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The relationship between hydrophobicity and surfactant properties of proteins Keshavarz, Elaheh 1977

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THE RELATIONSHIP BETWEEN HYDROPHOBICITY AND SURFACTANT PROPERTIES OF PROTEINS  by ELAHEH KESHAVARZ B.Sc,  University of Tehran, Iran, 1967  Pharm. D., University of Tehran, Iran, 1969 M.Sc,  University of B r i t i s h Columbia, 1974  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  Sciences  We accept this thesis as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA NOVEMBER 1977 ^cT)  ELAHEH KESHAVARZ, 1977  In p r e s e n t i n g t h i s an  thesis  advanced degree at  the  the L i b r a r y s h a l l make it I  further  in p a r t i a l  fulfilment  of t h e r e q u i r e m e n t s f o r  University  of  Columbia,  freely  British  available  for  I agree  r e f e r e n c e and  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s  that  study. thesis  f o r s c h o l a r l y purposes may be granted by the H e a d o f my D e p a r t m e n t o r by  his  representatives.  of  this  thesis for  It  financial  i s understood that c o p y i n g gain shall  written permission.  Department The  U n i v e r s i t y o f B r i t i s h Columbia  2075  Wesbrook  Vancouver, V6T  Date  T~QOV  of  Place  Canada  1W5  \0,  \q 1  7  or publication  not be allowed without my  i ABSTRACT  The c o m p o s i t i o n ,  s t r u c t u r e and f u n c t i o n a l p r o p e r t i e s o f  p r o t e i n s a r e t h o u g h t t o be i n t e r r e l a t e d b u t t h e n a t u r e relationship  i s n o t c l e a r l y known.  of  this  I n most s t u d i e s o f t h e s t r u c t u r e -  f u n c t i o n r e l a t i o n s h i p s o f p r o t e i n s , more a t t e n t i o n h a s b e e n on t h e p o l a r r e s i d u e s w h i l e the p r o t e i n molecule study  focused  the importance of the n o n p o l a r i t y of  has been o v e r l o o k e d .  I t seems n e c e s s a r y  to  t h e r e l a t i o n s h i p s , i f any, between h y d r o p h o b i c i t y and f u n c t i o n a l  p r o p e r t i e s which are important  The p u r p o s e o f t h i s establishment  o f methods  i n food  systems.  study has been t w o - f o l d .  f o r determination  First,  of hydrophobicity,  and  second, to c o r r e l a t e the h y d r o p h o b i c i t i e s w i t h the s u r f a c t a n t properties of proteins.  The h y d r o p h o b i c i t i e s c a l c u l a t e d s o f a r  (Bigelow,  1962; Waugh, 1954) f r o m t h e amino  1967; T a n f o r d ,  compositions  do n o t a p p e a r t o c o r r e l a t e w i t h t h e p r o p e n s i t y o f p r o t e i n  to form hydrophobic  interactions.  I n an a t t e m p t t o d e t e r m i n e t h e e f f e c t i v e (the c a p a c i t y to p a r t i c i p a t e i n hydrophobic  and a r o m a t i c  S e p h a r o s e 4B.  However,  of hydrophobicity.  amines were coupled these  T h i s may  charges or to the exceedingly t h e l i g a n d and t h e p r o t e i n s .  hydrophobicity  interactions),  c h r o m a t o g r a p h y o n s u b s t i t u t e d S e p h a r o s e g e l s was aliphatic  acid  employed.  column Oleic  acid,  t o t h e CNBr a c t i v a t e d  g e l s were n o t s u i t a b l e f o r d e t e r m i n a t i o n be due t o t h e p r e s e n c e o f u n d e s i r a b l e strong hydrophobic  i n t e r a c t i o n s between  C h r o m a t o g r a p h y o n S e p h a d e x G-150 was a l s o e m p l o y e d i n t h e p r e s e n c e o f T r i t o n X-100 a n d t h e amount o f t h e n o n i o n i c d e t e r g e n t t o t h e p r o t e i n was d e t e r m i n e d . determination  of the detergent  bound  However, l a c k o f r e p e a t a b i l i t y i n t h e bound t o t h e p r o t e i n p r o h i b i t e d the  a p p l i c a t i o n o f t h i s method f o r d e t e r m i n a t i o n  s  of the effective  hydrophobicity.  Alkylepoxy synthesized.  d e r i v a t i v e s o f S e p h a r o s e 4B ( C 4 , C6 a n d C8) w e r e  H y d r o p h o b i c chromatography on t h e b u t y l and h e x y l  d e r i v a t i v e s was s u c c e s s f u l i n d e t e r m i n a t i o n since octylepoxy-Sepharose,  of hydrophobicity.  However,  because of i t s h i g h h y d r o p h o b i c i t y ,  tightly  i n t e r a c t e d w i t h t h e p r o t e i n s i t was f o u n d t o b e i m p r a c t i c a l f o r t h e purpose of measuring hydrophobicity. these  adsorbents  I n an aqueous s o l v e n t  d e m o n s t r a t e some o f t h e p r o p e r t i e s o f a n o i l / w a t e r  interface,  i n c l u d i n g the p o s s i b i l i t y  Therefore,  i t i s assumed t h a t t h e p r o t e i n s were d e n a t u r e d  octylepoxy-Sepharose  of denaturing  some p r o t e i n s . on t h e  gel.  The h y d r o p h o b i c  p a r t i t i o n m e t h o d was a l s o e m p l o y e d .  phase polymer systems o f p o l y e t h y l e n e  of the  hydrophobic  b i n d i n g o f t h e p r o t e i n s t o t h e p a l m i t a t e g r o u p was e x p r e s s e d c o e f f i c i e n t " of the proteins.  of p r o t e i n s determined by hydrophobic  determination  A l l of these  as t h e  The e f f e c t i v e h y d r o p h o b i c i t i e s  chromatography w i t h b u t y l - and  h e x y l e p o x y - S e p h a r o s e s and by hydrophobic w i t h each o t h e r .  Two  g l y c o l / d e x t r a n and p o l y e t h y l e n e  g l y c o l p a l m i t a t e / d e x t r a n were used and t h e e x t e n t  "hydrophobic  phase,  partition significantly correlated  t h r e e methods were found s u i t a b l e f o r  of the e f f e c t i v e hydrophobicity.  