t i e s from s t u d i e s gen A .  on p o r c i n e p e p s i n A and i t s  (Northrop, and  d a t a on t h e  of pepsin zymogen,  H i g h l y p u r i f i e d or c r y s t a l l i n e pepsin  have been o b t a i n e d from the g a s t r i c  fish  1933)t  1940s  a proteinase  mucosa o f man, cow  Sprissler,  (or protease)  Unlike other types of proteases sulfhydryl proteases),  (serine,  pepsin functions  v e r y a c i d i c medium o f a b o u t pH 2-3* enzymes  are therefore  1942). which  It  contains  and  optimally i n a  P e p s i n and p e p s i n proteases.  o f p o r c i n e p e p s i n was  (Tang e t a l . , 1973;  327 a m i n o a c i d r e s i d u e s .  catalyses  proteins.  raetallo,  r e f e r r e d t o as a c i d  The p r i m a r y s t r u c t u r e mined r e c e n t l y  pepsino-  preparations  the c l e a v a g e of p e p t i d e bonds i n the s u b s t r a t e  like  are  c h i c k e n (Levchuk and O r e k h o v i c h , 1963)  ( N o r r i s and E l a m ,  Pepsin i s  gastric  deter-  Sepulveda et a l . , Pepsin structure  u n i q u e i n t h a t o n l y one l y s i n e a n d two a r g i n i n e s a r e w i t h i n t h e t e r m i n a l 20 r e s i d u e s protein. acid,  Along the  is  found  o f t h e c a r b o x y l end o f  The o t h e r 307 r e s i d u e s  histidine-97.  1975).  the  c o n t a i n o n l y one b a s i c  same s t r e t c h a r e 4 4 a c i d i c  residues,  i n c l u d i n g a phosphoserine.  structure  o f p o r c i n e p e p s i n h a s b e e n r e s o l v e d a t 0.27 nm  resolution  (N. Andreeva,  amino  The t h r e e - d i m e n s i o n a l  c i t e d by Tang,  1976).  16  The a c t i v e - s i t e  residues  f i e d a s Asp-32 ( B a y l i s s and Tang,  1972).  binding cleft Table p e p s i n and  o f p e p s i n have been  e t a l . , 1969) a n d  i n the  three-dimensional  1 summarizes  the precursor  activation is s t i l l  models have been proposed  porcine  of pepsin, i s a c t i v a t e d  the removal of the  at  4l-residue  The mechanism- f o r  n o t known, a l t h o u g h  ( T a n g , 1970}  several  A l - J a n a b i et  al..  K a s s e l l a n d K a y , 1973). Pepsin i s a protease (Hill,  1965).  w i t h broad side chain  The s e n s i t i v e  amino a c i d r e s i d u e and methionine phenylalanine  one  such as p h e n y l a l a n i n e ,  (Tang, (Inouye  ( K n o w l e s e t a l . , 1969)  1963).  Peptides  and F r u t o n ,  specifi-  bonds are g e n e r a l l y  i n d i p e p t i d y l u n i t s containing at l e a s t  1965)  of  pepsinogen.  a m i n o - t e r m i n a l p o r t i o n o f t h e zymogen.  city  apparent  structure.  some p h y s i c a l p r o p e r t i e s  pH b e l o w 5 t o f o r m p e p s i n u p o n  1972;  Asp-2l5, ( C h e n  T h e y a r e f o u n d t o be l o c a t e d i n a n  Pepsinogen,  pepsinogen  identi-  present  hydrophobic  tyrosine,  leucine  containing p-nitro-  1967)» 3 , 5 - d i n i t r © t y r o s i n e  and d i i o d o t y r o s i n e  (Jackson et  al..  a r e h y d r o l y s e d b y p e p s i n and have been u s e d i n k i n e t i c  investigations. I n a d d i t i o n to h y d r o l y s i n g peptide bonds, catalyses transpeptidation e t a l . , 1959;  of the a m i n o - t r a n s f e r  Fruton e t . a l . ,  1961).  pepsin type  Pepsin can a l s o  as an e s t e r a s e , h y d r o l y s i n g e s t e r l i n k a g e s  in  (Neumann act  suitable  17  Table 1.  Some p h y s i c a l p r o p e r t i e s of p o r c i n e p e p s i n and pepsinogen.  Pepsinogen  Reference  2.96-3.0  3.2-3.35  a) Blumenfeld and Perlmann, .1959. b) Arnon and P e r l mann, 1963.  8.70  7.54  35,000  41,000  Pepsin  s 20, w (sec X 1 0  D2 0 ,  1 3  )  w  (cm /sec X 1 0 2  Molecular Weight A  c  (nm)  1 3  c)  Orekhovich e t a l . , 1956.  )  216  236  d) R y l e ,  i960.  e) R y l e , 1965.  -M366 „278 nm Tflol  nm  212'  232  50,990 (MW=35000) 1  51,300 (MW=41000) 1  f ) Ryle and Port e r , 1959.  18  substrates  ( L o k s h i n a e t a l . . . , 1964;  I n o u y e a n d F r u t o n , 1967).  P e p s i n has been demonstrated t o p a r t i c i p a t e i n "plastein reaction" lyses  (Wasteneys  p e p t i d e bond s y n t h e s i s  a m i n o a n d »c - c a r b o x y l g r o u p s strate  concentration Pepsin belongs  (e.g.,  organisms, (e.g.,  of o l i g o p e p t i d e s  t o a g r o u p o f enzymes  at high  closely  1975)» a p p a r e n t l y a r e s u l t  of divergent  common a n c e s t r a l p r o t e i n ( H o f m a n n , 1974;  enzymes  are  These f a c t s  of the a c i d proteases  proteases  i n b l o o d plasma and acid  (Sepulveda et  al.,  e v o l u t i o n , from a Tang,  1976).  inhibitors.  i n t h i s group have a s i m i l a r  c a t a l y t i c m e c h a n i s m , a l t h o u g h t h e i r s p e c i f i c i t y may v a r y to differences (Tang,  1976).  The  a r e a l s o a l i k e a n d thea'e  i n h i b i t e d b y t h e same s i t e - s p e c i f i c s u g g e s t t h a t enzymes  en-  from m i c r o -  f r o m p l a n t s , mammalian l y s o s o m a l  s t u d i e d so f a r a r e homologous  sites  sub-  similar  s e m i n a l p l a s m a . . The a m i n o a c i d s e q u e n c e s o f a l l t h e  active  «<-  These i n c l u d e o t h e r g a s t r i c  c a t h e p s i n D and E ) and p r o t e a s e s  proteases  cata-  by d i r e c t condensation of  g a s t r i c s i n and c h y m o s i n ) , p r o t e a s e s  proteases  It  1930).  ( D e t e r m a n n e t a l . , 1965).  i n s t r u c t u r e and a c t i v i t y . zymes  and B o r s o o k ,  the  i n the topography of t h e i r b i n d i n g  sites  due  19 Chemical Modification of Pepsin Chemical modification has contributed greatly to the i d e n t i f i c a t i o n of a c t i v e - s i t e residues i n pepsin as well as other amino acid side groups e s s e n t i a l f o r pepsin a c t i v i t y . Diazoacetyl norleucine methyl ester, a derivative of diazoacetic acid, was  found to inactivate pepsin by e s t e r i -  f i c a t i o n of only one carboxyl group (Rajagopalan 1966a;  Lundblad and Stein, 1969).  et a l . ,  I s o l a t i o n and sequence  analysis of a peptide containing the diazo modified residue indicates that the e s t e r i f i e d aspartyl group i s located at residue 215 (Bayliss e t . a l . , 1969). A second carboxyl group i n the active s i t e of pepsin was  determined by the use of a substrate-like epoxide i n -  a c t i v a t o r , l,2-epoxy-3-(p-nitrophenoxy) propane (EPKP). Sequence determination of the EPNP-containing peptide placed the aspartyl residue at p o s i t i o n 32!(Chen and Tang, 1972). A t h i r d carboxyl group i n pepsin was modified by bromophenacyl bromide (Erlanger et al... 1965).  However,  the fact that the f u l l y reacted enzyme retained about 20% of i t s a c t i v i t y and that other acid proteases were unaffected suggests that t h i s modified group was not d i r e c t l y involved i n c a t a l y s i s (Clement, 1973). The presence of an a r g i n y l residue near the active centre of pepsin was  demonstrated by modification with  phenylglyoxal (Kitson and Knowles, 1971)  and butanedione  20 (Huang and Tang, 1972), and was  found to be A r g - 316.  The  f u l l y r e a c t e d enzyme r e t a i n e d about 20$ o f a c t i v i t y .  The  r e a c t i o n was  r e t a r d e d by the presence  of p e p t i d e s u b s t r a t e s  i n d i c a t i n g the p r o x i m i t y of the m o d i f i e d r e s i d u e t o the a c t i v e c e n t r e of the enzyme (Huang and Tang, 1972). S e l e c t i v e m o d i f i c a t i o n of c a r b o x y l groups i n p e p s i n was  c a r r i e d out u s i n g a c o l o r e d amine, N - ( 2 , 4 - d i n i t r o p h e n y l ) -  hexamethylenediamine and a w a t e r - s o l u b l e c a r b o d i i m i d e yash e t a l , , 1973). p e r molecule 40$.  (Mat-  I n c o r p o r a t i o n of one amine molecule  of p e p s i n l e d to a drop of a c t i v i t y of about  Three c a r b o x y l groups were modified*  /& - c a r b o x y l group  of a s p a r t a t e , r - c a r b o x y l group of glutamate. and  the  c a r b o x y l group of C - t e r m i n a l a l a n i n e . N-Bromosuccinimide has been used to modify r e s i d u e s i n p e p s i n (Lokshina and Orekhovich, and Witkop, 1964). amounted t o 85-90%.  tryptophan  1964;  Green  P e p s i n i n a c t i v a t i o n by the reagent Dopheide and Jones (1968) used  hydroxyl-5-nitrobenzylbromide  2-  f o r tryptophan m o d i f i c a t i o n .  I n c o r p o r a t i o n of two r e s i d u e s of the reagent was  observed,  r e s u l t i n g i n a l o s s of o n l y 25-30$ of p r o t e o l y t i c and peptidase  activity. Methionine  residues, i n p e p s i n were a l k y l a t e d by! i o d o -  a c e t i c a c i d w i t h no observable change i n a c t i v i t y , t h a t methionine  indicating  r e s i d u e s were not important f o r the a c t i o n  of p e p s i n (Lokshina and Orekhovich,  1964).  The amino groups  21  o f p e p s i n were a l s o  shown t o be n o n - e s s e n t i a l  for  enzyme  activity.  A c e t y l a t i o n o f t h e amino groups w i t h k e t e n e  not affect  protease  T h i s was f u r t h e r acid  activity  supported by deamination w i t h n i t r o u s  ( M e l c h i o r and F a h r n e y , change i n  1970)  and i o d i n a t i o n . observed  tidase  b o t h o f w h i c h caused no  after  acetylation  o f 11-12  activity  t y r o s i n e - O H groups  The e s t e r a s e a n d p e p t i d a s e  activity,  ( L o k s h i n a a n d O r e k h o v i c h , 1966).  i o d i n a t i o n l e d to a decrease i n protease,  and e s t e r a s e a c t i v i t i e s  Sequence a n a l y s i s  of the  In  pep-  ( H o l l a n d a n d F r u t o n * 1968).  iodinated peptides  modified tyrosine residues  e t a l . , 1973).  appre-  acetylation  A 95% i n h i b i t i o n i n p r o t e o l y t i c  was i n c r e a s e d  comparison,  N-ethoxyformylation  have been m o d i f i e d by  i n the pepsin molecule. however,  and  activity.  Tyrosine residues  was  ( H e r r i o t t a n d N o r t h r o p , 1934).  ( P h i l p o t a n d S m a l l , 1938)  ciable  did  located  a t p o s i t i o n s 9 a n d 175  the  (Mains  22  MATERIALS AND METHODS  Biochemicals All specified  and S p e c i a l  chemicals  used were o f r e a g e n t grade  unless  otherwise.  Porcine pepsin mucosa,  Chemicals  ( E . C . 3.4.23,.l|from h o g  2X c r y s t a l l i z e d )  stomach  was p u r c h a s e d f r o m S i g m a C h e m i c a l  C o . and W o r t h i n g t o n B i o c h e m i c a l C o r p .  The l o t f r o m S i g m a  h a d an a s s a y o f 3»520 u n i t s / m g p r o t e i n , w h i l e t h e l o t W o r t h i n g t o n h a d a n a s s a y o f 2,700 u n i t s / m g p r o t e i n . pepsin sin  (1»10,000), p e p s i n o g e n  (from hog stomach)  ( r e n n i n , - E . C . 3.4.23.4; f r o m c a l f  o f Sigma C h e m i c a l C o .  Rennet  from ICN P h a r m a c e u t i c a l s ,  (NF r e n n i n ) was  Crude  and chymo-  were  products  purchased  Inc.  The two w a t e r - s o l u b l e project,  stomach)  from  c a r b o d i i m i d e s used i n  l-cyclohexyl-3-(2-morpholinyl-4-ethyl)  imide metho-p-toluenesulfonate  this  carbodi-  and l - e t h y l - 3 < 3 - d i m e t h y l -  a m i n o p r o p y l ) c a r b o d i i m i d e were purchased from Sigma C h e m i c a l Co.  G l y c i n e m e t h y l e s t e r h y d r o c h l o r i d e was a p r o d u c t  A l d r i c h Chemical C o . , inine,  leucine,  lysine,  Sigma Chemical Co. taurine  I n c . , w h i l e the methyl e s t e r s  of  arg-  t y r o s i n e and t r y p t o p h a n were from  E t h y l e n e d i a m i n e d i h y d r o c h l o r i d e and  (2-aminoethanesulfonic  Chemical Co.  of  a c i d ) were a l s o from Sigma  23  The s y n t h e t i c diiodo-L-tyrosine, N-carbobenzoxy  (p-nitrophenoxy)  N-acetyl-D-phenylalanyl-L-tyrosine  Two p e p s i n i n h i b i t o r s ,  propane  and bromophenacyl  f r o m Eastman Kodak C o .  determination of proteolytic hemoglobin ical  N-acetyl-L-phenylalariyl-  - L - g l u t a m y l - L - t y r o s i n e were p r o d u c t s  Sigma Chemical Co.  purchased  dipeptides,  (bovine  serum),  of  l,2-epoxy-3bromide were  The s u b s t r a t e f o r  activity,  and  the  acid-denatured  was f r o m W o r t h i n g t o n B i o c h e m -  Corp. 3-(2-Aminoethyl)  were p r o d u c t s  i n d o l e and p - t o l u e n e s u l f o n i c  of Matheson Coleman & B e l l ,  N i n h y d r i n and h y d r i n d a n t i n were purchased Kodak C o .  Norwood,  acid Ohio.  from Eastman  24  Methods 1.  M o d i f i c a t i o n of carboxyl The c a r b o x y l g r o u p s  groups  i n p e p s i n , pepsinogen  and  chy-  m o s i n were m o d i f i e d b y t h e c a r b o d i i m i d e - p r o m o t e d amide f o r m ation.  The m e t h o d was  essentially  that  d e s c r i b e d by Hoare  a n d K o s h l a n d (1967) f o r t h e d e t e r m i n a t i o n o f c a r b o x y l tent  i n proteins.  However,  sufficiently  lower  con-  concentrations  o f c a r b o d i i m i d e and n u c l e o p h i l e were u s e d t o a c h i e v e tive modification.  Denaturants  g u a n i d i n e h y d r o c h l o r i d e , as  such as 8 M u r e a o r 6 M  s u g g e s t e d by the above  f o r t o t a l covering of carboxyl groups, present  were ommitted i n  (10 mg/ml) a n d n u c l e o p h i l e w e r e  i n d i s t i l l e d water. w i t h 1 N NaOH t o 5 . 5 .  The pH o f t h e s o l u t i o n was The w a t e r - s o l u b l e  added as a s o l i d t o b r i n g i t s  a d d i t i o n o f 0.1  adjusted  concentration to the  90 m i n u t e s .  r e a c t i o n m i x t u r e was k e p t c o n s t a n t  at  temperatue  The pH o f  5.5 by  Denmark).  The r e a c t i o n was  which reacted w i t h excess n u c l e o p h i l e .  r e a g e n t s were removed by e x h a u s t i v e  dialysis  the  automatic  N H C l , u s i n g a Radiometer type TTTIc  (Copenhagen,  was  desired  titr-  terminated  by the a d d i t i o n of excess 3 M sodium a c e t a t e b u f f e r 5*5  the  dissolved  carbodiimide  The r e a c t i o n m i x t u r e was k e p t a t room  and s t i r r e d c o n t i n u o u s l y f o r  ator  workers  investigation. The p r o t e i n  level.  selec-  The  a t pH  residual  against  dis-  25 t i l l e d water f o r 48 hours at 4° C and the modified protein was recovered by l y o p h i l i z a t i o n . To determine the extent of carboxyl modification, the modified protein was subjected to amino acid a n a l y s i s . The protein was hydrolysed f o r 24 hours at 110° C with 3(2-aminoethyl) indole and p-toluenesulfonic acid, as described by L i u and Chang (1971).  The hydrolysate was  then  neutralized and applied to an amino acid analyser column. From the chromatogram, the peak area of the p a r t i c u l a r amino a c i d which had been incorporated was measured and compared to that of the c o n t r o l .  The increase i n peak area was  pro-  p o r t i o n a l to the number of molecules of amino acid methyl ester incorporated into the protein, or the number of carboxyl groups modified.  2.  Determination of milk c l o t t i n g a c t i v i t y The milk c l o t t i n g a c t i v i t y of pepsin and chymosin  was  determined by the methods of Berridge (1945) and F o l t -  mann (1970) with some modifications. Commercial spraydried skimmilk powder was used as substrate and was desiccated  at  4°C.  