No c o r r e l a t i o n was f o u n d  between the e f f e c t i v e h y d r o p h o b i c i t y (Bigelow,  and t h e " a v e r a g e  1967) n o r w i t h t h e m o l e c u l a r  The i n t e r f a c i a l  tensions  hydrophobicity"  weights of the p r o t e i n s .  o f t h e 0.2% p r o t e i n  solution/  c o r n o i l i n t e r f a c e s were d e t e r m i n e d as a p a r a m e t e r o f t h e s u r f a c t a n t properties of p r o t e i n s .  A negative  c o r r e l a t i o n was  found to e x i s t  between t h e e f f e c t i v e h y d r o p h o b i c i t i e s and t h e i n t e r f a c i a l of the p r o t e i n s . in  This r e s u l t suggests that hydrophobicity  the surfactant properties of p r o t e i n s .  protein,  the e m u l s i f y i n g  process.  i s involved  The m o r e h y d r o p h o b i c t h e  the b e t t e r the surface a c t i v e p r o p e r t i e s ,  facilitating  tensions  thereby  iv  T A B L E OF  CONTENTS Page  INTRODUCTION  LITERATURE  . .  SURVEY  1  6  MATERIALS AND METHODS  31  1.  Activation  o f Sepharose  4B w i t h  2.  Coupling  amines  3.  Coupling  of 4-phenylbutylamine  4.  Coupling  of oleic  5.  Chromatography  6.  Synthesis  of g l y c i d y l  7.  Treatment  o f Sepharose  8.  Coupling  9.  Determination suspensions  to Sepharose  acid  cyanogen  bromide  . . . .  4B  32 (PBA) t o S e p h a r o s e  . . .  to Sepharose  glycidyl  .  34 35  t o Sepharose  o f the dry weight  column  34  4B  ethers  33 33  on a l k y l a m i n o - S e p h a r o s e s ethers  32  35  o f Sepharose g e l 36  10.  Hydrophobic  chromatography  36  11.  G e l chromatography  with  37  12.  Removal o f the T r i t o n  13.  Quantitation of Triton  14.  Hydrophobic p a r t i t i o n  15.  Synthesis  16.  Interfacial  detergents  X-100  37  X-100  of polyethylene  37 38  glycol  palmitate  39  t e n s i o n measurements  40  Continued.  TABLE OF CONTENTS  (Continued) Page  RESULTS AND 1.  DISCUSSION  41  C h r o m a t o g r a p h y on S e p h a r o s e s s u b s t i t u t e d w i t h a l k y l amine  41  2.  Chromatography on o l e i c  3.  C h r o m a t o g r a p h y on a l k y l a m i n o - S e p h a r o s e s  4.  Chromatography w i t h d e t e r g e n t s  5.  Hydrophobic p a r t i t i o n  69  6.  Interfacial  78  SUMMARY AND  S e p h a r o s e 4B  t e n s i o n measurements  CONCLUSION  50. 51 .  62  84  BIBLIOGRAPHY  88  APPENDIX. •  93  The scheme o f t h e CNBr a c t i v a t i o n a n d c o u p l i n g o f S e p h a r o s e 4B  94  T h e scheme o f t h e r e a c t i o n s i n v o l v e d i n t h e p r e p a r a t i o n of a l k y l epoxy Sepharoses  95  vx LIST OF TABLES Table I  II  III  IV  Page R e t e n t i o n c o e f f i c i e n t s o f p r o t e i n s determined by hydrophobic column chromatography (butylepoxy- Sepharose 4B)  .  65  R e t e n t i o n c o e f f i c i e n t s of p r o t e i n s determined by hydrophobic column chromatography (hexylepoxy- Sepharose 4B)  .  66  H y d r o p h o b i c i t y o f p r o t e i n s determined by the hydrophobic p a r t i t i o n technique I n t e r f a c i a l t e n s i o n of 0.2% corn o i l i n t e r f a c e .  protein  70  solution/ 79  vii L I S T OF  FIGURES  Figure 1  2  3  4  5  6  7  8  9  10  11  12  Page B e h a v i o u r o f p r o t e i n s o n (15 x 1 cm) o f b u t y l - , h e x y l - , and o c t y l a m i n o S e p h a r o s e 4B  columns  B e h a v i o u r o f b o v i n e s e r u m a l b u m i n on S e p h a r o s e 4B and 4 - p h e n y l b u t y l a m i n o - S e p h a r o s e  42  4B  . . . .  47  E l u t i o n o f a m i x t u r e o f g - l a c t o g l o b u l i n and Y - g l o b u l i n f r o m p h e n y l b u t y l a m i n o - S e p h a r o s e 4B b y s u b s e q u e n t a d d i t i o n o f 0.1 N a C l a n d 50% e t h y l e n e g l y c o l i n b u f f e r  49  R e t e n t i o n volume o f b o v i n e serum a l b u m i n on h e x y l e p o x y - S e p h a r o s e 4B a t d i f f e r e n t s a l t concentrations  56  R e t e n t i o n v o l u m e o f b o v i n e s e r u m a l b u m i n and g l u c o s e pn h e x y l e p o x y - S e p h a r o s e 4B  58  C o r r e l a t i o n between h y d r o p h o b i c i t y d e t e r m i n e d on a h e x y l e p o x y - S e p h a r o s e 4B c o l u m n a n d m o l e c u l a r weight of proteins  60  C o r r e l a t i o n between e f f e c t i v e h y d r o p h o b i c i t y m e a s u r e d o n a h e x y l e p o x y - S e p h a r o s e 4B c o l u m n and t h e "average h y d r o p h o b i c i t y " c a l c u l a t e d by B i g e l o w (1967)  61  R e t e n t i o n volume o f p r o t e i n s on b u t y l e p o x y S e p h a r o s e 4B  63  R e t e n t i o n volume o f p r o t e i n s on h e x y l e p o x y S e p h a r o s e 4B  64  C o r r e l a t i o n between r e t e n t i o n c o e f f i c i e n t s measured b y two h y d r o p h o b i c c h r o m a t o g r a p h y t e c h n i q u e s . . . . . .  67  C o r r e l a t i o n between h y d r o p h o b i c i t i e s measured h y d r o p h o b i c c h r o m a t o g r a p h y on b u t y l e p o x y S e p h a r o s e 4B a n d h y d r o p h o b i c p a r t i t i o n  by 74  C o r r e l a t i o n between h y d r o p h o b i c i t i e s measured h y d r o p h o b i c c h r o m a t o g r a p h y on h e x y l e p o x y S e p h a r o s e 4B and h y d r o p h o b i c p a r t i t i o n  by 75 Continued....  vili LIST  OF F I G U R E S  (Continued)  Figure 13  Page C o r r e l a t i o n between h y d r o p h o b i c i t y determined on b u t y l e p o x y - S e p h a r o s e 4B a n d i n t e r f a c i a l t e n s i o n o f 0.2% p r o t e i n s o l u t i o n / c o r n o i l interface  14  80  C o r r e l a t i o n between h y d r o p h o b i c i t y determined o n h e x y l e p o x y - S e p h a r o s e 4B a n d i n t e r f a c i a l t e n s i o n o f 0.2% p r o t e i n s o l u t i o n / c o r n o i l interface  15  81  C o r r e l a t i o n between h y d r o p h o b i c i t y of p r o t e i n s determined by the hydrophobic p a r t i t i o n m e t h o d a n d i n t e r f a c i a l t e n s i o n o f 0.2% protein solution/corn  o i l interface  82  ACKNOWLEDGEMENT  I  wish  Dr.  S.  his  supervision  of  Nakai,  this  Dr.  Dr.  B.  their  express  Professor, and  my  sincere  Department  invaluable  of  advice  appreciation  to  Food  ,  Science  throughout  for  the  course  study.  My and  to  appreciation  P.M.  Townsley  Roufogalis invaluable  I Department  of  of the  Food  the preparation  My encouragement  of  Faculty  gratitude and  and  indebted  Science the  also  extended  the Department  suggestions  am g r e a t l y of  is  for  of  of  to  Dr.  Food  W.D.  Powrie  Science,  and  Pharmaceutical  Sciences  guidance.  to Dr. his  B.  Skura  invaluable  of  the  advice  during  manuscript.  also  advice  extends  during  this  to Dr. work.  F.  Jamali  for  his  to for  1 INTRODUCTION  The In for of  fact,  this  example,  type  of relationship  function.  governing  flexibility  conformation  under  applications  (Franks,  has  been  related  as  that  t h e amino  the primary  milk  acid  structure  i s the ability  structure (Scott  is  change  structure  food  f o r only  to It  1967).  (Kinsella,  common  determined  to  thought  et a l . ,  proteins  o f most  i n many  i n functional  and quaternary food  to a  systems.  i s important  of the proteins  can lead  be c r i t i c a l  i n food  The primary  composition  Investigation  between  must  cases,  may b e  1976).  proteins  i s  a few, such  and egg p r o t e i n s .  such  properties  of  solutions.  protein  properties  as e m u l s i f i c a t i o n  surfactant  may  be r e l a t e d  For  example,  is  conditions,  of several  i n many  information which  of proteins  the tertiary  be i n t e r r e l a t e d .  1968).  of relationship  sequence, h a s been  Functional systems,  Bernhard,  functionality  1975).  t o the behaviour  Although known,  certain  should  confirmed  of protein, which  the conformation suggested  has been  1967;  of the type  their  of protein  provides  The conformation  The  determine  (Baker,  of proteins  understanding  mechanisms  and function  i n enzymes  the structure  better and  structure  There  i s evidence  (Davis  food  as tensions  that  surface  activity  (Davis  e_t a l . ,  1973)  and surface  active,  of a protein  active  i n  and i n t e r f a c i a l  to suggest  a r e hydrophobic  surface  are important  can be categorized  to the surface  to the hydrophobicity  and also  that  and foaming,  and r e l a t e d  apolipoproteins  hydrophobic  of proteins  e_t a l .  , 1973).  .  g-casein  2 Almost of  amino  acids  and  the b e n z y l  low  affinity  in  an  many  have  of  water,  factors  they  important  relation  acid  in  amino are  acids  are  not,  were  surface. The  expected  have  (1974) s u g g e s t e d  a pronounced  such  as  The  molecular  sites  of  on  the  another,  of  the  most  configuration can  a correlation  proteins  character  also  of  between  nonpolar out  amino  that  nonpolar  i n t e r i o r because  to  model  occur  should  or  they  bonded  on be  contain  subsites  d i s t r i b u t i o n p a t t e r n of  effect  a  one  i n t e r n a l l y hydrogen  or  have  properties.  content  this  valine  to  configuration  therefore  of  chains  folded  (1959) p o i n t e d  whether  that  one  of  the  protein  modified.  exposed  the  hydro-  crevices  hydrophobic  or  sites  and  behaviour  of  these  to be  the major  factor  at should  macro-  ( T a n f o r d , 1973).  between  (however  the  and  side  adhere  the  the p r o t e i n  number  binding  interactions  lipid-protein binding  action  in  Chothia  Hydrophobic to  occur  considerable  and  Kauzmann  hydrophilic  junctions.  molecules  existence  to be  a  to  concentration isopropyl  probably  the  thought  of  is  functional  residues,  "pockets"  subunit  to  tendency  the  protein  the  the nonpolar  1959). T h i s  Polar  surfaces  phobic  to  of  as  s t a b i l i z a t i o n of  the molecule.  hydrophobic.  the  such  form what  (1964) s u g g e s t e d properties  r e l a t i v e l y high  Since  (Kauzmann,  some p h y s i c a l residues  in  a  chains  have  and  involved  proteins  Fisher  side  phenylalanine.  environment,  native  an  contain  with nonpolar  for  aqueous  important in  a l l proteins  weak)  lipids between  level.  The  and  (Gurr  appear  and  proteins  these  two  James, is  taken  components  s t a b i l i z a t i o n of  1971). A s s o c i a t i o n  any  t o mean  the  so  contact  such  that  emulsion  or  contributing inter-  formation of  or  is  made  bonds at  structure  the must  3 be  critically  dependent upon the  Although formation between the stability  o i l droplets  of the  i s s u g g e s t e d t o be  surfactants  (Overbeek,  d e p e n d s on  responsible  for  not  the  owe The  their  of  the little  (Friberg>  1976).  stabilization  coalescence of  stabilities  repulsion  their  the o i l  surrounding  film  1952).  Proteins  f o r m an  important c l a s s of  e a r l i e r b e e n t r e a t e d m a i n l y as water i n t e r f a c e . b o t h the  do  to double l a y e r e f f e c t s .  