The substrate solution was  kept pre-  pared by r e c o n s t i t u t i n g 12 g of skimmilk powder i n 100 ml of 0.01 M CaClg with vigorous s t i r r i n g f o r 5 minutes without foaming.  The reconstituted skimmilk had a pH of about  26  6.3.  The pH can be adjusted by varying the concentration  of C a C l from 0.001 M (pH=6.5) to 0.08 M (pH=5.7) and the 2  addition of small amount of HCl or NaOH. The reconstituted skimmilk substrate was l e f t at room temperature f o r one hour to e q u i l i b r a t e ,  The skim-  milk (10 ml) was pipetted into a stoppered test tube and incubated i n a water bath at 30° C f o r 10 minutes.  The  enzyme was diluted with c i t r a t e buffer to an a c t i v i t y corresponding to a c l o t t i n g time of 4-5 minutes.  The diluted  enzyme s o l u t i o n (1 ml), pre-incubated to 30°C, was pipetted into the test tube with thorough mixing. was started.  A stopwatch  The milk was kept flowing from one end of the  stoppered tube to the other.  The  moment when the t h i n  f i l m of milk broke into v i s i b l e p a r t i c l e s was  recorded as  the c l o t t i n g time. As defined by Berridge (1945), one unit of milk c l o t t i n g a c t i v i t y was the amount of enzyme which would c l o t 10 ml of reconstituted skimmilk i n 100 seconds at 30°C.  The s p e c i f i c a c t i v i t y was expressed as milk c l o t -  t i n g a c t i v i t y per mg  protein.  The enzyme concentrations  were determined by the method of Lowry et a l . (1951) using bovine serum albumin as  a protein standard.  27  3.  Determination of p r o t e o l y t i c a c t i v i t y The assay method of Anson (1938) was followed with  modifications. strate.  Acid-denatured hemoglobin was used as sub-  Substrate s o l u t i o n was f r e s h l y prepared by d i s -  s o l v i n g hemoglobin powder i n d i s t i l l e d water.  Hydrochloric  a c i d was added to y i e l d the desired pH, and the concentrat i o n was adjusted to 2.0% (w/v) protein by the addition of distilled  water.  The enzyme (0.5 ml), i n suitable d i l u t i o n , was added to a stoppered test tube containing 0.5 ml of 0.1 M KCl/HCl b u f f e r ( c i t r a t e buffer f o r pH's above 2.5) pH.  of appropriate  For routine analysis, the optimum pH f o r pepsin a c t i v i t y  (pH 2.0) was used.  The enzyme and substrate solutions  were equilibrated i n a water bath at 3 7 ° C f o r 10 minutes. The substrate solution (0.5 ml) was added to the assay tubes with vigorous mixing using a vortex mixer. incubation at 37° C f o r exactly 10 minutes,  After  10 ml of  5% (w/v) t r i c h l o r o a c e t i c acid (TCA) were added to the assay tubes.  TCA stopped the enzyme reaction and precipitated  the unhydrolysed substrate.  The samples were f i l t e r e d  through Whatman No. 2 papers and the absorbance at 280 nm of the f i l t r a t e was read with a Beckman DB  spectrophotometer  using 1 cm cuvettes. A blank was run i n which TCA was added before the enzyme s o l u t i o n .  The absorbance of the blank  was measured and deducted from the sample absorbance.  All  28  d e t e r m i n a t i o n s were r u n i n d u p l i c a t e used  f o r c a l c u l a t i n g the p r o t e o l y t i c One u n i t o f p r o t e o l y t i c  amount o f enzyme t h a t minute at  37°C.  the p r o t e o l y t i c  activity.  a c t i v i t y was d e f i n e d a s  produced an absorbance  The s p e c i f i c  The p e p t i d a s e  per  a c t i v i t y was e x p r e s s e d  as  activity  activity o f p e p s i n was d e t e r m i n e d  N-acetyl-L-phenylalanyl-diiodo-L-tyrosine strate.  o f 0.001  the  a c t i v i t y p e r mg p r o t e i n .  Determination of peptidase  4.  and t h e a v e r a g e s were  1  The a s s a y m e t h o d o f J a c k s o n e t  (APDT) a s  al.  using  sub-  (19^5) was  l o w e d w i t h some m o d i f i c a t i o n s a s d e s c r i b e d b y R y l e  fol-  (1970).  N i n h y d r i n r e a g e n t was p r e p a r e d b y d i s s o l v i n g 20 n i n h y d r i n a n d 6 g h y d r i n d a n t i n i n 750 m l o f To t h i s was a d d e d 250 m l o f a c e t a t e b u f f e r 3 H 0 + 2  100 m l o f g l a c i a l a c e t i c  acid).  s t o r e d under n i t r o g e n i n a dark b o t t l e penser.  The s u b s t r a t e  when k e p t  2-methoxyethanol. (544 g CH^COO.  The r e a g e n t  was  equipped w i t h a  s o l u t i o n (1.0 mM) ,  d i s s o l v i n g APDT i n 0.01  g  N NaOH , was s t a b l e  prepared  disby  for several  weeks  i n the c o l d room.  The enzyme s o l u t i o n (0.5 m l ) was a d d e d t o t u b e s w i t h 0.25  m l o f H C l o f a p p r o p r i a t e n o r m a l i t y and i n c u b a t e d i n a  water b a t h a t 37°C.  A f t e r 10 m i n u t e s ,  0.25  m l o f APDT  s o l u t i o n was a d d e d t o t h e r e a c t i o n t u b e s a n d 1.0 m l o f  29  n i n h y d r i n r e a g e n t was a d d e d t o t h e b l a n k s . 10 m i n u t e s ,  exactly  1.0 m l o f n i n h y d r i n r e a g e n t was p i p e t t e d t o  r e a c t i o n t u b e s a n d a t a n y t i m e 0.25 the  After  the  m l o f APDT was a d d e d  to  blanks. All  the tubes,  stoppered  by m a r b l e s , were p l a c e d  a b o i l i n g w a t e r b a t h f o r 15 m i n u t e s t o d e v e l o p t h e a n d were t h e n c o o l e d w i t h c o l d w a t e r . t u b e s w e r e d i l u t e d w i t h 5 m l o f 60$ mixing,  the absorbance  against  the b l a n k s .  and b l a n k s , peptidase  color,  The c o n t e n t s o f (v/v)  ethanol.  read  samples  and t h e a v e r a g e s were used t o c a l c u l a t e  activity,  expressed  the  After  o f t h e s o l u t i o n a t 570.nm was  D u p l i c a t e s were r u n f o r a l l  in  the  a s APDT u n i t s .  One APDT u n i t i s d e f i n e d a s t h e q u a n t i t y o f  enzyme  w h i c h l i b e r a t e s 1.0 m i c r o m o l e o f d i i o d o t y r o s i n e p e r m i n u t e at  37° C .  tyrosine  Since E i s 22.8,  Q  ^ fo  r  *  n e  color reaction of  diiodo-  t h e v o l u m e o f t h e s o l u t i o n was 7 m l a n d  t h e t i m e o f i n c u b a t i o n was 10 m i n u t e s , A  £ 1 7 n  was  converted  prO nm t o APDT u n i t s b y m u l t i p l y i n g b y 0.0306. a c t i v i t y was e x p r e s s e d  5.  peptidase  a s APDT u n i t s / m g p r o t e i n .  Assay of pepsin w i t h x-casein P e p s i n a c t i v i t y was a s s a y e d  c a s e i n as s u b s t r a t e  Specific  and « s - c a s e i n 3 l  b y a method u s i n g  K-  ( D o u i l l a r d a n d R i b a d e a u Dumas, 1970).  - O a s e i n m i x t u r e s were a l s o u s e d as  substrate.  30 A : - C a s e i n and « : - c a s e i n s l  of  Zi t t i e  casein  **  g l  -casein  the  w / v ) w e r e made b y d i s s o l v i n g t h e p r o t e i n s  M citrate buffer,  0.05  0.2%  pH 5*3.  s o l u t i o n s , w h i c h gave a f i n a l  t i o n s were  of  g l  -  in  A m i x t u r e o f K - c a s e i n and  ( l i l ) was p r e p a r e d b y m i x i n g e q u a l v o l u m e s  X - c a s e i n a n d 0.1$  0.195  method  (1963). S o l u t i o n s o f J < - c a s e i n a n d  and C u s t e r  (0.2%  were p r e p a r e d b y the  «* -casein. s l  of  concentration The s u b s t r a t e  incubated i n stoppered t e s t tubes  at  of solu-  37° C .  Enzyme s o l u t i o n o f a p p r o p r i a t e c o n c e n t r a t i o n was a d d e d w i t h immediate m i x i n g .  The t i m e a t w h i c h v i s i b l e  a p p e a r e d was t a k e n as t h e  clotting time.  particles  One u n i t  a c t i v i t y was a r b i t r a r i l y d e f i n e d a s t h e amount o f which would c l o t at  37°C.  10 m l o f c a s e i n s o l u t i o n i n 100  Specific  a c t i v i t y was e x p r e s s e d  as  of enzyme  seconds  clotting  a c t i v i t y p e r mg p r o t e i n .  6.  Determination of r a t e The r a t e  of release  of casein  hydrolysis  of n o n - p r o t e i n n i t r o g e n from  w h o l e c a s e i n b y p e p s i n was d e t e r m i n e d b y a m e t h o d b y G r e e n (1972).  The s u b s t r a t e  s o l u t i o n c o n t a i n e d 1%  whole  c a s e i n i n 0.1 M s o d i u m c i t r a t e b u f f e r ,  After  incubation at  At time i n t e r v a l s , 10%  30° C  described  pH 5.3  f o r 10 m i n u t e s , enzyme was  samples were w i t h d r a w n and  ( w / v ) TCA was a d d e d t o g i v e  (w/v)  or  6.5.  added.  sufficient  a f i n a l TCA c o n c e n t r a t i o n  31  of 3$.  After f i l t e r i n g through Whatman No. 2 papers, the  nitrogen content of the f i l t r a t e was procedure of Lang (1958). soluble nitrogen was  7.  determined by the  The rate of l i b e r a t i o n of  TCA-  expressed as jig N released/mg casein.  Agarose g e l electrophoresis Electrophoresis of enzymes and milk proteins  was  c a r r i e d out with the agarose f i l m cassette system of Analyt i c a l Chemists, Inc. (Palo A l t o , C a l i f o r n i a ) The agarose f i l m s contained 1.2$ and 0.035$ EDTA i n 0.05  agarose, 10$ sucrose  M b a r b i t a l buffer, pH 8.6.  The  plate consisted of a t h i n layer of agarose gel adhered to a transparent  plastic  backing.  Samples (1-2 jul) containing  20-40 jag proteins were pipetted into the pre-castrsample wells with a Hamilton m i c r o l i t e r sample dispenser and a disposable t i p . The agarose plate was sette cover.  Electrophoresis was  f i t t e d into a c a s -  started by i n s e r t i n g the  film-loaded cassette onto the electrophoresis c e l l /power supply u n i t . 0.05  The electrophoresis c e l l s contained 200 ml of  M b a r b i t a l buffer, pH 8.6,  with 0.035$ EDTA.  f i l m c a r r i e d a voltage gradient of 15 V/cm heat buildup, no cooling was  Since  the  with n e g l i g i b l e  necessary.  A f t e r electrophoresis which took 35-50 minutes, the plate was  removed from the cassette cover and stained with  32  0.2$  (w/v)  a m i d o b l a c k 10B  i n 5$ ( v / v )  acetic  acid for  15  minutes i n a s t a i n i n g bath w i t h continuous s t i r r i n g by a magnetic acetic  stirrer.  The s t a i n e d f i l m was r i n s e d w i t h  a c i d and d r i e d a t  72°C  5$  f o r 15 m i n u t e s i n a n  oven.  The d r i e d f i l m was d e s t a i n e d b y w a s h i n g t w i c e w i t h 5$ acid,  acetic  a n d t h e d e s t a i n e d g e l p l a t e was d r i e d a g a i n i n t h e  oven. For electrophoresis d i s s o l v e d i n the  o f m i l k c u r d s , t h e c u r d s were  electrophoretic  buffer  The a g a r o s e f i l m was e q u i l i b r a t e d f o r urea-containing barbital buffer  8.  one m i n u t e w i t h  before  Determination of i s o e l e c t r i c  containing 6 M urea.  sample a p p l i c a t i o n .  point  The i s o e l e c t r i c p o i n t o f p e p s i n s a m p l e s was mined by g e l e l e c t r o p h o r e s i s  at d i f f e r e n t  pH*s.  o f a p p r o p r i a t e pH ( K C l / H C l b u f f e r  agarose M  buffer  citrate  buffer  A f t e r s a m p l e a p p l i c a t i o n , . e a c h s t r i p was  nected by b u f f e r  c o n t a i n e d i n two 5 - m l b e a k e r s  p l a c e d i n t h e two e l e c t r o d e was f i l l e d w i t h s u f f i c i e n t connected to the b u f f e r strip.  f o r pH 1-2,  deter-  The  f i l m was c u t u p i n t o s t r i p s a n d e q u i l i b r a t e d i n 0.1  f o r pH 3-5)•  the  compartments. 0,2  w h i c h were  E a c h compartment  1 N a C l s o l u t i o n w h i c h was  i n the beakers  by a s m a l l paper  F i v e s a m p l e s w e r e r u n a t t h e same t i m e i n  w i t h p H ' s o f 1.0,  2.0,  3.0,  4.0  con-  and  5.0.  buffers  33  A f t e r e l e c t r o p h o r e s i s , the pH*s of the b u f f e r s were measured t o d e t e c t pH change t h a t may  occur d u r i n g the  run.  A f t e r s t a i n i n g , the m o b i l i t i e s of the enzyme bands were measured and p l o t t e d a g a i n s t the pH*s. curve  The  straight  obtained by l i n e a r r e g r e s s i o n a n a l y s i s was  line  extraplot-  ated t o cut the pH a x i s which corresponded to zero m o b i l i t y . T h i s was  9.  the estimated  Determination The  of  i s o e l e c t r i c p o i n t of the enzyme.  and k.  i n i t i a l velocity  (V ) q  h y d r o l y s i s of APDT, expressed a t e d per minute, was trations.  ^ o f the  as jmoles  pepsin-catalysed diiodotyrosine l i b e r -  measured a t d i f f e r e n t s u b s t r a t e concen-  The M i c h a e l i s c o n s t a n t , K , was m  determined from  the Lineweaver-Burk p l o t which i s the r e c i p r o c a l of s u b s t r a t e c o n c e n t r a t i o n a g a i n s t the r e c i p r o c a l of v . Q  s i o n a n a l y s i s was The  molecular  ^ c a t * was  v where  0  Linear regres-  employed t o y i e l d the b e s t - f i t  a c t i v i t y c o e f f i c i e n t or c a t a l y t i c  c a l c u l a t e d by the Michaelis-Menten  - * c a t [sum  £E] was  /(K  m +  constant,  equation!  rsj)  the t o t a l enzyme c o n c e n t r a t i o n and [S] was  the s u b s t r a t e c o n c e n t r a t i o n at zero time. o f p e p s i n was  curves.  estimated  The  concentration  from the absorbance a t 280  nm,  assuming a molar a b s o r p t i v i t y of 50,990. K  m  and k .j. were determined a t both pH 2.0 a  and  4.5.  34  10.  Assay of pepsinogen The p r o t e o l y t i c and milk c l o t t i n g a c t i v i t i e s of  porcine pepsinogen were determined after a c t i v a t i o n of the zymogen.  Pepsinogen was dissolved i n 0.01 M KCl/HCl buffer,  pH 2.0 at a concentration of 1 rag/ml. The solution was kept at room temperature overnight when a l l the zymogen was converted to the active enzyme (Herriott, 1938).  11.  Determination of pepsin s t a b i l i t y near neutral pH The pH s t a b i l i t y of both c r y s t a l l i n e and crude pepsins  was determined by the methods described by Lowenstein (1974) and Green (1972). The enzyme s t a b i l i t y i n buffer was measured by incubation of a suitable concentration of pepsin i n 0.05 M phosphate buffer, pH 6.5, at 3 0 ° C .  At time i n t e r v a l s , an a l i -  quot of sample was removed and the milk c l o t t i n g and proteol y t i c a c t i v i t i e s were determined.  The changes i n a c t i v i t i e s  were followed f o r 2-4 hours. The s t a b i l i t y of pepsin i n milk u l t r a f i l t r a t e s also studied.  was  Reconstituted milk (from skimmilk powder) was  passed through an u l t r a f i l t r a t i o n c e l l (Model 5 2 , Amicon Corp., Lexington, filter.  MA.)  equipped with a PM Diaflo u l t r a -  The f i l t r a t e collected was stored at 4°C  within two days a f t e r preparation.  and used  35 The enzyme was incubated at 30° C i n the milk u l t r a f i l t r a t e which has been previously adjusted to pH 6.60 1 N lactic acid.  The pH of the incubation mixture  with  was  lowered by adding l a c t i c acid at 15-minute i n t e r v a l s , at approximately the same rate at which i t was lowered during cheese-making.  The scheme suggested by Green (1972) was  followed (Table 2).  Samples of enzyme were taken at various  time i n t e r v a l s f o r assay of milk c l o t t i n g and p r o t e o l y t i c activities.  12.  Determination of thermal s t a b i l i t y of pepsin The thermal s t a b i l i t y of pepsin was determined  by  measuring both milk c l o t t i n g and proteolytic a c t i v i t i e s of the enzyme at increasing temperatures.  