rather  d o u b l e l a y e r and  s t a b i l i z a t i o n of w a t e r / o i l emulsions  Moreover, nonionic  droplets  of a d i e l e c t r i c  o i l / w a t e r e m u l s i o n s , t h i s mechanism i s of  importance to the  capacities  above-mentioned i n t e r a c t i o n s .  o i l and  e m u l s i f i e r s w h i c h have  two-dimensional molecules at  Weak i n t e r a c t i o n s b e t w e e n t h e water molecules are  p r o t e c t i v e m u l t i l a y e r s around the  emulsifier molecule  important i n the  o i l droplets  the o i l /  formation  ( K r o g and  and  of  Lauridsen,  1976).  I n an  o i l / w a t e r i n t e r f a c e , a more h y d r o p h o b i c p r o t e i n  thought to have a g r e a t e r of  the  o i l droplets  c o a l e s c e n c e and in  the  tendency to u n f o l d  providing  a t h i c k l a y e r and  adherence of the  o i l globules.  e m u l s i f i e r m o l e c u l e have been r e p o r t e d  provide  better  It hydrophobicity possible  a s s o c i a t i o n b e t w e e n o i l and  seemed n e c e s s a r y t o s t u d y t h e and  to p r e d i c t  surfactant the  and  properties  l a t t e r from the  o r i e n t on thereby  the  was surfaces  preventing  Longer hydrocarbon t o be  water  more a c t i v e  (Baret,  information  so  and  1969).  r e l a t i o n s h i p , i f any,  of p r o t e i n s ,  chains  between  that i t would  which i s provided  be on  the  former.  A l t h o u g h t h e r e h a v e b e e n many s p e c u l a t i o n s  r e l a t i o n s h i p between h y d r o p h o b i c i t y properties  (Kinsella,  i n f a n c y and Therefore,  no  o f a p r o t e i n and  1976), r e s e a r c h  i n this field  c l e a r c u t c o n c l u s i o n has  further research  b e e n made on  i s required  mechanisms r e s p o n s i b l e f o r s p e c i f i c  attempted  their  constituent apolar  1962;  t h a t i n most c a s e s even the  contributes  to the  formation  the  flexibility  be  and  s t e r i c hindrance or other  and with  the nonionic  the h y d r o p h o b i c i t y  The their  Bigelow, through However,  s i d e c h a i n o f a p o l a r amino a c i d  not  take p a r t  used f o r s e p a r a t i o n  approaches  Moreover, there  do on  might  i n i n t e r a c t i o n s due  and  Series of ligands study.  T r i t o n X - 1 0 0 , was  affinity  purification  to determine the  were used i n t h i s  detergent, of  proteins.  to  reasons.  of p r o t e i n s .  palmitic moieties)  physicochemical  of p r o t e i n s mainly  of the moleclue.  were s u c c e s s f u l l y m o d i f i e d  hydrophobicity  subject.  F i s h e r , 1964;  h y d r o p h o b i c c h r o m a t o g r a p h y and  methods, which are w i d e l y molecules,  in its  e f f e c t i v e h y d r o p h o b i c i t i e s w h i c h a l s o depend  conformation  study  the  of hydrophobic bonds, t h e i r  h y d r o p h o b i c a m i n o a c i d s w h i c h do  In t h i s  is still  amino a c i d s o r s o l u b i l i t y p r o p e r t i e s .  considering  seem t o m e a s u r e t h e  i t s functional  to e l u c i d a t e the  to measure the h y d r o p h o b i c i t y  not  the  f u n c t i o n a l p r o p e r t i e s of  Many i n v e s t i g a t o r s ( T a n f o r d , 1967)  on  partition of  macro-  effective  (amines, a l c o h o l s , I n t e r a c t i o n of a l s o employed to  oleic  protein determine  proteins.  effective hydrophobicity  o f p r o t e i n s was  s u r f a c t a n t p r o p e r t i e s i n the hope o f r e a c h i n g  correlated with  a better  understanding  5 of  4  the complicated  system of emulsion formation,  groundwork f o r f u t u r e terms of p r o t e i n  This hydrophobicity  i n t e r p r e t a t i o n of food  thus l a y i n g the  emulsion formation  structure.  research  was  of proteins  initiated: as i n v o l v e d  1) t o d e t e r m i n e t h e e f f e c t i v e i n hydrophobic i n t e r a c t i o n s ;  a n d 2) t o c o r r e l a t e t h e e f f e c t i v e h y d r o p h o b i c i t i e s surfactant properties  The  f o l l o w i n g methods were a p p l i e d  with  2.  Chromatography  on o l e i c - s u b s t i t u t e d S e p h a r o s e  3.  Chromatography  on a m i n o a l k y l - s u b s t i t u t e d  detergents.  a.  A l i p h a t i c amines: b u t y l - , h e x y l - and  b.  A r o m a t i c amines:  4B.  Sepharose  4B:  octylamines;  4-phenylbutylamine.  chromatography  on:  a.  Butylepoxy-Sepharose  4B;  b.  Hexylepoxy-Sepharose  4B;  c.  Octylepoxy-Sepharose  4B.  Hydrophobic  the  of the p r o t e i n s :  Chromatography  Hydrophobic  the  to determine  1.  5.  p a r t i t i o n method.  Interfacial was  with  of p r o t e i n s .  effective hydrophobicity  4.  in  tension of p r o t e i n s o l u t i o n / c o r n o i l i n t e r f a c e  measured f o r the s u r f a c t a n t  activity  of the p r o t e i n s .  6 LITERATURE SURVEY  Knowledge of the i n t e r r e l a t i o n s h i p s between c o m p o s i t i o n , s t r u c t u r e , and mostly  f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s has been o b t a i n e d  t h r o u g h b i o c h e m i c a l s t u d i e s c o n c e r n i n g enzyme r e a c t i o n ,  p r o t e i n i n t e r a c t i o n s , and  ligand-protein binding.  