For proteolysis,  the a c t i v i t y was measured at 30, 40, 50, 60 and 70° C. For milk c l o t t i n g , the a c t i v i t y was determined at 25t 35, 40, 45 and 50°C.  30,  The pH was 2.0 f o r proteolysis and  6.3 f o r milk coagulation.  The Q  10  values were calculated  which represent the r a t i o of the a c t i v i t y at (T + 10)° to that at T°.  temperature  36  Table  Adjustment  2.  stability  I n c u b a t i o n Time  o f pH o f m i l k u l t r a f i l t r a t e test  of  for  pepsin.  pH  (minute)  Corresponding Cheese-making Stage  1  6.60  Enzyme  addition  50  6.50  Cutting  120  6.40  Maximum s c a l d  160  6.25  210  5.90  Pitching  37  13.  Determination of curd t e n s i o n The f i r m n e s s  o r c u r d t e n s i o n o f the coagulum from  p e p s i n - c u r d l e d m i l k was d e t e r m i n e d b y a m e t h o d by Hehir  (1968), l a t e r i m p r o v e d b y E l l i s  technique  involves  f e r when a w e i g h t milk  t h e measurement  developed This  (1972).  of the weight  i s p l a c e d on t h e s u r f a c e  of a  transcoagulated  sample. P l a s t i c beakers  (50 m l ) w e r e u s e d f o r c u r d t e n s i o n  d e t e r m i n a t i o n s and were r e d u c e d t o a s t a n d a r d w e i g h t shaving the r i m s . (+ 0.01  g)  U s i n g t h e t a r e f a c i l i t y on a 1,200  capacity,  3704) t h e w e i g h t  d i g i t a l top-pan balance  of the beakers  was r e d u c e d t o  m l ) was p i p e t t e d i n t o a t a r e d b e a k e r . i n a water bath at  30°C.  zero. (19.8  The b e a k e r was  placed  A f t e r incubation for 5 minutes,  m l o f a p p r o p r i a t e l y d i l u t e d enzyme was a d d e d t o  milk with mixing.  g  (Sartorius  A s t a n d a r d volume of r e c o n s t i t u t e d s k i m m i l k  0.2  by  The c o n c e n t r a t i o n o f enzyme was minutes  m i l k s a m p l e was i n c u b a t e d a t  30° C f o r f u r t h e r 30 m i n u t e s  At the precise  time to give time  (T +30  The  such  t h a t m i l k was c l o t t e d i n 4-5  which i s a convenient  (T).  the  coagulated  curd formation. minutes), the beaker  of  c o a g u l u m was g e n t l y t a k e n f r o m t h e w a t e r b a t h , d r i e d on the outside with tissue  p a p e r a n d p l a c e d on t h e  pan.  E x a c t l y 15 s e c o n d s  flat  b o t t o m e d 10 g w e i g h t  after  balance  removal from the b a t h ,  a  s u p p o r t e d b y a m e t a l c h a i n was  38 placed on the surface of the curd* the nearest 10 mg  The scale was read to  (centigram) at exactly 15 seconds (to  minimize surface tension e f f e c t ) and 60 seconds a f t e r the weight was and 31.25  i n contact with the curd, i . e . , 30.5 minutes minutes a f t e r curd formation.  The difference  between the two readings, i n centigrams, was taken as an a r b i t r a r y measure of the curd tension which was the amount of weight transferred from the curd to the chain i n 45 seconds. A constant displacement  of the hanging weight was  set by p l a c i n g 20 ml d i s t i l l e d water i n a standardized beaker.  By adjusting the height of the point of support  of the weight, the balance was  set a r b i t r a r i l y at 20.70 g  when about 70% of the weight's volume was immersed.  This  operation ensured a constant distance between the point of chain suspension and the curd surface.  14.  Determination  of the rate of syneresis  The rate of exudation of whey from curd was measured by the discontinuous method of Lawrence (1959) with some modifications. Samples of reconstituted skimmilk (100 ml) at pH  6.3  were added to 250-ml beakers and incubated at 30° C i n a water bath. An appropriate amount of enzyme was added to the  39  milk.  The g e l was c u t i n t o b l o c k s  utes after  clotting.  of uniform size  30 m i n -  The c u r d was t h e n h e l d a t 30° C  with-  out s t i r r i n g . At different on t o a s i e v e by the sieve  time i n t e r v a l s ,  e a c h b e a k e r was t i p p e d  placed over a f u n n e l .  The c u r d was r e t a i n e d  a n d t h e whey was d r a i n e d i n t o a  c y l i n d e r under the f u n n e l . cylinder  30 s e c o n d s  after  graduated  The v o l u m e o f whey i n t h e t i p p i n g was r e c o r d e d .  Volume  o f t h e c u r d was c a l c u l a t e d b y s u b t r a c t i n g t h e v o l u m e t h e whey f r o m t h e i n i t i a l v o l u m e o f m i l k . expressed  as percentage  Percentage  syneresis  of  The r e s u l t was  syneresisi -  ^ 1 ^ 0 1 ^ 1 ^ 1 1 *  x  ">°  40 RESULTS AND DISCUSSION  Choice of Enzyme Source The pepsin samples used i n the present i n v e s t i g a t i o n were 2X c r y s t a l l i z e d and l y o p h i l i z e d enzyme from hog stomach mucosa.  Two l o t s , one from Sigma Chemical Co. (Lot No.  67C-8195) and the other from Worthington Biochemical Corp. (Lot No. PM 37C6?5)» were used.  Both enzymes appeared as  one sharp band when applied to agarose g e l electrophoresis, and were eluted as one sharp peak on DEAE c e l l u l o s e column with 0.1 M sodium acetate buffer (pH 4.2) and increasing concentration of NaCl as eluents.  These observations sug-  gest that the enzyme preparations used were e s s e n t i a l l y homogeneous and were therefore used d i r e c t l y without further purification. Rajagopalan et a l . (1966b) proposed a method to prepare homogeneous pepsin from pepsinogen.  The procedure i n -  volved a c t i v a t i o n of the zymogen at 14° C and pH 2 f o r 20 minutes and the separation of the enzyme from the peptide by passage through a column of s u l f o e t h y l Sephadex  C-25.  The high cost of pepsinogen prohibited preparation of large amount of pepsin by t h i s method. In some experiments, crude pepsin (1:10,000) was used.  The i n d u s t r i a l pepsin i s the s t a r t i n g material from  which c r y s t a l l i n e pepsin has t r a d i t i o n a l l y been prepared.  41  This i s the enzyme preparation that can he used, together with c a l f rennet as 50*50 mixtures to make cheese.  Hence,  i n experiments designed to t e s t the s u i t a b i l i t y of modified pepsin f o r cheese-making, 1x10,000 pepsin i s a more approp r i a t e enzyme than the c r y s t a l l i n e pepsin. In t h i s thesis, the term pepsin refers to the crys t a l l i n e porcine pepsin while the impure enzyme w i l l be s p e c i f i e d as crude or 1x10,000 pepsin.  Choice of Carbodiimides and Nucleophiles Two commercially available water-soluble carbodiimides, l-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide metho-p-toluenesulfonate  (CMC) and l-ethyl-3-(3-dimethyl-  aminopropyl) carbodiimide (EDC) were used to modify the carboxyl groups i n pepsin.  Both carbodiimides were found  to modify pepsin carboxyl groups with similar changes i n enzymatic  a c t i v i t i e s and properties. However, EDC was  found to be more e f f e c t i v e than CMC a t the same molar concentration.  This could be due to the smaller size of EDC  (M.W.=191.7) than CMC (M.W..423.6) enabling EDC to react with p a r t i a l l y buried carboxyl groups i n pepsin.  EDC was  therefore used subsequently. Three nucleophiles, ethylenediamine, methyl esters of various amino acids and 2-aminoethanesulfonic  acid  42  (taurine) were used f o r carboxyl modification.  The d i f f e r e n t  charges carried by these nucleophiles caused considerable difference i n the i o n i c character of the modified enzymes (see F i g . 2). Ethylenediamine  and taurine were found to  cause extensive loss i n milk c l o t t i n g a c t i v i t y of pepsin even at low concentrations (Table 3 ) .  Hence, methyl esters  of various amino acids, mainly glycine, were used i n l a t e r experiments.  E f f e c t of Nucleonhile Concentration on the Extent of Carboxyl Modification The number of pepsin carboxyl groups covered i n the carbodiimide reaction was determined by amino acid a n a l y s i s . EDC and glycine methyl ester at various concentrations were used.  The number of carboxyl groups modified increased as  the concentration of nucleophile was increased ( F i g . 3 ) . The concentration of EDC was found to be less c r i t i c a l i n a f f e c t i n g the amount of modification.  However, EDC concen-  t r a t i o n s lower than 10 mM ( 3 3 - f o l d excess to pepsin) were found to be i n e f f e c t i v e i n modifying pepsin. For most of the subsequent experiments, unless specif i e d otherwise, the concentrations of reagents used were* 3 3 - f o l d excess of EDC and 174-fold excess of glycine methyl  T a b l e 3.  Carboxyl modification of pepsin w i t h  different  nucleophiles.*  Milk clotting activity (fo c o n t r o l )  Concn. (mM)  Proteolytic activity (% c o n t r o l )  Gly methyl ester  50  94  17  Ethylenediamine  50  85  3  Taurine  50  90  5  Nucleophile  Pepsin  (10 mg/ml) was m o d i f i e d w i t h 3 3 - f o l d e x c e s s  EDC a n d 1 7 4 - f o l d e x c e s s o f n u c l e o p h i l e 90 m i n u t e s .  a t pH 5.5  of  for  44  r\h  i  .05  Nucleophile FIGURE 3.  i  l  .10  Concn.,  .15  M  EFFECT OF NUCLEOPHILE CONCENTRATION ON THE EXTENT OF CARBOXYL MODIFICATION OF PEPSIN.  1  .20  45 ester,  corresponding  nucleophile  Activity 1.  Milk  p e r mole  clotting  pepsin i s  carboxyl modification.  shown i n F i g . 4.  The  i n the  A d r o p o f 70-90%  clotting  extent  i n milk  with glycine  methyl ester alone,  milk clotting a c t i v i t y .  When t h e  milk c l o t t i n g a c t i v i t y with a corresponding number o f m o d i f i e d c a r b o x y l g r o u p s treated  35% ( s e e  Table  Table  methyl ester  the  When  4).  o f p e p s i n was a l s o  nucleodecr-  4).  (87-fold excess to  a n d EDC ( 3 3 - f o l d e x c e s s t o p e p s i n ) , about  60% o f  decrease i n  When c r u d e p e p s i n was m o d i f i e d w i t h a l o w a t i o n of g l y c i n e  concen-  w i t h 10 mM EDC i n t h e a b s e n c e o f  the milk c l o t t i n g a c t i v i t y  eased by about  (see  pepsin  carbodiimide  t r a t i o n was 50 mM), t h e m o d i f i e d enzyme h a d a b o u t  phile,  of  clotting  c o n c e n t r a t i o n was l o w e r e d t o 5 mM ( t h e n u c l e o p h i l e  p e p s i n was  activity  observed.  When t r e a t e d 100$  Pepsins  o f c a r b o x y l m o d i f i c a t i o n on m i l k  was f o u n d t o d r o p r a p i d l y w i t h i n c r e a s e  retained  of  activity  of porcine  a c t i v i t y was  moles  pepsin.  o f N a t i v e and C a r b o x y l M o d i f i e d  The e f f e c t activity  t o a n i n c o r p o r a t i o n o f 5.2  5®?* i n m i l k c l o t t i n g a c t i v i t y .  concentrpepsin)  t h e r e was a d r o p With a higher  of nucleo-  46  No. of FIGURE 4.  COOH  G p . Modified  EFFECT OF CARBOXYL MODIFICATION OF PEPSIN ON MILK CLOTTING ACTIVITY.  Table 4.  E f f e c t of carboxyl modification on pepsin a c t i v i t i e s .  Glv methvl ester  EDC Concn. Excess to pepsin (mM) (-fold)  Concn. Excess to pepsin (mM) (-fold)  No. C00H gp. modified/mole pepsin  Milk c l o t t i n g activity (# control)  Proteolytic activity (% control) pH 2.0 PH3.5  10  33  0  •0  0  65  100  100  10  33  25  87  2.8  27  88  210  10  33  50  174  5.2  17  94  340  10  33  100  348  8.6  13  105  330  10  33  200  706  11.2  9  90  340  0  0  50  174  0  100  100  100  50  174  1.0  60  100  120  5  16.5  48  phile concentration (174-fold excess to pepsin), the drop i n a c t i v i t y was 70$. This indicates that compared to crys t a l l i n e pepsin, crude pepsin retained higher milk c l o t t i n g a c t i v i t y when modified with the same quantity of reagents. Amino acid analysis of the native and modified crude pepsin d i d not y i e l d consistent r e s u l t s , probably due to impurities present i n the enzymes.  Hence, the extent of  carboxyl modification was not determined f o r the crude pepsin.  2.  Proteolytic activity The proteolytic a c t i v i t y of pepsin at pH 2.0 was found to  decrease s l i g h t l y a f t e r carboxyl modification ( F i g . 5 ) » Even with extensive modification (11.2 moles of carboxyl groups covered/mole pepsin), the enzyme s t i l l retained 90% of i t s a c t i v i t y , showing that the carboxyl groups blocked were not d i r e c t l y involved i n proteolysis of hemoglobin. In contrast to the present r e s u l t , carboxyl modif i c a t i o n of porcine pepsin with CMC and a colored amine caused about 40$ drop i n a c t i v i t y against hemoglobin, a l though only one amine molecule was incorporated per molecule of pepsin (Matyash et a l . , 1973).  The carboxyl groups modi-  f i e d by the colored amine could.be more d i r e c t l y r e l a t e d to pepsin c a t a l y s i s .  o  3  No. of  6  9  COOH G p .  Modified  EFFECT OF CARBOXYL MODIFICATION OF PEPSIN ON PROTEOLYTIC ACTIVITY,  • , PH 2 . 0 ;  o,  pH  3.5,  12  50  When t h e a c t i v i t y a t pH 3 » 5 » t h e  h e m o g l o b i n was  than the  m a i n l y due t o t h e f a c t proteolytic  that  control  at  (Fig. 5).  pH 3 . 5 t  the  had h i g h e r  This indicates  may b e a s h i f t  that  there  Similar results modified with 33-fold methyl e s t e r .  90$ of i t s the that  proteolytic  activity of  the  This  was  activity  at  at  i n the  spe-  pH 2 . 0 ,  pH 3 . 5 . pH p r o f i l e .  w e r e o b t a i n e d when c r u d e p e p s i n  was  e x c e s s o f EDC a n d 8 7 - f o l d e x c e s s  of  The m o d i f i e d enzyme r e t a i n e d  about  activity  pH 3 . 5 ,  a t pH 2 . 0 , w h i l e a t  o f t h e m o d i f i e d enzyme was  about  times  2.5  control.  T a b l e k summarizes  the  effect  of carboxyl  modifi-  c a t i o n o f p e p s i n on m i l k c l o t t i n g and p r o t e o l y t i c i n r e l a t i o n to the  3.  have  c o n t r o l had  a c t i v i t y much l o w e r t h a n t h a t  w h i l e t h e m o d i f i e d enzyme  glycine  measured  c a r b o x y l m o d i f i e d p e p s i n was f o u n d t o  a c t i v i t y much h i g h e r  cific  against  extent of  Milk clotting*proteolytic  activity  modification.  activity  ratio  W i t h a marked d e c r e a s e i n m i l k c l o t t i n g a c t i v i t y no s i g n i f i c a n t  changes i n p r o t e o l y t i c  m o d i f i c a t i o n of pepsin r e s u l t e d milk clotting:proteolytic extensive pepsin),  modification t h e r a t i o was  activity,  carboxyl  i n a dramatic decrease  activity ratio  (11.2 carboxyl only 10$ that  (Fig. 6).  groups of the  and  in  With  modified/mole control.  FIGURE 6 ,  EFFECT OF CARBOXYL MODIFICATION OF PEPSIN ON MILK CLOTTING:PROTEOLYTIC ACTIVITY RATIO.  52  The milk c l o t t i n g : p r o t e o l y t i c a c t i v i t y r a t i o i s an i n d i c a t i o n of the s p e c i f i c i t y of proteases.  Chymosin, the  major enzyme component of c a l f rennet, was found to have the highest c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o among other p r o t e o l y t i c enzymes (Ernstrom,  1974),  i n d i c a t i n g that  chymosin has the highest s p e c i f i c milk c l o t t i n g  activity.  The present data show that r e l a t i v e to general proteolysis, the s p e c i f i c a c t i v i t y of pepsin towards milk c l o t t i n g  was  lowered a f t e r carboxyl modification.  4.  Peptidase a c t i v i t y The peptidase a c t i v i t y of pepsin was measured using  APDT as substrate. Result shows that the peptidase a c t i v i t y at pH 2.0 and  37°C  was decreased by carboxyl modification  (Pig. 7 ) . Unlike milk c l o t t i n g , however, the decrease i n peptidase a c t i v i t y was moderate. tained about  40-60$  The modified pepsin r e -  of a c t i v i t y against the dipeptide sub-  strate . Pepsin modified with CMC  and a colored amine also  showed a drop of 5 0 $ i n peptidase a c t i v i t y ,  when N-acetyl-  L-phenylalanyl-L-tyrosine was used as substrate (Matyash et  al.,  1973).  