protein-  These s t u d i e s p r o v i d e  t h e f o o d c h e m i s t w i t h t h e t e c h n i q u e s and b a s i c i n f o r m a t i o n t h a t c o u l d a p p l i c a b l e to research designed  t o e l u c i d a t e p h y s i c o c h e m i c a l phenomena  underlying p r o t e i n functions i n foods. generally  and  However, f o o d systems  are  complex to p e r m i t a p p l i c a t i o n of  classical  p h y s i c o c h e m i c a l t e c h n i q u e s i n the study of the m o l e c u l a r changes  (chemical  or  too heterogeneous  be  conformational) that occur during t e c h n i c a l  In food systems, f o r c e s and  the r e l a t i v e  t h e i r v a r i a b l e behaviour  actions  c o n t r i b u t i o n of s e v e r a l types  i n d i f f e r e n t environments  the f u n c t i o n a l behaviour of p r o t e i n s . h y d r o g e n and  processes.  influence  These c o n t r i b u t i n g f o r c e s are  i o n i c b o n d s , e l e c t r o s t a t i c f o r c e s and h y d r o p h o b i c  (Sheraga,  1961).  The  influential  extent of i n t e r - m o l e c u l a r i n t e r a c t i o n s .  inter-  e f f e c t s are e x e r c i s e d through  c o n f o r m a t i o n a l changes, m o l e c u l a r ' f l e x i b i l i t y ,  and b y  influencing  A g g r e g a t i o n by  the  hydrophobic  a s s o c i a t i o n o r f o r m a t i o n o f i n t e r f a c i a l membrane l a y e r s i n e m u l s i o n s foams a r e examples o f t h e s e k i n d s o f  1.  Types of  and  interaction.  bonding:  I s h i n o and  Okamoto  ( 1 9 7 5 ) showed t h e i m p o r t a n c e  i n t e r a c t i o n s on t h e p h y s i c a l b e h a v i o u r Farrell  of  of soy p r o t e i n .  (1973) d i s c u s s e d t h e c o n t r i b u t i o n o f h y d r o p h o b i c  i n t e r a c t i o n s , hydrogen bonding  and  of  hydrophobic  Thompson and  and  electrostatic  d i s u l f i d e bonds t o the s t r u c t u r e  and  behaviour  of m i l k caseins.  Hegg a n d L o f q u i s t ( 1 9 7 4 ) a d o p t e d a new a n d  p r a c t i c a l approach, t o q u a n t i f y t h e t h e r m a l This to  aggregation  p r o t e i n was u s e d a s a m o d e l f r o m w h i c h d a t a  help minimize thermal  refining.  denaturation  a t a l k a l i n e pH.  o v a l b u m i n m o l e c u l e s by i n t e r n a l through a combination  2.  Surface  of ionic  of ovalbumin  of t h e i r  and h y d r o p h o b i c  Franks  of three proteins having  different  g l o b u l a r and f l e x i b l e ; (1975) u s e d s u r f a c e surface adsorption.  chemical  techniques  I t was o b s e r v e d  adsorption.  up.  Heat induced  flexible  coil.  Franks  to monitor the k i n e t i c s of and  relative  by 3 - c a s e i n , r e s u l t e d i n r a p i d  T h i s was f o l l o w e d b y a p a r t i a l d e s o r p t i o n  to form a monolayer  multilayer built  properties  b o v i n e serum a l b u m i n , w h i c h i s  that the f l e x i b i l i t y  o f polymers, as t y p i f i e d  (1975) s t u d i e d  These p r o t e i n s were:  and 3 - c a s e i n , w h i c h i s a f l e x i b l e  equilibrium configuration.  like  stabilize  interactions.  w i t h a slow rearrangement of the polymer molecules,  fashion.  particularly  s t r u c t u r e and s u r f a c e  structures.  l y s o z y m e , w h i c h i s g l o b u l a r and r i g i d ;  surface  low  polypeptides  ( 1 9 7 5 ) a n d Graham a n d P h i l i p s  the r e l a t i o n s h i p s between the m o l e c u l a r  initial  i s o l a t i o n and  at very  that anionic detergents  stabilization  extrapolated  p r o p e r t i e s and e m u l s i f i c a t i o n :  Recently,  hydrophobicity  detergents,  denaturation  I t was c o n c l u d e d  c o u l d be  of proteins during  These workers found t h a t i o n i c  concentrations, minimize thermal  of ovalbumin.  associated  while attaining  an  I n c o n t r a s t , lysozyme adsorbed s l o w l y on t h e of native, unfolded  p r o t e i n , and t h e n a  B o v i n e serum a l b u m i n behaved i n an  intermediate  u n f o l d i n g o f the l a t t e r p r o t e i n caused i t t o p e r f o  3-casein molecules.  The r h e o l o g i c a l b e h a v i o u r  of the  adsorbed The  protein  f i l m was  flexible p-casein  albumin  and  reached  a maximum  to  stability.  foam  lysozyme  aggregation the  formed films  value  at  the  were  of  of  emulsification  capacity  and  play  Also,  1976). follow  the  important  and  roles  in  phase,  and  emulsified  phase  the  A  low  sufficient" agents  liquids.  the  of  that  was  the  amino  in  acid  serum  which  turn  solution  related  and  residues  of  reduce  However,  solid of  the  has on  of  in  the  the  largely  emulsion  energy  to  salt  phase  to at  are  (Lynch  of  stability  required  the the of  prime  and  of  the  of  the be a  Chen, two  between in  1974).  emulsion  as  the  importance  Griffin,  cannot  between for  temperature  interface  classed  (Ross  and  surface  abandoned,  be  emulsions  Viscosity  and  formation  different  concentration,  correlated  been  may  be  emulsifier  multilayers  i n t e r f a c i a l tension the  pH,  particles  emulsion  for  explain  The mechanisms  activities.  i n t e r f a c i a l tension condition  to  and w a t e r / o i l  p r i m i t i v e view which  energy  Consequently,  suggested  oil/water  continuous  i n t e r f a c i a l tension  disregarded.  active  and  the  interfacial free  not  of  formation  stability  Although  but  bovine  seem  emulsification  emulsified  the  the  stability  concentration  adsorption  low  This  specific  been  emulsion  formation  phase,  with  stage.  surfactants.  same p a t t e r n .  structure  determining  whereas  structure.  viscoelasticity,  stated  to  have  the  continuous  the  also  related  theories  capacity  viscosity,  a marked  monolayer  (1975)  emulsification  does not  liquid-like films,  the molecular  molecule.  A number  (Friberg,  correlated with  possessed  Franks  properties  3-casein  closely  stability  effect  of  wholly "necessary 1959).  Surfai  non-miscible  emulsification  of  the  two  liquids  i s reduced.  Therefore, a reduction  of the i n t e r f a c i a l  tension  t h r o u g h t h e a d d i t i o n o f an e m u l s i f i e r f a c i l i t a t e s  the  formation  of an e m u l s i o n .  Repulsion from the e l e c t r i c double l a y e r , the l a t e r a l interadhesive the  energy of the s u r f a c t a n t  s t r u c t u r e of water are described  important s t a b i l i z i n g forces  stabilize  It  m o l e c u l e s and t h e i r  by D a v i e s  Supposedly,  h a s l o n g b e e n known t h a t p r o t e i n s , of emulsified  character,  as e m u l s i f i e r s , form a  droplets  (Friberg  of the i n t e r f a c i a l  tension.  The a b i l i t y  a pronounced  of proteins  s h o u l d be v e r y i m p o r t a n t f o r s u c h a p p l i c a t i o n s as meat  extenders  (Rand 1 9 7 6 ) .  also lower surface  tension,  Generally,  of  e m u l s i f i c a t i o n process.  show s u p e r i o r  the peptide chains with  greater  This  binding  i s influenced  the l i p i d  droplets.  volume/surface area of protein  emulsifying  e f f i c i e n c y of the p r o t e i n  i o n i c bonding.  replacers  of l i p o p h i l i c  which  material.  involved i n  by h y d r o p h o b i c  association  The r e s u l t i s t h a t  a much  i s made a v a i l a b l e a n d t h e i s enhanced  (Kinsella  Hydrophobic i n t e r a c t i o n s a r e n o t p r e c l u d e d by favoring  to bind  more h y d r o p h o b i c p r o t e i n s ,  T h e p r o t e i n m o l e c u l e i s u n f o l d e d due t o s h e a r i n g the  1976).  these substances are f o r c e f u l l y  a d s o r b e d t o t h e i n t e r f a c e between o i l and w a t e r , c a u s i n g  and  these  the "thick s k i n " at the interface.  Owing t o t h e i r a m p h i p h i l i c  lipid  on  e_t a l . ( 1 9 7 3 ) a s t h e m o s t  factors i n emulsification.  t h i c k s k i n around the surface  reduction  influence  1976).  conditions  M o r e o v e r , t h e l a t t e r may b e n e c e s s a r y i n o r d e r  to get the p r o t e i n molecules c l o s e  enough f o r s u b s e q u e n t s h o r t e r  range  10 interactions  to  with  and w a t e r  the  o i l  structures liquid  called  crystals  emulsifier in  part  of  presence highly  1976). of  small  systems  at  one  crystalline and  phases  (1969)  The  of  against which layers  and  also  mixture  due (Krog  emulsified type,  to  and  be to  the  and  in  should  be  The  aid  of  is  associated  several at to  the the  an  phases with  packing  on  their  emulsion, to  of  for  of  studying  stability  consist  of the  surface  of  an  the  substances other is  years  ago  by  crystalline  phase  and equilibria.  emulsions hindrance,  the  ordered  factors  can  hydrocarbon  be  chains  oil-water-emulsifier  film  an  the  crystal  steric  These  of  possible  liquid  of  interface.  whether  a  Friberg  affecting  The  an  than  liquid  by  surrounding  oil/water  association  .  formation  seventeen  factors  of  increase  as  liquid of  These  the  sudden  more  the  so-called  conditions  at  amphiphilic  presence  the  behaviour  1976).  and  influence  methods  oil-water  considered  The  a  emulsion  demonstrated  independent  o v e r a l l phase  is  the  1969).  c h a r a c t e r i z e d by  recognized  was  association  phases  phenomena  viscosity  specific  emulsions  the  to  often  these  was  of  emulsifiers  d e f i n i t i o n of  crystalline  water.  the  and,  formed,  emulsifiers  The of  is  present  are  on  emulsions  Lauridsen,  droplets  depend  crystalline  of  due  neutral  that  emulsion  contributes  which  1976).  formation  the  The  liquid  which  (1960).  emulsifiers  considered  of  times  liquid  composed  of  includes  in  of  ( F r i b e r g j2t al.,  phase  experienced.  nature  with  coalescence  is  this  s t a b i l i t y of  co-workers effect  which  emulsions  the  crystals  at  system  mesophases  the  liquid  phase  (Berger,  hundred  determine  third  amounts in  Weak i n t e r a c t i o n s  a  also  Goldemberg  on  is  Food  hydrophobic  least  as  dispersion  emulsion  James  appear  therefore,  (Friberg,  molecules  lyotropic  stability  the  1976).  (Rand,  concentration  emulsion  emulsion,  occur  or  the  water/oil  structure  between t h e t h r e e components: o i l , e m u l s i f i e r and w a t e r Lauridsen,  1976).  Bull  (1947) s u g g e s t e d t h a t t h e i o n i c groups a r e  r e s p o n s i b l e f o r s u r f a c e s p r e a d i n g and s t a b i l i t y film. the  isoelectric point of proteins.  A c o n s i d e r a b l e r e d u c t i o n i n f r e e e n e r g y was s u g g e s t e d t o b e  t o p o l a r i n t e r a c t i o n s and h y d r o g e n b o n d i n g between p o l a r groups and  water the  ( F r i b e r g e t a l . , 1969).  a l c o h o l i c - O H group  E v e n a weak h y d r o p h i l i c g r o u p  aqueous phase  g i v e s about  12 K c a l / m o l e due t o t h e s t r u c t u r a l  o f t h e e m u l s i o n s and t o f a c i l i t a t e  (Friberg,  1976).  For  about  and s u r f a c e  two d e c a d e s  f o r a g i v e n system.  solubility  The  to influence the  the e m u l s i f i c a t i o n process  tension.  h y d r o p h i l e - l y p o p h i l e b a l a n c e (HLB) h a s  been used as t h e most u s e f u l c r i t e r i a emulsifiers  changes  I t seems t o b e d i f f i c u l t t o d i s c e r n a d i r e c t c o n n e c t i o n  between emulsion s t a b i l i t y  it  i n water.  