53  FIGURE  7.  EFFECT  OF  CARBOXYL MODIFICATION  ON P E P T I D A S E  ACTIVITY.  OF  PEPSIN  54  Effect  o f C a r b o x y l M o d i f i c a t i o n o n pH P r o f i l e s  M i l k c l o t t i n g PH  1.  The pH p r o f i l e s  profile of milk c l o t t i n g f o r native  carboxyl modified pepsins  a r e shown i n F i g . 8.  enzymes, the m i l k c l o t t i n g a c t i v i t y pH 6.0-to 6.5. activity  and  For  both  dropped r a p i d l y  from  The m o d i f i e d enzyme r e t a i n e d s l i g h t l y  a t pH 6.3  a n d 6.5  than the  control.  Crude p e p s i n and the c o r r e s p o n d i n g m o d i f i e d also  showed s i m i l a r pH p r o f i l e s ,  indicating that  m o d i f i c a t i o n d i d n o t c h a n g e t h e pH p r o f i l e in  2.  higher  enzyme  carboxyl  of milk  clotting  pepsin.  Proteolytic  PH  The e f f e c t lytic  pH p r o f i l e  profile o f c a r b o x y l m o d i f i c a t i o n on the  of pepsin i s  i l l u s t r a t e d i n F i g . 9.  p e p s i n h a d a pH optimum a t a b o u t  hemoglobin decreased s h a r p l y towards  pH's.  carboxyl modification,  t o about  3.5.  The p l a t e a u  observed  Native  and t h e s p e c i f i c  2.0  v i t y against After  proteo-  acti-  higher  t h e pH optimum was a t pH 1.5-2.0 f o r  m o d i f i e d enzyme c o u l d be a t t r i b u t e d t o i n c o m p l e t e  shifted the  modifi-  cation. It teolytic  is interesting  to note  p H - a c t i v i t y curve  that  closely  chymosin had a p r o -  resembling t h a t  m o d i f i e d p e p s i n , w i t h a pH optimum n e a r 3.5  (Pig.  of 9).  the  55  FIGURE  8.  MILK CLOTTING  PH  CARBOXYL MODIFIED NATIVE  PEPSINj  P R O F I L E S OF  NATIVE  AND  PEPSINS. O , C A R B O X Y L MODIFI ED P E P S I N  01 1.5  — i  2.0  I 3.0  i  2.5  i• 3.5  • 4.0  i 4.5  pH FIGURE  9.  PROTEOLYTIC  PH  P R O F I L E S OF  CARBOXYL MODIFIED CHYMOSIN A  ,  CRYSTALLINE  CRYSTALLINE  PEPSIN  o ,  PEPSIN AND  57  A s i m i l a r s h i f t i n p r o t e o l y t i c pH p r o f i l e s was also observed when crude pepsin was modified with EDC and glycine methyl ester ( F i g . 10). The native enzyme had a pH optimum at about 2.0, and a f t e r modification the optimum s h i f t e d to between pH 3.5-4.0.  The modified crude pepsin had a wider  pH optimum than the modified pure enzyme. A s h i f t i n pH p r o f i l e had been noted i n some immob i l i z e d enzymes and was d i r e c t l y related to the "microenvironment" effect r e s u l t i n g from the embedment of enzymes within the c a r r i e r s (Silman and Katchalski, 1968).  Binding  of charged c a r r i e r s to enzymes may also produce changes i n the d i s t r i b u t i o n of charges on the enzyme molecules.  Con-  sequently, the pH i n the domain of the enzymes w i l l be d i f f e r e n t from the external bulk solution, thus creating an apparent s h i f t i n pH p r o f i l e (Goldstein, 1970).  Since  the nucleophile (glycine methyl ester) attached to pepsin was of small molecular size when compared to the c a r r i e r s used i n enzyme immobilization, the s h i f t i n pH p r o f i l e i n the modified pepsin was u n l i k e l y due to "micro-environment" effect.  I t was probably related to changes i n the charge  d i s t r i b u t i o n on the enzyme as a r e s u l t of covering of the negatively-charged carboxyl groups by the neutral nucleophile.  58  FIGURE  10.  PROTEOLYTIC CARBOXYL  PH  P R O F I L E S OF  MODIFIED  ®, NATIVE PEPSIN  CRUDE  NATIVE  PEPSINS.  (1:10,000);  O, CARBOXYL MODIFIED PEPSIN  (1:10,000)  AND  59 3' PH-Activitv curves f o r the hydrolysis of APDT When the synthetic dipeptide, APDT, was used as subs t r a t e , the pH-activity curves of both native and carboxyl modified pepsin were found to be s i m i l a r except that the modified pepsin had s l i g h t l y higher a c t i v i t y at more alkal i n e pH  (Fig. 11). Both enzymes had a pH optimum around  2.0 and the a c t i v i t y decreased r a p i d l y towards higher pH. However, the decline i n a c t i v i t y was more gradual i n the modified  enzyme.  The r e s u l t shows that unlike hemoglobin, the pHa c t i v i t y curve on dipeptide substrate was not s i g n i f i c a n t l y s h i f t e d by carboxyl modification. The pH-activity curves f o r APDT hydrolysis were not determined f o r the crude pepsins.  Electrophoretic Mobility of Native and Carboxyl Modified Pepsins on Agarose Gel Agarose g e l electrophoresis was used to measure •the electrophoretic mobility of native pepsin and the carboxyl modified enzymes.  Both native and modified enzymes app-  eared as one band on the agarose f i l m (Plate I ) .  Hetero-  geneity of chemically modified proteins, r e s u l t i n g from s u b s t i t u t i o n of d i f f e r e n t numbers of carboxyl groups, might be observed i n electrophoresis as a diffused band.  The  pH FIGURE  11,  PH AND  ACTIVITY  C U R V E S FOR  CARBOXYL MODIFIED  THE ACTION PEPSINSON  PHENYLALANYL-D MODO-L-TYROSINE.  • , NATIVE PEPSIN; A , CARBOXYL MODIFIED PEPSIN.  OF  NATIVE  N-ACETYL-L-  PLATE  I.  AGAROSE G E L ELECTROPHORETIC  OF  N A T I V E AND C A R B O X Y L  PATTERNS  MODIFIED  PEPSINS.  NUMBER OF CARBOXYL GROUPS MODIFIED/MOLE ENZYME: lj 0 (CONTROL); 2, 5.2; 3, 8.7; ^  11,2;  5, 15.6.  61  +  •  origin  62  sharpness  of the enzyme bands shown i n the present r e s u l t  suggest that the derivatives were p r a c t i c a l l y free of unmodified pepsin, and the modified products were essentially  homogeneous. The electrophoretic mobility of pepsin was found to  decrease progressively with increases i n the extent of carboxyl modification (Plate I ) . Electrophoretic separation of proteins on g e l matr i c e s such as starch and polyacrylamide i s based on both e l e c t r i c charge and molecular size difference of the proteins.  However, owing to the low g e l concentration and  large pore size of the agarose g e l used, the molecular s i e v i n g effect i s minimal.  Furthermore, as glycine methyl  ester has a molecular weight of 125» the incorporation of ten molecules of nucleophile would increase the molecular weight of pepsin by less than % .  Gel f i l t r a t i o n chromato-  graphy of the most extensively modified pepsin (11.2 carboxyl groups modified/mole pepsin) on Sephadex G-200 (Superfine) confirmed that the change i n molecular size was not marked, as the modified enzyme was eluted at the same elution volume as the native pepsin (0.1 M acetate buffer, pH 4.5). r e s u l t also indicates that there was no aggregation tween the modified pepsin molecules.  This be-  Hence, the drop i n  electrophoretic mobility a f t e r carboxyl modification was  63  mainly attributed to a decrease i n the net negative charge on the modified enzyme r e s u l t i n g from the blocking of negat i v e l y charged carboxyl groups.by glycine methyl ester. When the number of carboxyl groups modified/mole pepsin was plotted against logarithm of the r e l a t i v e electrophoretic mobility, R^, a l i n e a r r e l a t i o n s h i p was obtained (Fig. 12). This indicates that the net charge of the peps i n molecule i s d i r e c t l y proportional to the electrophoretic mobility.  I t has been shown that provided the molecular  weight and molecular shape of a protein are known, the net charge of the protein can be calculated from i t s e l e c t r o phoretic mobility (Shaw, 1969).  The present r e s u l t i s i n  agreement with the above f i n d i n g . Presumably, the decrease i n negative charge on peps i n was not only a r e s u l t of the blocking of carboxyl groups by nucleophile but also of the f i x a t i o n of p o s i t i v e l y charged carbodiimide residues as N-acylurea on other carboxyl groups of the pepsin molecule.  Matyash et a l . (1973) observed  that pepsin treated with radioactive CMC alone contained 2-3 residues of l a b e l l e d carbodiimide.  In the present study,  pepsin treated with 10 mM EDC i n the absence of nucleophile had a R a R  f  f  value of O.89 (native pepsin was assumed to have  of 1.0), showing that the contribution of carbodiimide  to the decrease i n net negative charge on pepsin was s i g n i ficant .  63a  0  FIGURE  5  10  NO. OF COOH GROUPS 12.  EFFECT  OF C A R B O X Y L  MODIFIED  MODIFICATION  OF  15  P E P S I N ON  NATIVE PEPSIN WAS ASSUMED TO HAVE A R VALUE OF 1.0. RELATIVE  ELECTROPHORETIC  F  MOBILITY,  64 I s o e l e c t r i c Point of Native and Carboxyl Modified Pepsins The i s o e l e c t r i c point of native pepsin, determined by electrophoresis of the protein on agarose g e l at d i f f e r ent pH's, boxyl  was  found to be below 0 . 5 .  car-  modification ( 1 1 . 2 carboxyl groups modified/mole  pepsin), the i s o e l e c t r i c point was 0.7  A f t e r extensive  (Fig.  found to r i s e to about  13).  The present r e s u l t i s i n accordance with that of other workers who  observed that at pH 1.0, highly p u r i f i e d  pepsin  s t i l l migrated as an anion, i n d i c a t i n g that the i s o e l e c t r i c point of porcine pepsin i s below 1.0 ( T i s e l i u s et a l . , 1938j  H e r r i o t t et a l . ,  1940).  The i s o e l e c t r i c point of proteins determined by zone electrophoresis may be subject.  to errors such as absorp-  t i o n , c a p i l l a r y flow and electro-osmosis.  More superior  techniques would be moving boundary electrophoresis isoelectrofocusing.  and  However, while moving boundary e l e c t r o -  phoresis requires expensive instruments and  complicated  experimental procedures, the pH range of commercially a v a i l able ampholytes for isoelectrofocusing i s between 3 « 5 and 1 0 . 0 , well above the i s o e l e c t r i c point of pepsin. has been shown to exhibit l i t t l e electro-osmosis  Agarose and  ad-  sorption, and electrophoretic m o b i l i t i e s determined by agarose gel electrophoreis are found to be s i m i l a r to those determined by moving boundary technique (Shaw,  1969).  65  FIGURE 13,  DETERMINATION OF ISOELECTRIC POINTS OF NATIVE AND CARBOXYL MODIFIED PEPSINS.  ®, NATIVE PEPSIN; O, CARBOXYL MODIFIED PEPSIN (11,2CARBOXYL GROUPS MODIFIED/MOLE ENZYME)  66  Thus, agarose g e l electrophoresis was employed to provide information on changes i n the i s o e l e c t r i c point of pepsin a f t e r chemical modification. The  increase i n i s o e l e c t r i c point i n the carboxyl  modified pepsin further indicates that there was a decrease i n the net negative  charge i n the enzyme as a r e s u l t of  modification.  Carboxyl Modification of Pepsin Bv  other Amino A c i d  Methyl Esters Methyl esters of amino acids other than glycine were used as nucleophiles to modify pepsin.  The extent of car-  boxyl modification, the a c t i v i t i e s and the r e l a t i v e e l e c t r o phoretic mobility of the modified enzymes were measured and the r e s u l t s are presented i n Table 5» A l l nucleophiles were found to incorporate into pepsin, but the extent of modification was v a r i a b l e . The milk c l o t t i n g a c t i v i t y of a l l modified enzymes was markedly reduced, p a r t i c u l a r l y those modified with tyrosine and tryptophan methyl esters.  At pH 2.0, the p r o t e o l y t i c a c t i v i t y against  hemoglobin was not changed i n pepsin modified with arginine and lysine methyl esters  while pepsins treated with leucine,  tyrosine and tryptophan methyl esters showed a drop i n a c t i vity.  The s p e c i f i c  proteolytic  activity  at pH 3 . 5 was  67  Table 5 . Carboxyl modification of pepsin amino acid methyl e s t e r s .  Methyl ester  a  No. CGOH gp. Milk c l o t t i n g modified/mole activity pepsin ($ control)  by  different  a  Proteolytic activity (% control) pH2.0 pH3.5  Arg  4.7  14  100  300  0.50  Lys  5.1  12  100  330  0.48  Leu  7.3  10  75  200  0.68  Tyr  8.0  7  50  100  0.68  Try  3.5  4  40  25  0.70  Gly  5.2  94  340  17  0.56  Pepsin (10 mg/ml) was modified with 3 3 - f o l d excess of EDC and 174-fold excess of methyl ester at pH 5.5 f o r 90 minutes. Native pepsin was assumed to have a R_ value of 1.0.  68  s i g n i f i c a n t l y increased i n most modified enzymes, i n d i c a t i n g a s h i f t i n pH p r o f i l e .  However, tyrosine methyl ester-  treated pepsin did not show a change i n p r o t e o l y t i c a c t i v i t y at pH 3.5 and the tryptophan derivative even showed a marked decrease i n a c t i v i t y at t h i s  pH.  The difference i n the response of pepsin to d i f f e r e n t types of amino acid methyl esters could be a t t r i b u t e d to solubility.  Incorporation of hydrophobic amino acids such  as tyrosine and tryptophan into pepsin would increase the hydrophobicity and decrease the s o l u b i l i t y of the enzyme, leading to an apparent drop i n a c t i v i t y . A l t e r n a t i v e l y , the discrepancy could be due to the covering of the hydrophobic binding s i t e i n pepsin by the hydrophobic methyl esters.  Apart from the c a t a l y t i c s i t e ,  several investigators advocated a hydrophobic binding s i t e i n pepsin which plays an important role i n pepsin a c t i v i t y (Tang,  1965$  Jackson et. a l . ,  1966).  The hydrophobic methyl  esters would have a greater tendency to bind to t h i s secondary substrate binding s i t e , thus rendering i t unavailable f o r further i n t e r a c t i o n with other substrates and r e s u l t i n g i n a loss of a c t i v i t i e s . The electrophoretic mobility of a l l modified enzymes was  found to decrease when compared to the native enzyme.  Arginine and lysine methyl esters, being c a t i o n i c , the electrophoretic mobility to a greater extent.  decreased  69  Caseinolytic Properties of Native and Carboxyl Modified Pepsins 1.  Rate of casein hydrolysis The rate of hydrolysis of 1 $ (w/v) whole casein by  pepsin, determined by the release of non-protein (NPN)  soluble i n 3% (w/v)  by carboxyl modification.  TCA,  nitrogen  was not s i g n i f i c a n t l y affected  As shown i n F i g . 14 and F i g . 1 5 ,  at both pH 5 » 3 and 6 . 5 » there was  an i n i t i a l rapid r i s e i n  the release of nitrogen followed by a gradual increase.  The  extent of casein hydrolysis was  6.5  than at pH 5 * 3 . hydrolysed  At pH 5 » 3 t the carboxyl modified enzyme  casein at a rate s l i g h t l y higher than that of the  control (Fig. 14). hydrolysis was  2.  considerably lower at pH  At pH 6 . 5 , however, the rate of casein  s l i g h t l y decreased a f t e r modification ( F i g . 1 5 ) .  Electrophoretic patterns of hydrolysed  casein  At time i n t e r v a l s when the release of NPN mined, casein samples were also withdrawn  was  deter-  simultaneously,  s o l u b i l i z e d i n 6 M urea and subjected to agarose g e l e l e c t r o phoresis.  The r e s u l t i n g patterns were e s s e n t i a l l y s i m i l a r  (Plate I I ) .  They a l l showed an i n i t i a l conversion of Jt-  casein to para-x-casein.  This was  followed by some break-  down of ^-casein, the rate being f a s t e r with the pepsin.  There was no perceptible breakdown of  modified  e>c -casein. ol  70  60  120  180  Time, min.  FIGURE 14.  R A T E OF MODIFIED  CASEIN  HYDROLYSIS  P E P S I N S AT  PH  BY  5.3.  •, NATIVE PEPSIN; o, CARBOXYL MODIFIED PEPSIN. « NPN>  NON-PROTEIN NITROGEN  N A T I V E AND  CARBOXYL  71  30r  o  !20  180  Time, min.  FIGURE 1 5 .  RATE OF C A S E I N  H Y D R O L Y S I S BY N A T I V E AND  B O X Y L M O D I F I E D P E P S I N S A T PH  6.5.  •, NATIVE PEPSIN; o, CARBOXYL MODIFIED PEPSIN. NPN, NON-PROTEIN NITROGEN.  CAR-  71a  PLATE  II.  AGAROSE G E L ELECTROPHORETIC OF WHOLE C A S E I N AND  SAMPLE:  1, 1%  PATTERNS  HYDROLYSED BY  CARBOXYL MODIFIED  NATIVE  PEPSINS.  (W/V) WHOLE C A S E I N ;  2,  CASEIN  HYDROLYSED BY NATIVE P E P S I N (30  MIN.);  3, CASEIN HYDROLYSED BY NATIVE P E P S I N (180 M I N . ) ;  CASEIN HYDROLYSED BY  CARBOXYL MODIFIED P E P S I N (30  MIN.);  5, CASEIN HYDROLYSED BY CARBOXYL MODIFIED P E P S I N (180  MIN.)  72  73 3.  Electrophoretic patterns of milk curds Reconstituted  skimmilk was  c l o t t e d with both native  and carboxyl modified pepsins at pH 6 . 3 .  At time i n t e r v a l s  a f t e r c l o t t i n g , a sample of curd was withdrawn, s o l u b i l i z e d i n 6 M urea and applied to agarose plate f o r electrophoresis. The electrophoretic patterns, shown i n Plate I I I , appeared to be i d e n t i c a l f o r both native and modified enzymes. both instances, there was  In  a decline i n the amount of x-casein  together with the appearance of a para-,£-casein band.  This  indicates that the nature of the c l o t t i n g reaction was  not  changed by the modification.  The above r e s u l t s indicate that the c a s e i n o l y t i c properties of pepsin were not s i g n i f i c a n t l y affected by carboxyl modification. v i t y was  The decrease i n milk c l o t t i n g a c t i -  therefore not due to the blocking of carboxyl  group(s) e s s e n t i a l f o r the hydrolysis of caseins i n milk, since the rate of release of nitrogen from caseins was  not  decreased a f t e r modification. In the present investigation, chymosin was  found to  have c a s e i n o l y t i c properties very s i m i l a r to those of pepsin, since both the rate of casein hydrolysis and the e l e c t r o phoretic patterns of hydrolysed. caseins and curdled milk were s i m i l a r .  The r e s u l t i s consistent with the reports of  other workers who  compared the proteolysis of whole casein  PLATE  III.  AGAROSE G E L ELECTROPHORETIC  PATTERNS  OF MILK  PRODUCED BY N A T I V E AND C A R B O X Y L M O D I F I E D  CURDS  PEPSINS.  SAMPLES  1,2,3,&  4:  DISSOLVED CURDS FORMED WITH NATIVE PEPSIN;  SAMPLES  5,6,7,&  8:  DISSOLVED CURDS FORMED WITH MODIFIED PEPSIN.  TIME AFTER CLOTTING: 1 & 5 , 0 HR.; 3  & 7,  2  HR.;  2 & 6 , 1 / 2 HR.; 4  & 8,  24  HR.  para-x-casein  75  and i n d i v i d u a l casein fractions by porcine pepsin and c a l f rennet under different conditions.  In most instances, pepsin  was found to have approximately the same a c t i v i t y as rennet (Mickelsen and Fish, 1 9 7 0 ; Hansen, 1 9 7 0 ; Green,  1971;  Itoh and Thomasow,  1972).  Coagulation of X--Casein and gc^-Casein by Native and Carboxyl Modified Pepsins Since X-casein has been known to be the protein component responsible f o r s t a b i l i z a t i o n of the caseinate mic e l l e s (Waugh and von Hipp e l , and Walter, the  1963),  diminished  1956;  Zittle, .1961;  Zittle  an experiment was designed to test whether  c l o t t i n g a c t i v i t y of the carboxyl modified  pepsin was due to a decreasejin i t s a b i l i t y to coagulate Xcasein. The r e s u l t shows that 0.2$ (w/v) x-casein at pH 5-3 and an i o n i c strength of 0.05 was coagulated by both native and modified pepsins.  Omission of calcium i n the reaction  solution d i d not a f f e c t the r e s u l t s .  The s p e c i f i c a c t i v i t y  was markedly increased a f t e r modification (Table 6 ) . A 1:1 (w/w) mixture of X-casein and <* -casein was gl  also used as substrate since  -casein i s the ealciumsl  sensitive  component which i s normally protected by X-casein  against coagulation ( Z i t t l e ,  1961).  In this case, carboxyl  76  Table 6.  Effect  of carboxyl modification on c l o t t i n g  a c t i v i t y of pepsin to Jc-casein and * - * ] _ g  casein mixture.  Enzyme  Pepsin COOH-modif i e d pepsin  Pepsin  0.2$ pH  K-casein  K  1.2  5.3  0.2$ /c-casein pH 5 . 3  x-« -^-casein s  ( 1 : 1 ) , pH  COOH-modif i e d pepsin  Specific activity  Substrate  14.3  11.9  1.5  0.5  5.3  /c-<* ^ c a s e i n s  ( 1 : 1 ) , pH  3.0  5.3  The casein c l o t t i n g a c t i v i t y i s defined as described under Method.  Specific  a c t i v i t y i s expressed as c l o t t i n g  a c t i v i t y per mg protein.  77 modification greatly reduced the a b i l i t y of pepsin to coagulate the casein mixture (Table 6 ) . The result also shows that native pepsin had a much higher s p e c i f i c a c t i v i t y against the casein mixture than against X-casein, with an a c t i v i t y r a t i o 12.  (K+<*/K)  of about  In contrast, the carboxyl modified pepsin had an  a c t i v i t y r a t i o of only 0.5, i n d i c a t i n g that the enzyme had greater s p e c i f i c i t y towards X-casein than  X-cx^^-casein  mixture (Table 6 ) . When the s p e c i f i c a c t i v i t i e s against casein f r a c t i o n s were compared to those against skimmilk and hemoglobin, i t can be seen that the r i s e i n s p e c i f i c a c t i v i t y against  K-  casein coincided with an increase i n proteolytic a c t i v i t y at pH 3'5t with the exception of the most extensively modif i e d enzyme which showed a drop of 70% i n x-casein c l o t t i n g activity.  The s p e c i f i c a c t i v i t y against X-«: ^-casein mixg  ture was found to decrease with a trend very similar to the drop i n milk c l o t t i n g a c t i v i t y (Table 7). The r e s u l t s obtained from the above experiments suggest that the increase i n s p e c i f i c a c t i v i t y against X-casein was  probably attributed to a s h i f t i n pH optimum of the  enzyme s i m i l a r to that observed when hemoglobin was used as substrate. f a r from the  Since the a c t i v i t y was measured at a pH ( 5 . 3 ) not optimum pH ( 3 * 5 ) of the modified enzyme f o r  proteolysis, the modified pepsin would hydrolyse x-casein  Table 7.  Comparisionof a c t i v i t i e s of carboxyl modified pepsin to different substrates.  Enzyme  Pepsin COOH modif i e d pepsin t» n n  a c t i v i t y ($ control) hemoglobin skimmilk K-PC " -casein pH 6.3 pH2.0 pH3.5 |5H 5 . 3 Specific  No. of COOH groups modified  x-casein pH 5 . 3  0  100  100  100  100  100  2.8  320  20  27  88  210  5.2  250  12  17  94  340  8.6  200  10  13  11.2  30  5  9  S  105  90  330 340  CO  79  at a rate faster than the native enzyme which has low prot e o l y t i c a c t i v i t y at higher pH s (see F i g . 9). The coaf  gulation of i s o l a t e d x-casein by an enzyme has been demonstrated to be due to the aggregation  of insoluble p a r a - x -  casein s p l i t o f f from x>casein (Cheeseman, 1962) and i s therefore e s s e n t i a l l y p r o t e o l y t i c i n nature.  An increase  i n p r o t e o l y t i c a c t i v i t y against X-casein would therefore increase the rate of x-casein coagulation. The dramatic drop i n X-casein c l o t t i n g a c t i v i t y .when pepsin was extensively modified  (11.2 moles of glycine  methyl ester incorporated/mole pepsin) .suggests that a carboxyl group(s) e s s e n t i a l f o r x-casein hydrolysis may be blocked.  This group(s), however, was not e s s e n t i a l f o r  hydrolysing hemoglobin since the s p e c i f i c a c t i v i t y of t h i s derivative against hemoglobin s t i l l remained high when compared to the native enzyme. The high a c t i v i t y r a t i o (K+et/x) observed f o r "the native pepsin may suggest that i n t e r a c t i o n between X-casein and <?i i -casein i n the micelles could lead to changes that sl enhance the a f f i n i t y of the native enzyme f o r the substrate. A c t i v a t i o n of X-casein hydrolysis by <*-casein and ,g-casein was reported i n chymosin (Kanamori et a l . , 1977) and was attributed to the association of x-casein with other proteins .  80  The extensive loss i n pepsin a c t i v i t y against  K-  casein/c<: -casein mixture a f t e r carboxyl modification gl  suggests that i n t e r a c t i o n between the two caseins on the micelles may retard the binding between the micelles and the modified enzyme.  Changes i n the charge d i s t r i b u t i o n  on the modified enzyme may  i n t e r f e r e with substrate-enzyme  i n t e r a c t i o n through e l e c t r o s t a t i c or ionic repulsion. This would a f f e c t the primary (enzymatic) phase of milk coagulation and slow down the secondary phase of m i c e l l a r aggregation.  The observed drop i n milk c l o t t i n g a c t i v i t y  i n the carboxyl modified pepsin could therefore be  attri-  buted to a change i n the i o n i c c h a r a c t e r i s t i c of the enzyme.  S p e c i f i c i t y of Carboxyl Modification To investigate whether the carboxyl modification by the carbodiimide reaction i s s p e c i f i c f o r pepsin, pepsinogen and chymosin were modified under s i m i l a r conditions ( 3 3 - f o l d excess of EDC,  7 l 6 - f o l d excess of glycine methyl  ester, pH 5 . 5 , 90 minutes incubation). The r e s u l t , presented i n Table 8, indicates that the carbodiimide reaction was not s p e c i f i c f o r pepsin. Both pepsinogen and chymosin were modified to about the same extent as pepsin with a s i g n i f i c a n t decrease i n milk clotting activity.  The decrease i n proteolytic a c t i v i t y  Table 8.  E f f e c t of carboxyl  modification on a c t i v i t i e s  of pepsin, pepsinogen and chymosin.  Enzyme  No. of COOH ^ m  o  d  i  f  i  e  d  M  S p e c i f i c a c t i v i t y (% control) i clotting Proteolytic pH 6.3 pH2.0 pH3.5 l  k  Pepsin  11.2  9  90  340  Pepsinogen  12.1  30  70  450  Chymosin  13.0  15  50  50  The enzymes (10 mg/ml) were modified with 3 3 - f o l d excess of EDC and 706-fold excess of glycine methyl ester at pH 5.5 f o r 90 minutes.  82  of the modified zymogen and chymosin at pH 2.0 was not as extensive as the drop i n milk c l o t t i n g a c t i v i t y .  Like  the modified pepsin, the p r o t e o l y t i c a c t i v i t y of the modif i e d pepsinogen at pH 3*5 was  s i g n i f i c a n t l y increased,  suggesting a s h i f t i n pH p r o f i l e .  In contrast, modified  chymosin only had 5 0 $ proteolytic a c t i v i t y at pH 3.5 when compared to the native enzyme. t e o l y t i c pH optimum near 3»5  Native chymosin has a pro-  (Berridge,  19^5),  a shift in  pH optimum, i f occurred i n the modified chymosin, would be detected at pH lower or higher than  3«5«  Both carboxyl modified pepsinogen and chymosin exh i b i t e d a marked retardation i n electrophoretic mobility i n agarose gels with R  f  values of 0.70 and 0.63 respectively  (compared to the native proteins which were assumed to have a R^, value of 1 . 0 ) .  This indicates a decrease i n net nega-  t i v e charge on the modified protein molecules. The results show that the carbodiimide reaction was not s p e c i f i c f o r pepsin.  Both the pepsin precursor and  other acid proteases such as chymosin, when modified, showed s i m i l a r responses.  In contrast to the present f i n d i n g ,  modification with CMC  and a colored amine was found to be  s p e c i f i c to pepsin (Matyash et a l . ,  1973).  No amine was  incorporated into pepsinogen and an acid protease from Aspergillus awamori.  The zymogen and fungal protease  retained 1 0 0 $ p r o t e o l y t i c a c t i v i t y .  This suggests that  83  CMC and the colored amine are only e f f e c t i v e i n modifying the carboxyl groups i n pepsin.  On the other hand, EDC  and glycine methyl ester are able to modify the carboxyl groups i n pepsin and related enzymes, showing that they are less s e l e c t i v e than the CMC-colored amine system.  E f f e c t of Carboxyl Modification on 1 ^ and i r  c a t <  of Pepsin  For k i n e t i c studies, the synthetic dipeptide APDT was used as substrate.  The Michaelis constant ( 1 ^ ) and  molecular a c t i v i t y c o e f f i c i e n t ( k ^ ) were determined at c a  pH  2.0 and 4 . 5 .  APDT was chosen because i t i s among the  most sensitive substrates f o r pepsin (Fruton, 1 9 7 0 ) and the k i n e t i c parameters of pepsin on t h i s substrate have been determined  (Jackson et a l . ,  1965).  F i g . 1 6 and F i g . 1 7 depict the Lineweaver-Burk plots f o r native and carboxyl modified pepsins at pH 4.5 r e s p e c t i v e l y .  The K  M  values determined from the i n t e r -  cepts are presented i n Table 9. from the Miehaelis-Menten  2.0 and  The k ^. values, calculated ca  equation, are also shown i n  Table 9. The  KJJJ  and k.  : cat  values determined f o r native pepsin  were found to compare favorably with the published data (Jackson et a l . ,  1965).  The r e s u l t shows that a f t e r car-  boxyl modification, 1 ^ was markedly increased at both pH  -15 S]~ X 1 0 FIGURE 17.  LINEWEAVER-BURK PLOTS DIIODO-L-TYROSINE AT  PH  4,5.  BY  OF T H E . H Y D R O L Y S I S NATIVE  (*)  AND  OF  3  , fVI  1  N-ACETYL-L-PHENYLALANYL-  CARBOXYL MODIFIED  (A)  PEPSINS  CO  86  Table 9«  Kinetics of the hydrolysis of N-acetyl-L-,phenylalanyl-L-diiodotyrosine by native and carboxyl modified pepsins.  Enzyme  Concn.  pH  m M X 10  cat min." K  6  Pepsin  0.51  2.0  0.?8  COOH modif i e d pepsin  0.76  2.0  1.33  Pepsin  0.76  4.5  12.0  4.1  1-52  4.5  37.0  4.2  ™ « 7 „  f i e d pepsin  12.0  13.8  1  87  2.0  and 4 . 5 , while k  c a t  The increase i n K  was not markedly a l t e r e d . m  suggests that carboxyl modifi-  cation of pepsin decreased the a f f i n i t y of the enzyme f o r the dipeptide substrate, i . e . , the enzyme-substrate binding step was hindered.  On the other hand, the c a t a l y s i s of  the enzyme-substrate complex was not affected, as the  k ^. c&  values were not changed markedly after carboxyl modification. The p a r t i a l loss i n peptidase a c t i v i t y i n the modified pepsin could be a t t r i b u t e d to a blocking of the active s i t e on the enzyme. A change i n k i n e t i c parameters was also reported i n arginine modified pepsin which has 45$ of the p r o t e o l y t i c a c t i v i t y of the native pepsin (Kitson and Knowles, 1 9 7 1 ) . In t h i s case, however, K  m  was not changed while k ^. ca  was  s i g n i f i c a n t l y lowered, i n d i c a t i n g that the i n a c t i v a t i o n took place not by the blocking of the enzyme*s active s i t e , but by an effect on the c a t a l y t i c process subsequent to enzyme-substrate binding.  88  E f f e c t of Synthetic Dipeptides on Carboxyl Modification Carboxyl modification of pepsin was carried out i n the presence of some synthetic dipeptides.  These include  a s e n s i t i v e substrate (APDT), a poor substrate (N-carbobenzoxy-L-glutamyl-L-tyrosine,  Z-glu-tyr) and a non-sub-  strate (N-acetyl-D-phenylalanyl-L-tyrosine, APT).  Modi-  f i c a t i o n s were carried out under the same conditions ( 3 3 f o l d excess EDC, 174-fold excess glycine methyl ester, pH 5 « 5 and 9 0 minutes).  The extent of modification, milk  c l o t t i n g a c t i v i t y and electrophoretic mobility of pepsins a f t e r treatments were determined (Table 10).  Result shows that i n the presence of APDT, a sens i t i v e pepsin substrate, the extent of carboxyl modification was s i g n i f i c a n t l y decreased when compared to pepsin modif i e d i n the absence of substrate.  