r e d u c t i o n i n s u r f a c e energy has been s a i d  stability  the  Adsorption to the  t h e h y d r o c a r b o n c h a i n s when t h e y a r e d i s s o l v e d  pronounced  such as  l o s e s a b o u t 3.4 K c a l / m o l e when b e i n g a d s o r b e d  from a hydrocarbon to the o i l / w a t e r i n t e r f a c e .  of  o f t h e monomolecular  He f o u n d t h e b e s t s p r e a d i n g g e n e r a l l y o c c u r s a t pH c l o s e t o  ' . due  ( K r o g and  f o roptimal selection of stable  The HLB c o n c e p t g i v e s i n f o r m a t i o n  of the emulsifier i n either  t h e o i l o r t h e w a t e r phase and  may b e u s e d a s a g u i d e t o p r e d i c t w h i c h t y p e o f e m u l s i o n w i l l b e  HLB number d e p e n d s d i r e c t l y  narrow,  formed,  on t h e m o l e c u l a r s t r u c t u r e o f t h e e m u l s i f i e r .  However, t h i s v a l u e h a s , i n f a c t , o n l y a l i m i t e d food i n d u s t r y because  about  application within the  such a c l a s s i f i c a t i o n o f food e m u l s i f i e r s i stoo  and i t does n o t a c c o u n t f o r o t h e r p r o p e r t i e s o f e m u l s i f i e r s  such  as  complex f o r m a t i o n w i t h s t a r c h components o r p r o t e i n s (Krog  Lauridsen,  1976).  M o r e o v e r , HLB  d e t e r m i n a t i o n c a n n o t be  evaluate e m u l s i f i c a t i o n p r o p e r t i e s of p r o t e i n s . suggested HLB  replacement  o f t h e l a b o r i o u s and  w i t h t h e i r method.  emulsion  may  be  interfacial w i t h t h e HLB  and  The  to the spreading  spreading  of the e m u l s i f i e r s . prepared  fluid.  R o s s and  t h e o i l d r o p l e t s , and  negative  The  spreading  Those o i l s  which  in  water,  surfactant layers,  the a d j a c e n t  coalescence  that  more w a t e r s o l u b l e  the adsorbed  into  correlate  interstitial  of the o i l d r o p l e t s  1974).  It  i s often d i f f i c u l t  p r o t e i n , an  t o e s t a b l i s h w h e t h e r , upon m i x i n g  i n t e r a c t i o n occurs o r the l i p i d s  coexist separately. molecular  to r a p i d  an  from  the g r e a t e s t s o l u b i l i t i e s  into  of  Chen ( 1 9 5 9 ) r e p o r t e d  coefficients.  to penetrate  This would g i v e r i s e  (Rehfeld,  and  tend  of  c o e f f i c i e n t o f w a t e r and o i l  w i t h o i l s w h i c h had  p o s s i b l y p o s i t i v e spreading  h y d r o c a r b o n s may  (1959)  imprecise determination  a p p e a r e d t o be m o s t s t a b l e t o c r e a m i n g .  which s t a b i l i z e  Chen  c o e f f i c i e n t s were c a l c u l a t e d  s p r e a d m o s t e a s i l y on w a t e r a l s o h a d and  Ross and  to  s u r f a c e t e n s i o n m e a s u r e m e n t s and w e r e shown t o  o i l / w a t e r emulsions coefficients  used  T h e s e w o r k e r s showed t h a t t h e s t a b i l i t y  related  soluble solutes.  and  The  proteins simply  i n t e r f a c e b e t w e e n t h e b u l k p h a s e s must i n v o l v e  a s s o c i a t i o n s ( G u r r and  (1970) c o n s i d e r e d  and  lipid  James, 1971).  Gonzalez  and  MacRitchie  t h e o i l / w a t e r i n t e r f a c e as a " g o o d s o l v e n t " f o r p r o t e i n  i n w h i c h t h e p o l a r g r o u p s i n t e r a c t w i t h t h e p o l a r p h a s e and groups w i t h the nonpolar c o n f i g u r a t i o n of very  phase.  the  nonpolar  T h i s a l l o w s t h e p r o t e i n t o t a k e up  low f r e e energy.  If a lipid-protein  a  interaction  took be  place, hydrophobic  involved  3.  (Rand,  describe from  an  the  aqueous  suggested that  i t  that  is  in  The  the  the  increased  disorder  protein molecule  has  been  nonpolar  Kauzmann  of  This bonds  groups,  occurs or  the  in  probable  such  term used  the  term s t a b i l i z e s hydrophobic  types  of  bond  to  cluster  the  a way  so  f r e e volume,  as  not  upon w h i c h  to  is  and  water  the  capacity  ice-like  water  1968).  of  over  of  the  surrounding  the by  waterthe  nonpolar  t h e number  entropy  due  of  heat  dominated  diminish  the  in  to melt  the nature  of  to be  molecules  increase  and  accomodation  Kauzmann  consequence  water  that  to  residues  aggregation  seems  and R a t n e r ,  r e l a t i v e l y simple,  with  a  needed  (Crothers  (1959)  nonpolar  protein.  bond  The  heat  of  protein  is  of  1975).  the excess groups  which  Kauzmann  bonding.  the hydrophobic  the  by  transfer  i n t e r i o r of  (1959) p o s t u l a t e d  interface is  charged  on  the  (Hjerten,  a t t r i b u t e d to  the  increase,  within  the  around  to  of  of  entropy  is  energy  result  existence  to  protein  free  entropic a  basically  formed  bond  environment  mainly  the most  bonding:  hydrophobic gain  are  1976).  Hydrophobic  The  bonds  solvation areas.  of  hydrogen  the water  strongly  depends.  Klotz  (1958) h a s  view which proposes small  shells  of  that  been  the major  spokesman  the protein induces  water molecules  around  a  for  general  proteins.  When  a more  moderate  ordering  effect  a  hydrophobic  on  r e s i d u e i s i n t r o d u c e d i n t o water the degree of s t r u c t u r e i n the  layer  o f w a t e r n e x t t o t h e h y d r o c a r b o n i n c r e a s e s a n d becomes i c e - l i k e .  