The milk c l o t t i n g a c t i -  v i t y and electrophoretic mobility, markedly decreased by carboxyl modification, were only moderately lowered when modification took place i n the presence of APDT. In the presence of a poor substrate and a non-subs t r a t e , pepsin was also modified, but to a l e s s e r extent than the f u l l y modified c o n t r o l .  The milk c l o t t i n g a c t i -  v i t y and R^ value were increased r e l a t i v e to the f u l l y modified pepsin.  However, the changes were not as marked  as those that occurred i n the presence of APDT.  89  Table 1 0 .  E f f e c t of dipeptides on carboxyl modification of pepsin.  Addition  3.  EDC + gly methyl ester  a  a  100  1.00  5.2  17  0.56  2.3  50  0.90  3.6  35  0.77  4.8  22  0.65  10 50  0.3  2  +  (2)  APT  0  +  (2)  Z-glu-tyr ^  5.  Milk c l o t t i n g activity (% control)  +  (2)  APDT 4.  No. of COOH gp. modified  -  1. None 2.  Concn. mM  C  2  N-acetyl-L-phenylalanyl-diiodo-L-tyrosine. N-carbobenzoxy-L-glutamyl-L-tyrosine.  c  N-acetyl-D-phenylalanyl-L-tyrosine.  90 The present r e s u l t indicates that the effect of carboxyl modification on pepsin properties were less prominent i n the presence of a dipeptide substrate.  This  could be due to one of the following: . F i r s t l y , the b i n ding of the dipeptide substrate to the enzyme may protect the carboxyl groups, mainly those i n the v i c i n i t y of the pepsin active centre from modification by carbodiimide and nucleophile.  Secondly, substrate-enzyme binding may  lead to conformational changes i n the pepsin molecule which i n d i r e c t l y a f f e c t subsequent modification.  F i n a l l y , the  substrate may interact with carbodiimide and nucleophile and less reagents would be available f o r modifying The r e s u l t shows that even non-substrate  pepsin.  can hinder  carboxyl modification, though to a lesser extent than the two dipeptide substrates.  This suggests that dipeptides  can react with the reagents.  APDT caused more pronounced  changes than Z-glu-tyr probably because i t i s a better substrate and can bind to pepsin more e f f i c i e n t l y .  Hence,  substrate-enzyme binding would be the major factor i n causing such moderation i n carboxyl modification.  91  Response of Native and Carboxyl Modified Pepsins to Inhibitors The reactions of the native and carboxyl modified pepsins with i n h i b i t o r s were studied. l,2-epoxy-3-(p-nitrophenoxy) a c y l bromide were used.  Two pepsin i n h i b i t o r s ,  propane (EPNP) and bromophen-  EPNP i s a substrate-like epoxide  i n a c t i v a t o r modifying Asp-76 i n pepsin with almost complete loss of p r o t e o l y t i c  a c t i v i t y (Chen and Tang,  1972),  while  bromophenacyl bromide reacts with a carboxyl group i n pepsin not d i r e c t l y involved i n c a t a l y s i s (Erlanger et a l . , Clement,  1965*  1973).  F i g . 18 shows the i n a c t i v a t i o n of native and carboxyl modified pepsins by EPNP.  Native pepsin was inactivated  more r a p i d l y and the treated enzyme retained only 1 0 $ of p r o t e o l y t i c a c t i v i t y a f t e r 1 2 0 hours of incubation.  The  modified enzyme reacted less promptly with EPNP, and retained 3 0 $ of i t s a c t i v i t y at the end of incubation. Table 1 1 shows the reaction of pepsin bromophenacyl bromide.  with EPNP and  Bromophenacyl bromide produced a loss  of 7 0 $ p r o t e o l y t i c a c t i v i t y i n the native pepsin, but only 3 5 $ i n the modified enzyme. The r e s u l t indicates that carboxyl modified pepsin was s t i l l reactive to s i t e - s p e c i f i c i n a c t i v a t o r s , confirming that the carboxyl groups blocked were not the a c t i v e - s i t e residues of pepsin.  However, the modified pepsin was less reactive  to the i n a c t i v a t o r s than the native enzyme.  This suggests  92  24  48  °72  Time,  FIGURE  18.  INACTIVATION MODIFIED  96  hour  OF N A T I V E AND C A R B O X Y L  P E P S INSBY  EPNP.  • , NATIVE P E P S I N j o , CARBOXYL MODIFIED  PEPSIN.  ™~120  Table 1 1 . Response of native and carboxyl modified pepsins to i n h i b i t o r s .  Enzyme  Proteolytic activity ($ remaining)  Inhibitor  Concn.  Pepsin  EPNP  1 mg/mg protein  10  COOH modif i e d pepsin  EPNP  1 mg/mg protein  27  Pepsin  BPB  200  joM  30  COOH modif i e d pepsin  BPB  200  JJM  65  a  a  b  1,2-epoxy-3-(p-nitrophenoxy) bromophenacyl bromide.  propane.  94  that the carboxyl groups reactive to the i n h i b i t o r s were protected i n the modified enzyme, probably as a r e s u l t of conformational change or s t e r i c hindrance.  S t a b i l i t y of Native and Carboxyl Modified Pepsins Near Neutral pH The s t a b i l i t y of pepsin near neutral pH was studied. In the f i r s t series of experiments, pepsin was incubated i n d i l u t e b u f f e r (0.05 M phosphate buffer) at pH 6.5 30  C.  and  Both milk c l o t t i n g and p r o t e o l y t i c a c t i v i t i e s of  pepsin were determined at selected time i n t e r v a l s .  The  percentage changes i n milk c l o t t i n g and proteolytic a c t i v i t i e s were found to be s i m i l a r . milk c l o t t i n g a c t i v i t y was Fig.  Subsequently,  only  determined.  19 shows the s t a b i l i t y curves of native and  carboxyl modified c r y s t a l l i n e pepsins i n buffer.  The milk  c l o t t i n g a c t i v i t y of native pepsin dropped rapidly, following a f i r s t order k i n e t i c with a h a l f - l i f e ( t - ^ ) v a t i o n of about 15 minutes.  f o r  inacti-  Less than 10% a c t i v i t y  was  retained a f t e r one hour of incubation. With mild modification (incorporation of 2.8 moles of nucleophile /mole pepsin), the s t a b i l i t y was markedly improved, and the enzyme retained 20% of milk c l o t t i n g a c t i v i t y after 4 hours. When pepsin was more extensively modified (incorporation of 5.2 moles of nucleophile), the s t a b i l i t y was improved further.  95  120 TIME,  FIGURE  19.  STABILITY  MSN,  OF C R Y S T A L L I N E  MODIFIED  P E P S I N S IN  PH 6.5.  A,  0.05  N A T I V E AND C A R B O X Y L M PHOSPHATE  NATIVE P E P S I N ;  BUFFER,  A, P E P S I N MODIFIED  WITH 8 7 - F O L D EXCESS OF NUCLEOPHILE; • ,  P E P S I N MODIFIED WITH 1 7 4 - F O L D EXCESS OF  NUCLEOPHILE.  96  The curve started to l e v e l o f f a f t e r 2 hours and the loss i n a c t i v i t y was only about 5 0 $ a f t e r k hours of incubation. The s t a b i l i t y of crude pepsin i n d i l u t e buffer was also studied.  As shown i n F i g . 2 0 , the crude pepsin pre-  paration had a low s t a b i l i t y at pH 6.5.  The time-dependent  decrease i n milk c l o t t i n g a c t i v i t y also followed a f i r s t order k i n e t i c .  The h a l f - l i f e of i n a c t i v a t i o n was calcu-  l a t e d to be 1 0 minutes.  The s t a b i l i t y was again found to  increase markedly a f t e r carboxyl modification. The degree of improvement was also dependent on the extent of modification.  The less extensively modified enzyme retained 2 5 $  a c t i v i t y a f t e r 2 hours incubation while the more extensively modified pepsin retained about 5 Q $ a c t i v i t y . In the second series of experiments, pepsins were incubated i n milk u l t r a f i l t r a t e at 3 0 ° C with progressive adjustment  of pH according to that found i n cheese-making.  The r e s u l t f o r c r y s t a l l i n e pepsins i s presented i n F i g . 2 1 and that f o r crude pepsins i s shown i n F i g . 2 2 .  In both  cases, the s t a b i l i t y of the native enzyme was much higher i n milk u l t r a f i l t r a t e than i n d i l u t e buffer.  The milk  c l o t t i n g a c t i v i t y decreased r a p i d l y i n the f i r s t hour but s t a r t e d to l e v e l o f f a f t e r 2 hours.  For both c r y s t a l l i n e  and crude pepsins, about 2 0 $ a c t i v i t y was found to remain a f t e r incubation.  97  Time, FIGURE  20.  STABILITY  OF  PEPSINS IN 0 . 0 5  min.  CRUDE N A T I V E AND M  CARBOXYL  PHOSPHATE BUFFER,  PH  MODIFIED 6.5.  A , NATIVE PEPSIN; A , PEPSIN MODIFIED WITH 87FOLD EXCESS OF NUCLEOPHILE; • , PEPSIN MODIFIED WITH 174-FOLD EXCESS OF NUCLEOPHILE.  98  O  SR  °'  60  I20 Time,  FIGURE  21.  STABILITY MODIFIED SIMULATED  J80  240  min.  OF C R Y S T A L L I N E N A T I V E AND  CARBOXYL  PEPSINS IN MILK ULTRAFILTRATE CHEESE-MAKING  CONDITIONS.  9 , NATIVE PEPSIN; o, CARBOXYL MODIFIED PEPSIN,•'  UNDER  99  FIGURE  22.  STABILITY MODIFIED  OF  CRUDE N A T I V E AND C A R B O X Y L  P E P S I N S IN M I L K U L T R A F I L T R A T E  SIMULATED CHEESE-MAKING  CONDITIONS.  • , NATIVE P E P S I N ; o,  CARBOXYL MODIFIED P E P S I N .  UNDER  100  A f t e r carboxyl modification, the s t a b i l i t y of c r y s t a l l i n e or crude pepsin was markedly improved.  both The  rate of decline i n a c t i v i t y was more gradual when compared to the c o n t r o l . was  For both enzymes, the decrease i n a c t i v i t y  about 40$.  The results show that the s t a b i l i t y of pepsin near n e u t r a l pH was much improved by carboxyl modification. Pepsin was shown to be unstable at pH 6.0  (Herriott,  and was r a p i d l y denatured at pH 7.0 at 30°C Fox, 1974).  1955)  (O'Leary and  I t was suggested that an e l e c t r o s t a t i c expan-  sion of the negatively charged polypeptide chains may  lead  to denaturation of pepsin at pH values higher than 6  (Low-  enstein, 1974).  The decrease i n net negative charge on  the carboxyl modified pepsin molecule may  lower the extent  of e l e c t r o s t a t i c expansion and subsequent denaturation. An improvement of enzyme s t a b i l i t y at pH 6.3-6.5 was  also reported i n porcine pepsin covalently bound to  a soluble polyanionic c a r r i e r , ethylene maleic anhydride (Lowenstein, 1974).  By incubating the enzyme i n 0.05 M  phosphate buffer of jM* s 6.3 and 6.5 at 30°C, i t was observed that the s t a b i l i t y against the i r r e v e r s i b l e denaturation was  3-5 times better a f t e r modification.  The  enhanced s t a b i l i t y was attributed to d i f f e r e n t microenvironmental states of the enzymes.  101  The higher s t a b i l i t y of native pepsin i n milk u l t r a f i l t r a t e than i n d i l u t e buffer was probably due to the protection of the enzyme by other milk solids against denaturation.  The estimation of the amount of i n a c t i -  vation of pepsin i n milk u l t r a f i l t r a t e under simulated cheese-making conditions would therefore give a more v a l i d assessment of the s u i t a b i l i t y of the modified enzyme as a milk coagulant f o r cheese manufacture.  However, there were  differences between the simulated test and conditions that actually  exist  during cheese-making.  F i r s t l y , acid was added at i n t e r v a l s i n the simul a t i o n while i n the actual process, acid was produced continuously, although t h i s probably has l i t t l e influence on the r e s u l t . at 30°C  Secondly, the temperature was kept constant  i n the simulation but i s ; raised f o r scalding i n  cheese-making, although the r i s e i n temperature would be expected to reduce the amount of active enzyme to a greater extent.  T h i r d l y , the environment of the enzyme i n the  simulation was d i f f e r e n t from that i n cheese-making.  The  concentrations of enzyme were lower and other proteins were present i n higher concentrations i n the curd, which might increase the s t a b i l i t y of the enzyme.  F i n a l l y , the results  may not represent the amount of active enzyme retained i n the curd, since the d i s t r i b u t i o n of enzyme between whey and curd was not known.  102  However, i n spite of these provisions, the present r e s u l t s indicate that native pepsin was extensively i n a c t i vated under cheese-making, conditions while the carboxyl modified pepsin was a much more stable enzyme.  Consistent  with the present f i n d i n g , Green ( 1 9 7 2 ) reported that crude porcine pepsin ( 1 0 . 0 0 0 ) was almost completely denatured i n milk dialysate under simulated cheese-making conditions while rennet was found to be f a i r l y stable, r e t a i n i n g about 6 0 $ of p r o t e o l y t i c a c t i v i t y a f t e r treatment. report, Holmes and Ernstrom  (1973)  In another  were unable to recover  any active enzyme from Cheddar curd made with p i g pepsin whereas 5f» of the added c a l f rennet was recovered. I t has been observed that cheese made with porcine pepsin has a harder body, developed f l a v o r slowly and r e quired an aging period longer than was necessary with c a l f rennet (Maragoudakis et §X., 1 9 6 1 $ 1964$  Emmons et a l . , 1 9 7 1 $  Melachouris and Tuckey,  Green, 1 9 7 2 ) .  I t was suggested  that i n cheese made with c a l f rennet, the active enzyme retained i n the curd would a i d the s t a r t e r enzymes i n the ripening of cheese, while i n pepsin cheese, the p r o t e o l y t i c breakdown e s s e n t i a l f o r ripening would be almost e n t i r e l y dependent on s t a r t e r a c t i v i t y (Green, 1 9 7 2 $ 1972).  Lawrence ,et a l . .  Hence, the long aging period required i n pepsin  cheese has been attributed to a slow rate of proteolysis as a r e s u l t of extensive i n a c t i v a t i o n of the enzyme during  103  cheese-making (Thomasow,  1971;  Green and Foster,  1974).  The carboxyl modified pepsin might be a better milk coagulant than the native enzyme i n cheese manufacture. Since the s t a b i l i t y at pH above 6 was s i g n i f i c a n t l y improved, a greater amount of active enzyme would be retained i n the curd to aid i n the ripening of cheese,  with a  shortening i n the aging period and a reduction i n proA more v a l i d and complete comparision be-  duction cost.  tween native and carboxyl modified pepsins i n t h e i r performance i n cheese manufacture would await further i n v e s t i gations with small scale cheese-making t r i a l s .  Thermal S t a b i l i t y of Native and Carboxyl Modified Pepsins The s t a b i l i t y of pepsin at elevated temperatures was  studied.  Both milk c l o t t i n g and proteolytic a c t i v i t i e s  were measured. F i g . 23 and F i g . 24 show the thermal p r o f i l e s (milk c l o t t i n g ) of c r y s t a l l i n e and crude pepsins respectively. For c r y s t a l l i n e pepsin, both native and modified enzymes showed a maximum milk c l o t t i n g a c t i v i t y at about 40° C, and a rapid drop from 4 5 to 5 0 ° C.  The percentage maximum  a c t i v i t y was higher i n the native pepsin than i n the modif i e d enzyme at a l l temperatures  except at  50°C  (Fig. 23).  Temperature, °C  FIGURE  23.  THERMAL P R O F I L E S CRYSTALLINE  (MILK  CLOTTING)  N A T I V E AND C A R B O X Y L  PEPSINS.  • , NATIVE P E P S I N ; o , CARBOXYL MODIFIED P E P S I N .  OF MODIFIED  FIGURE  24.  THERMAL P R O F I L E S  (MILK  CLOTTING)  N A T I V E AND C A R B O X Y L M O D I F I E D  • , NATIVE P E P S I N ; o, CARBOXYL MODIFIED P E P S I N .  OF  CRUDE  PEPSINS.  106  The thermal p r o f i l e s f o r crude pepsin were s i m i l a r to those of the c r y s t a l l i n e enzymes. had a maximum a c t i v i t y at 40° C. maximum was s h i f t e d to 4 5 ° C .  The native crude pepsin After modification, the  The percentage maximum a c t i -  v i t y was considerably higher i n the native pepsin than i n the modified enzyme at temperatures between 25-40° C but lower at 4 5 - 5 0 ° C The Q  1Q  ( F i g . 24).  values (milk c l o t t i n g ) f o r both c r y s t a l l i n e  and crude pepsins are presented i n Table 12. The r e s u l t s indicate that the Q modification.  1Q  values increased a f t e r carboxyl  The increases were p a r t i c u l a r l y marked at  the higher temperature  range (40-50° C).  This shows that  modified pepsin had higher heat s t a b i l i t y than the native enzyme with regard to milk c l o t t i n g a c t i v i t y . The thermal p r o f i l e s f o r proteolysis are shown i n F i g . 2 5 and F i g . 2 6 .  For c r y s t a l l i n e pepsins, the percen-  tage maximum a c t i v i t y was higher i n carboxyl modified enzyme at lower temperatures (50-70°C),  (30-50°C).  At higher  temperatures  however, the p r o t e o l y t i c a c t i v i t y of the native  pepsin was greater than that of carboxyl modified pepsin. Both enzymes showed a rapid decrease i n a c t i v i t y from 6 0 - 7 0 ° C (Fig. 2 5 ) .  For crude pepsins, the thermal p r o f i l e s of the  native and modified enzymes were almost i d e n t i c a l .  Both  enzymes showed a gradual increase i n proteolytic a c t i v i t y with increasing temperature up to 6 0 ° C .  From 6 0 ° C  to  107  Table 12. E f f e c t of carboxyl modification of pepsin on Q  1Q  values (milk c l o t t i n g ) .  o Temperature ( C)  Enzyme  Pepsin ( c r y s t a l l i n e )  C00H modified pepsin ( c r y s t a l l i n e )  Pepsin (1:10,000)  COOH modified pepsin (1:10,000)  v 1 0  a  l  u  e  s  * 1 0  30-40  1.6  40-50  0.4  30-40 ^  1 Q  40-50  0.6  30-40  1.3  40-50  0.4  -, ,, "  3 0  n  4 0  n  40-50  * Q  Q  y  1  -. ^ ' 6  0.7  represent the r a t i o of milk c l o t t i n g a c t i v i t y  at (T+10)° to that at T ° .  108  FIGURE  25.  THERMAL P R O F I L E S  (PROTEOLYTIC)  N A T I V E AND C A R B O X Y L M O D I F I E D  •,  NATIVE P E P S I N ;  o , CARBOXYL MODIFI ED P E P S I N .  OF  CRYSTALLINE  PEPSINS.  109  a.  01 30  •  40  L_  50 60 Temperature, °C  FIGURE 26. THERMAL PROFILES (PROTEOLYTIC) OF CRUDE NATIVE AND CARBOXYL MODIFIED PEPSINS.  o,  NATIVE PEPSIN; CARBOXYL MODIFI ED PEPSIN.  110  70°C,  the a c t i v i t y decreased sharply to less than 2 0 $ of  the maximum l e v e l ( F i g . 2 6 ) . The Q^  0  Table 1 3 .  values f o r proteolysis are  presented i n  For both c r y s t a l l i n e and crude pepsins, the  native enzymes had higher Q^Q values than the modified enzymes at a l l temperature ranges. marked except at  60-70° C  when the  The difference was Q^Q  not  valuerof native  c r y s t a l l i n e pepsin was three times higher than that of the modified enzyme.  The r e s u l t shows that carboxyl modi-  f i c a t i o n caused a s l i g h t drop i n thermal s t a b i l i t y of pepsin when the a c t i v i t y was measured against hemoglobin. The rapid decline i n milk c l o t t i n g and p r o t e o l y t i c a c t i v i t i e s of pepsin at high temperatures naturation.  was due to de-  At pH 6.3 at which milk c l o t t i n g a c t i v i t y  was measured, denaturation occurred at temperatures 45° C  above  At pH 2.0 at which p r o t e o l y t i c a c t i v i t y was measured,  denaturation did not occur u n t i l the temperature exceeded . 60° C.  This indicates that pepsin was more stable against  heat at lower pH's. O'Keeffe et a l .  S i m i l a r phenomena were  (1977)  who  reported by  showed that denaturation of  pepsin was c r i t i c a l l y dependent on pH and temperature within very narrow l i m i t s . The improvement i n heat s t a b i l i t y of carboxyl modif i e d pepsin at m i l k ; c l o t t i n g  pH  may be of some p r a c t i c a l  Ill  Table 1 3 .  E f f e c t of carboxyl modification of pepsin on Q, values-(proteolytic). n  Enzyme Pepsin ( c r y s t a l l i n e )  o Temperature ( C)  Q  * 1 0  30-40  1.43  40-50  1.10  50-60  1.21  60-70  0.54  C00H modified  30-40  1.27  pepsin ( c r y s t a l l i n e )  40-50  1.07  50-60  0.90  60-70  0.17  30-40  1.37  40-50  1.04  50-60  1.11  60-70  0.16  30-40 J+0-50  1.31 1.00  50-60  1.09  60-70  0.15  Pepsin (1*10,000)  COOH modified pepsin (1*10,000)  *  o Q values represent the p r o t e o l y t i c a c t i v i t y at (T +10) to that at T ° . 1Q  112  importance.  In the making of Cheddar cheese, the curd i s  normally heated to temperatures near 40° C during cooking, while the pH remains  above 6.0.  An increase i n enzyme  s t a b i l i t y under these conditions would ensure that a higher proportion of active enzyme to be retained i n the curd and be available f o r aiding the ripening of cheese.  E f f e c t of Carboxyl Modification on Curd Tension and Rate of Syneresis The firmness of curds from milk clotted by native and carboxyl modified pepsins was measured by the method of Hehir  (1968).  The r e s u l t , shown i n Table 14 indicates  that f o r both c r y s t a l l i n e and crude pepsins, there was  no  s i g n i f i c a n t difference between the firmness of curds from native and modified enzymes.  For c r y s t a l l i n e pepsins,the  native enzyme produced a s l i g h t l y firmer curd than the modified enzyme, while the reverse was demonstrated f o r the crude pepsins.  According to the comparision of curd  tension measurements with subjective assessment of curd firmness (Hehir,  1968),  a curd tension higher than  units (eg) corresponds to a firm curd.  75  Hence, both cry-  s t a l l i n e and crude pepsins produced a firm curd and the curd tension was comparable to that of chymosin which gave a firmer curd (Table 14).  113  Table 14. Tension of curds produced by pepsin, carboxyl modified pepsin and chymosin.  C l o t t i n g time (min.)  Curd tension (cg)  Pepsin ( c r y s t a l l i n e )  3.5  125 + 5*  COOH modified pepsin ( c r y s t a l l i n e )  3.7  118+4  Pepsin  4.5  TTviQtrmA E  n  z  y  m  e  (1:10,000)  98 + 4  COOH modified pepsin ( 1 : 1 0 , 0 0 0 )  4.5  107 + 6  Chymosin  3.5  135+6  Curd tension values are presented as averages of f i v e determinations + S.E.  114  The e f f e c t of carboxyl modification of pepsin on the rate of syneresis was  studied.  sented i n F i g . 27 and F i g . 28. the rate of syneresis was fication.  The r e s u l t s are pre-  For c r y s t a l l i n e  pepsins,  not markedly affected by modi-  The native and modified enzymes produced almost  i d e n t i c a l curves ( F i g . 27).  The percentage syneresis i n -  creased r a p i d l y i n the f i r s t two hours and then l e v e l l e d off.  The modified pepsin produced syneresis at a rate  f a s t e r than the control, but the f i n a l percentage syneresis was  s l i g h t l y lower than that of the native enzyme.  was  found to y i e l d syneresis at a rate very s i m i l a r to  that of pepsin  (Fig. 27).  For crude pepsins, the percentage syneresis considerably lower than that of the c r y s t a l l i n e Syneresis did  Chymosin  was  pepsins.  . developed at a slower rate, and the curves ;  not l e v e l o f f u n t i l a f t e r three hours.  There was  no  s i g n i f i c a n t change i n the syneresis rate a f t e r carboxyl modification.  The modified enzyme produced syneresis at  a rate f a s t e r than the c o n t r o l , while the f i n a l percentage syneresis was  again s l i g h t l y lower than that of the native  enzyme ( F i g . 28). The above r e s u l t s show that i n the i n i t i a l  stages  of cheese-making, carboxyl modification of pepsin did not a f f e c t the q u a l i t y of the curd such as curd tension and rate of exudation of whey.  the  115  Time, hour FIGURE 27, SYNERESIS OF CURD BY CRYSTALLINE PEPSIN, CARBOXYL MODIFIED CRYSTALLINE.PEPSIN AND CHYMOSIN, A , NATIVE  PEPSIN;  A , CARBOXYL MODIFIED • ,CHYMOSIN.  PEPSIN;  70  T i m e , hour FIGURE 28,  SYNERESIS CARBOXYL  A,  OF CURD BY CRUDE MODIFIED  N A T I V E AND  PEPSINS.  NATIVE P E P S I N ;  A , CARBOXYL MODIFIED  PEPSIN.  117  GENERAL DISCUSSION  In the present i n v e s t i g a t i o n , selective modification of the carboxyl groups i n porcine pepsin was carried out at pH 5'5 using a water-soluble carbodiimide, EDC and an amino acid methyl ester as nucleophile.  The number of  nucleophile molecules incorporated per molecule of pepsin ranged from 2.8 to 11.2.  Profound changes i n the a c t i v i -  t i e s , s p e c i f i c i t y and physicochemical properties were observed i n the carboxyl modified pepsin. There was a s i g n i f i c a n t decrease i n milk c l o t t i n g a c t i v i t y while the p r o t e o l y t i c a c t i v i t y against hemoglobin was not affected.  The c l o t t i n g a c t i v i t y against x-casein  was increased by two to three-fold while the rate of coagulation of x-f< -casein mixture was decreased markedly gl  to 10-20$ of the c o n t r o l . There was a s h i f t i n p r o t e o l y t i c pH p r o f i l e with the pH optimum increased from 2.0 to about 3.5.  The r e l a t i v e electrophoretic mobility was  decreased  and the i s o e l e c t r i c point was s l i g h t l y increased. was a drop i n peptidase a c t i v i t y and the K while K ^ was not changed. ca  m  There  was increased  F i n a l l y , the pH s t a b i l i t y of  the enzyme was s i g n i f i c a n t l y increased. Several l i n e s of evidence suggest that the drop i n milk c l o t t i n g  a c t i v i t y of the modified pepsin was not  118  d i r e c t l y r e l a t e d to the modification of s p e c i f i c  carboxyl  group(s) involved i n the enzymatic c l o t t i n g of milk. F i r s t l y , the rate of casein hydrolysis was not affected by carboxyl modification, and both native and modified pepsin showed s i m i l a r c a s e i n o l y t i c properties. This indicates that the chemical procedure did not modify the carboxyl group(s) involved i n the enzymatic breakdown  of casein which i s the f i r s t step i n the coagulation of milk mediated by an enzyme. Secondly, pepsin treated with EDC alone showed a s l i g h t drop i n milk c l o t t i n g a c t i v i t y although the carboxyl residues were not modified.  The treated enzyme had a  s l i g h t l y lower electrophoretic mobility than the c o n t r o l , suggesting  a decrease i n net negative  molecule.  The incorporation of the p o s i t i v e l y charged  carbodiimide  charge on the enzyme  on the pepsin might lead to a change i n the  charge d i s t r i b u t i o n  of the enzyme which could a f f e c t the  milk c l o t t i n g a c t i v i t y . F i n a l l y , the carboxyl modification was not s p e c i f i c to pepsin.  I t caused s i m i l a r drop i n milk c l o t t i n g a c t i -  v i t y of pepsinogen and chymosin.  I f the decrease i n milk  c l o t t i n g a c t i v i t y of the modified pepsin was due to the blocking of s p e c i f i c carboxyl group(s) responsible f o r the enzymatic coagulation of milk, modification of other enzymes may not cause s i m i l a r changes.  This was i l l u s t r a t e d by  119  Matyash et a l . (1973) who found that modification of pepsin carboxyl groups by CMC and a colored amine resulted i n a drop i n proteolytic a c t i v i t y , while similar treatment on pepsinogen and another acid protease did not cause changes i n a c t i v i t y . In order to explain the decrease i n milk c l o t t i n g a c t i v i t y of the carboxyl modified pepsin, one has to  understand the mechanism of milk c l o t t i n g process.  Cur-  d l i n g of milk by c l o t t i n g enzymes i s a complex phenomenon. The f i r s t step or primary phase involves a highly s p e c i f i c a c t i o n of enzyme on x-casein to destroy the m i c e l l e - s t a b i l i z i n g power of the protein. is split  X-Casein, a glycoprotein,  s p e c i f i c a l l y at the Phe-Met bond.  This results  i n the release of an insoluble para-x-casein and a soluble glycomacropeptide. The sequence of events following the enzymatic action  on x-casein to d e s t a b i l i z e the micelles i s poorly  understood.  This i s p a r t l y due to an incomplete under-  standing of the structure of the caseinate m i c e l l e . e r a l models have been proposed (Waugh and Noble, G a m i e r and Ribadeau Dumas, 1970j  Sev-  1965;  Parry and C a r r e l l ,  1969;  S l a t t e r y and Evard, 1973) but no one i s absolutely s a t i s factory. However, from the available experimental data, some f a c t s about the secondary phase are now known.  Apparently,  120  coagulation requires the conversion of a minimum amount of X-casein to para-X-casein  (Green and Marshall,  1977).  The release of the highly negatively charged glycomacropeptides from micelles decreases the negative charge on the micelles (Green and C r u t c h f i e l d , 1971;  Pearce, 1976).  This results < i n the reduction of e l e c t r o s t a t i c repulsion between micelles and promotes aggregation C r u t c h f i e l d , 1971). m i c e l l a r surface,  Para-x-casein,  (Green and  present on the  increases the hydrophobicity of  the p a r t i c l e s , again promoting aggregation through hydrophobic interactions (Payens, 1966). that coagulation r e s u l t s  I t has been suggested  from s p e c i f i c i n t e r a c t i o n be-  tween micelles through enzymatically-modified areas on t h e i r surfaces (Waugh', 1971;  Knoop and Peters, 1975;  Green and Marshall, 1977). From the above observations, one may speculate that the decrease i n the rate of milk coagulation by carboxyl modified pepsin i s a t t r i b u t e d to a slow down i n the release of para-x-casein r e s u l t i n g from an interference with the enzyme-micelle i n t e r a c t i o n .  Since a defined amount of  para-x-casein i s required to i n i t i a t e coagulation (Green and Marshall, 1977)» a drop i n the rate of para-x-casein release would d i r e c t l y a f f e c t the aggregation of the micelles.  121 A l t e r n a t i v e l y , the decrease i n milk c l o t t i n g a c t i v i t y could be related to the secondary stage of coagulation, i . e . , interactions of the enzymatically modified micelles and the subsequent aggregation to form curd.  The carboxyl  modified pepsin may not be able to produce the suitable types of micelles f o r micelle-micelle i n t e r a c t i o n , which i s viewed as a highly s p e c i f i c reaction (Knoop and Peters, 1975;  Green and Marshall, 1977). Results from casein coagulation experiments indicate  that K.-casein was c l o t t e d by the modified pepsin at a rate f a s t e r than the native enzyme, while the K-o(  ^-casein  mixture was much more r e s i s t a n t to c l o t t i n g by the modified enzyme.  The c l o t t i n g a c t i v i t y against the casein mixture  was decreased  to the same extent as the drop i n milk c l o t -  t i n g a c t i v i t y a f t e r carboxyl modification (see Table 7). The data suggest that X - o C g ^ - c a s e i n i n t e r a c t i o n i n the micelles may cause a l t e r a t i o n s that increase the a f f i n i t y of native pepsin f o r i t s substrate, but hinder the binding between the micelles and the modified enzyme.  This i s  consistent with the view that the decrease i n milk c l o t t i n g a c t i v i t y i s r e l a t e d to an interference with the enzymemicelle i n t e r a c t i o n . The present results suggest that the c l o t t i n g of milk i s a process i n which charge may play an important role.  At least part of the forces involved i n m i c e l l e -  122  enzyme i n t e r a c t i o n i n the primary phase would be e l e c t r o s t a t i c i n nature, and changes i n the charge d i s t r i b u t i o n on the enzyme molecule could cause profound a l t e r a t i o n i n the v i t a l substrate-enzyme binding process.  This i s  consistent with the work of Green and Marshall (1977) who observed an increase i n the a f f i n i t y of c a l f rennet f o r the micelles and an acceleration i n the aggregation of caseinate micelles by the addition of cationic materials, i n d i c a t i n g that charge  plays  an important r o l e i n the  c l o t t i n g of milk. In contrast to milk c l o t t i n g , hydrolysis of proteins such as denatured hemoglobin by pepsin d i d not seem to be s i g n i f i c a n t l y influenced by charge e f f e c t .  Although the pH  p r o f i l e was s h i f t e d , the s p e c i f i c proteolytic a c t i v i t y at the optimum pH was r e l a t i v e l y unchanged a f t e r carboxyl modification.  The r e s u l t suggests that coagulation of milk by  pepsin, although i n v o l v i n g a v i t a l proteolytic step, i s d i f f e r e n t from general p r o t e o l y s i s , probably i n the mode of binding between enzyme and d i f f e r e n t substrates. Apart from the decrease i n milk c l o t t i n g a c t i v i t y , changes i n some physicochemical  properties i n the carboxyl  modified pepsin are also a t t r i b u t e d to an a l t e r a t i o n i n the net charge carried by the enzyme.  The s h i f t i n pH  a c t i v i t y curve when hemoglobin was used as substrate, the decrease i n r e l a t i v e electrophoretic mobility on agarose  123  g e l and the s l i g h t increase i n i s o e l e c t r i c point could a l l be related to a change i n the net ionic charge on the modif i e d enzyme. Modification of i n d i v i d u a l charged groups of  proteins  usually a f f e c t the net ionic charge i n a way which may disruptive to t h e i r c h a r a c t e r i s t i c properties.  be  Decreased  s o l u b i l i t y and changes i n conformation or i n the state of aggregation  frequently r e s u l t from such modifications  (Means and Feeney,  1971).  These changes were not observed  i n carboxyl modified pepsin, except when tyrosine and  try-  ptophan methyl esters were incorporated into the enzyme, which resulted i n a s i g n i f i c a n t decrease i n s o l u b i l i t y . This, however, was  probably not  attributed to the charge  e f f e c t but to an increase i n the hydrophobicity of the enzyme upon the incorporation of hydrophobic groups. K i n e t i c study on the modified pepsin suggests that the drop i n peptidase a c t i v i t y was  possibly due to a de-  crease i n the a f f i n i t y of the modified enzyme to the d i peptide substrate, as indicated by the increase i n  K. m  This may be due to e i t h e r a change i n the conformation or the charge d i s t r i b u t i o n on the enzyme, both of which could a f f e c t the binding step between the enzyme and the rate.  The fact that K ^. ca  subst-  remained unchanged indicates  that the decrease i n peptidase a c t i v i t y was not due to a change i n the c a t a l y t i c step.  124  The k i n e t i c data cannot he applied d i r e c t l y to exp l a i n the drop i n milk c l o t t i n g a c t i v i t y i n the modified pepsin since the substrates used were d i f f e r e n t . K  m  I f the  f o r milk coagulation can be calculated and found to  increase a f t e r carboxyl modification, one can conclude that the drop i n milk c l o t t i n g a c t i v i t y was due to an interference with the binding between the enzyme and the micelles.  Results from the present investigation show that carboxyl modification of pepsin greatly improves the s t a b i l i t y of the enzyme at pH around 6 . 5 .  This may have im-  portant p r a c t i c a l implication related to the use of t h i s enzyme as a milk coagulant i n cheese-making. The t r a d i t i o n a l coagulant used f o r cheese-making since p r e - h i s t o r i c time i s rennet extract from the abomasa of 10 to 30-day-old milk-fed calves. With a rapid r i s e i n world-wide cheese consumption, coupled with a decrease i n the practice to slaughter newly born calves, there has been a chronic world shortage of rennet during the l a s t two decades.  This has stimulated interest i n f i n d i n g s u i t -  able substitutes f o r rennet to use as coagulants i n the making of cheese. The basic function of a c l o t t i n g enzyme i s the conversion of l i q u i d milk to a g e l .  This process can be  125  catalysed by most proteases. Apart from c l o t t i n g milk, a suitable coagulant has to survive through cheese-making, and the r e s i d u a l enzyme incorporated i n the curd should contribute to proteolysis of the cheese during ripening (Lawrence et al..,  1972;  Green and Foster,  1974).  However,  many proteases are too p r o t e o l y t i c at the customary pH values of milk and cheese, hence reducing the y i e l d of cheese and retention of f a t by the curd (Veringa, 1 9 6 1 ; Ritter,  1970),  and generating b i t t e r peptides and poor  body (Green and Foster,  1974).  Thus, the most useful  milk coagulants have a high c l o t t i n g to p r o t e o l y t i c enzyme r a t i o (Ernstrom,  1974).  P r o t e o l y t i c enzymes from animals, higher plants and micro-organisms have been studied f o r t h e i r s u i t a b i l i t y to replace c a l f rennet i n making cheese, but only a few were successful commercially. pepsin, used as  50«50  These are swine  rennet-pepsin mixtures, and fungal  rennets from Endothia .paras i t i c a (Sardinas,1968), Mucor p u s i l l u s Lindt (Oka et a l , , 1 9 7 3 ; Arima et a l . , 1 9 7 6 ) and Mucor meihei (Sternberg,  1972).  Recent l i t e r a t u r e indicates that good q u a l i t y cheese can sometimes be produced using fungal rennets. In some cases, however, cheeses manufactured with microb i a l rennets were of a s l i g h t l y lower q u a l i t y than c a l f rennet cheeses (Martens and Naudts,  1976).  126  The use of pepsin, p a r t i c u l a r l y porcine and bovine pepsins i n cheese-making was considered a long time ago. In recent years, the use of c a l f rennet mixed with swine pepsin became wide-spread, and has been found to produce good q u a l i t y cheeses of various types including Cheddar (Phelan, 1976),  et a l . ,  1973).  Pharmigiano-Reggiano (Corradini et a l . .  Grana, Mozzarella and Taleggiano cheeses (Bottazzi 1976").  Recently, pure bovine pepsin was used on  an i n d u s t r i a l scale i n the production of Cheddar (Emmons et a l . , 1976;  1974$ 1 9 7 6 )  and other cheeses (Bottazzi et a l . ,  Corradini e t . a l . ,  1976).  As c a l f rennet substitute, porcine pepsin has some d i s t i n c t advantages.  I t i s considerably cheaper than  other rennet substitutes (Green,  1972} 1 9 7 7 ) .  In f a c t ,  the commercial success of the rennet-pepsin blends i s mainly due to the low cost of pepsin advantageously r e f l e c t e d i n the product price (Sardinas,  1976).  Porcine  pepsin i s commercially a v a i l a b l e , and the supply i s more stable than c a l f rennet and some rennet substitutes. However, when used alone i n cheese-making, porcine pepsin suffers from a number of disadvantages.  The curd  formed i s not as firm as c a l f rennet's, and there i s some l o s s of f a t i n the whey.  Organoleptic q u a l i t y of pepsin  cheese i s i n f e r i o r to that of rennet cheese (Green, Furthermore, a longer ripening peroid i s required i n  1972).  cheese made with pepsin alone (Melachouris and Tuckey, 1964). The l a s t drawback i s due to the i n s t a b i l i t y of porcine pepsin under cheese-making conditions, mainly high pH* s.  Green and Foster (1974) observed that coa-  gulating enzymes retained i n the cheese curd contribute s i g n i f i c a n t l y to casein hydrolysis, and that s t a r t e r enzymes and rennet are synergistic i n t h e i r action on caseins and t h e i r breakdown products (Ohimiya and Sato, 1972). 1977),  In spite of some controversy (0*Keeffe et a l . . the majority of available experimental evidence  indicates that porcine pepsin i s almost completely  de-  natured during cheese manufacture while at least some added c a l f rennet i s recovered and contributes to prot e o l y s i s i n cheese ripening (Green, 1972; Holmes and Ernstrom, 1973;  Green and Foster, 1974).  Consistent with the above reports, the present r e s u l t s also show that porcine pepsin was r a p i d l y i n activated i n b u f f e r at pH 6.5 and i n milk u l t r a f i l t r a t e under simulated cheese-making conditions. A f t e r carboxyl modification, however, the s t a b i l i t y of pepsin was s i g n i f i c a n t l y improved.  This could greatly enhance the  u l t i l i z a t i o n of porcine pepsin i n cheese manufacture. I f some active pepsin i s retained i n cheese curd, the ripening period  could be shortened thus lowering the  128  production cost.  In addition, some defects associated  with pepsin cheese such as harder body and slow f l a v o r development (Maragoudakis et a l . , 1961; Tuckey, 1964;  Melachouris and  Emmons et a l . , 1971) may also be overcome  since these defects are probably attributed to a slow rate of proteolysis i n the cheese. Although the ultimate conclusion on the s u i t a b i l i t y of a milk coagulant should be derived from large scale cheese manufacture, some simple screening procedures have been described.  These include non-protein nitrogen test  (DeKoning, 1972), measurement of the rate of firming of the milk g e l a f t e r coagulant addition (Stavlund and K i e r meier, 1973) and measuring the s t a b i l i t y of enzyme i n a simulation of the early stages of cheese-making (Green and Stackpoole, 1975).  The present results indicate that  the carboxyl modified pepsin has increased pH and heat s t a b i l i t y under cheese-making conditions. I t also has c a s e i n o l y t i c properties s i m i l a r to those of the native pepsin or chymosin.  The rate of syneresis and curd ten-  sion development were also s i m i l a r to those observed i n native pepsin and chymosin. One drawback of the modified pepsin i s the decreased milk c l o t t i n g a c t i v i t y .  However, modified 1«10,000 pepsin  seems to r e t a i n higher milk c l o t t i n g a c t i v i t y than the c r y s t a l l i n e enzyme while the increase i n s t a b i l i t y i s s t i l l  129  significant.  The degree of loss of milk c l o t t i n g a c t i v i t y  can be controlled by the extent of modification. A loss of 50$ a c t i v i t y would double the c l o t t i n g time which should not be a c r i t i c a l factor i n the making of cheese. Since porcine pepsin has r e l a t i v e l y high p r o t e o l y t i c a c t i v i t y , the active enzyme retained i n the curd may lead to extensive breakdown of proteins r e s u l t i n g i n bitterness and other t e x t u r a l defects.  However, as the s t a b i l i t y of  porcine pepsin i s dependent on the extent of carboxyl modif i c a t i o n , the amount of active pepsin retained can be adjusted by c o n t r o l l i n g the degree of modification on the enzyme. A p r i n c i p l e deterrent i n  u t i l i z i n g chemical modi-  f i c a t i o n i n food proteins i s the cost associated with proving to regulatory agencies that the products are nontoxic.  Carbodiimides have been known to be t o x i c , but as  they are just used as a coupling reagent i n the reaction and are removed by d i a l y s i s , the quantity retained i n the modified enzyme would be very small.  Furthermore, since  the c l o t t i n g enzymetmilk r a t i o i s generally low i n cheesemaking, and a large percentage  of coagulant i s l o s t i n the  whey, the concentration of carbodiimide i n the cheese, even i f bound to the enzyme, would be too low to be of any great s i g n i f i c a n c e .  The reagents used i n carboxyl modification are r e l a t i v e l y inexpensive and the reaction i s simple and mild.  Hence, the cost of producing carboxyl modified  pepsin would not be too high, and the price of the enzyme should be at l e a s t competitive to that of the c a l f rennet. The present data show the p o s s i b i l i t y ' o f modif y i n g the a c t i v i t y , s p e c i f i c i t y and physical properties of an enzyme by chemical d e r i v a t i z a t i o n .  This could  widen the scope of food-related enzymes i n the food i n dustry.  Problems associated with enzyme-catalysed  pro-  cesses may be solved i f the undesirable c h a r a c t e r i s t i c s of the enzyme are i d e n t i f i e d and can be corrected by chemical modifications. The present f i n d i n g also i n dicates the p o s s i b i l i t y of chemically modifying the properties of an enzyme to imitate another enzyme.  It  may be f e a s i b l e to substitute enzymes that are expensive and/or i n short supply with cheaper modified enzymes i f t h e i r properties are compatible with the enzymes.  substituted  131  CONCLUSIONS  Selective modification of carboxyl groups i n porcine pepsin by water-soluble EDC and amino acid methyl esters caused s i g n i f i c a n t physicochemical (1)  changes i n a c t i v i t i e s , s p e c i f i c i t y and  properties of the enzyme.  The milk c l o t t i n g a c t i v i t y was  significantly  decreased while the p r o t e o l y t i c a c t i v i t y against hemoglobin was not a l t e r e d .  Consequently, the milk clotting:proteo-  l y t i c a c t i v i t y r a t i o was markedly decreased.  The peptidase  a c t i v i t y against APDT was decreased by about 5 0 $ . (2)  The charge density of pepsin was altered by  carboxyl modification.  This was shown by a decrease i n  r e l a t i v e electrophoretic mobility, a s l i g h t increase i n i s o e l e c t r i c point and a s h i f t i n the p r o t e o l y t i c a c t i v i t y pH p r o f i l e . (3)  The c a s e i n o l y t i c properties were not affected.  However, the c l o t t i n g a c t i v i t y against /c-casein was i n c r eased while the c l o t t i n g a c t i v i t y against was s i g n i f i c a n t l y decreased.  /c-otg^-casein  Results suggest that the  drop i n milk c l o t t i n g a c t i v i t y may be attributed to a change i n the charge d i s t r i b u t i o n  on the modified enzyme,  thus hindering the i n t e r a c t i o n between pepsin and micelles. (4) that K  m  K i n e t i c study using dipeptide substrate shows  was increased while k  c a + <  was not s i g n i f i c a n t l y  132  altered.  This indicates that the lowering i n peptidase  a c t i v i t y was caused by an interference with the enzymesubstrate binding process, and the c a t a l y s i s of the enzymesubstrate complex was not affected. (5)  The presence of dipeptide substrates i n t e r f e r e d  with the carboxyl modification, suggesting that the modif i e d carboxyl groups were located near the enzyme-substrate binding s i t e . (6)  The carboxyl modification was not s p e c i f i c to  pepsin. Modification of carboxyl groups i n pepsinogen and chymosin caused s i m i l a r changes i n a c t i v i t i e s and properties. (7)  The modified pepsin remained reactive to two  s i t e - s p e c i f i c pepsin i n h i b i t o r s .  However, the modified  enzyme was less reactive to the inactivators than the native pepsin. (8)  The s t a b i l i t y of the modified enzyme was mark-  edly improved i n 0.05 M phosphate buffer at pH 6.5 and i n milk u l t r a f i l t r a t e under simulated cheese-making conditions. 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