If  two h y d r o p h o b i c c h a i n s , s u r r o u n d e d b y i c e - l i k e  structured water,  are  b r o u g h t c l o s e t o g e t h e r a p a r t o f t h i s s t r u c t u r e w h i c h i s b e t w e e n them must be d i s r u p t e d b e f o r e t h e h y d r o c a r b o n c h a i n s c a n c o a l e s c e . s t r u c t u r e i n water requires heat, j u s t l i k e melting i c e . positive  Disrupting  Thus, A H i s  and h e a t m u s t e n t e r t h e s y s t e m i n o r d e r t o m e l t t h e w a t e r  t h i s b r i n g s the hydrophobic chains together.  and  A f t e r c o a l e s c e n c e , the  q u a n t i t y o f s t r u c t u r e d w a t e r s u r r o u n d i n g them i s l e s s amount w h i c h p r e v i o u s l y s u r r o u n d e d t h e i n d i v i d u a l  than the  chains  total  (Stauffer,1972).  T h i s d e c r e a s e i n t h e t o t a l amount o f o r d e r ( s t r u c t u r e d w a t e r ) i n t h e s y s t e m g i v e s an i n c r e a s e i n A S , t h e  entropy of the system.  The  two  h y d r o p h o b i c r e s i d u e s come t o g e t h e r s p o n t a n e o u s l y b e c a u s e a l t h o u g h A H i s p o s i t i v e , T A S i s n u m e r i c a l l y e v e n l a r g e r , and negative  an  overall  value.  The is  so A F h a s  i n t e r a c t i o n b e t w e e n w a t e r and  g e n e r a l l y a g r e e d t o be a s i g n i f i c a n t  e x i s t e n c e and  stability  (Richardsj1963).  The  factor  contributing  h y d r o p h o b i c b o n d i s s u g g e s t e d t o be t h e  important than the hydrogen that hydrogen  protein  to the  o f t h e u n i q u e s t r u c t u r e o f most g l o b u l a r  determining f e a t u r e of p r o t e i n s t r u c t u r e ,  demonstrated  the a p o l a r groups of  proteins  major  i n d e e d p r o b a b l y much more  bond as a s t a b i l i z i n g  force.  Schellman  (1955)  bonding could not account f o r the s t a b i l i t y  g l o b u l a r p r o t e i n s i n aqueous s o l u t i o n s .  B i g e l o w (1967) f o u n d  generally  l o w h y d r o p h o b i c i t y v a l u e s f o r f i b r o u s p r o t e i n s and h i g h e r v a l u e s f o r  of  globular proteins.  He  pointed  average hydrophobicity the  globular  two  o r more s i d e c h a i n s  hydrophobic side chains  25-30% of are  the  f o r i t t o be  t h e h y d r o p h o b i c b o n d as  e s t a b l i s h i n g as many c o n t a c t s  length  of a c e r t a i n stable  in  conformation.  (1961) s t a r t s by  the  the n e c e s s i t y  f o r a p r o t e i n i n order  Formation of  on  out  and  as p o s s i b l e .  the  chains.  amino a c i d s i d e c h a i n s  typically hydrophilic  (Tanford,  by  Sheraga  approaching each other The  to the h y d r o p h o b i c i t y  s i z e of  described  and  c o n t r i b u t i o n of  of  the p r o t e i n s  the  depends  In water s o l u b l e proteins are  about  g e n e r a l l y h y d r o p h o b i c and  1973).  In the n a t i v e  45-50%  conformation  such a p r o t e i n , a s u b s t a n t i a l f r a c t i o n of the hydrophobic s i d e chains u s u a l l y b u r i e d w i t h i n the 1969).  i n t e r i o r of  Charged groups are not  presumably because the the nonpolar i n t e r i o r m o l e c u l e m u s t be s u c h as  i s too  (Tanford,  The  time,  surface  of  hydrophobic p a r t s of the  only  the  the  protein  f u l l y nonpolar side  of  side chains  f o u r methylene groups of  with  of the p r o t e i n surface  which the  chains contain  l y s i n e side  t h a t becomes b u r i e d  increasing molecular weight.  of p o l a r groups that  bonds i s e s s e n t i a l l y c o n s t a n t 1976).  The  such a group i n  1962).  the p r o p o r t i o n  (Chothia,  found i n s i d e macromolecules,  a l s o the nonpolar p a r t s  proportion  folding increases  great.  is  (Mohamadzadeh e t a l . ,  "thermodynamic c o s t " of b u r y i n g  c h a r g e d o r p o l a r g r o u p s , e.g., chain  generally  taken to i n c l u d e not  l e u c i n e , but  the m o l e c u l e  of  and  at the  form i n t r a m o l e c u l a r  i s independent of molecular  D i s t r i b u t i o n of p o l a r residues  the m a c r o m o l e c u l e can  But,  be  between the  by  same  hydrogen weight interior  searched f o r r e l a t i o n s h i p between  and  s t r u c t u r e and  function.  P r o t e i n s s h o u l d be  less soluble i n their  three  d i m e n s i o n a l s t r u c t u r e where p o l a r r e s i d u e s a r e b u r i e d and n o n p o l a r ones are a c c e s s i b l e to water.  For a p r o t e i n  t o be r e a s o n a b l y s o l u b l e i t m u s t  remove much o f t h i s h y d r o p h o b i c s u r f a c e f r o m c o n t a c t w i t h w a t e r b y b u r y i n g it  between the p i e c e s o f secondary s t r u c t u r e .  However, K l o t z  (1970)  e x a m i n e d t e n p r o t e i n s f o r w h i c h t h e t h r e e d i m e n s i o n a l s t r u c t u r e was and  showed t h a t t h e h y d r o p h o b i c g r o u p s , i n c l u d i n g  amino a c i d  the l a r g e r  s i d e c h a i n s s u c h a s t h o s e o f p h e n y l a l a n i n e and  known  hydrophobic  tryptophan, occur  more f r e q u e n t l y on t h e s u r f a c e o f t h e p r o t e i n t h a n h a d b e e n a s s u m e d .  4.  Protein-ligand  interaction:  In the f o r m a t i o n of a s t a b l e g l o b u l a r s t r u c t u r e , removal of hydrophobic s i d e chains from c o n t a c t w i t h water not possible. exposed  binding sites  i s generally  t h e y may  constitute  (1964)  s u g g e s t e d two  distinctly a small  nonpolar  a) a d i s s o l v e d n o n p o l a r m o l e c u l e c o u l d a t t a c h i t s e l f  a c c e s s i b l e s u r f a c e of a nonpolar c l u s t e r perhaps w i t h p a r t i a l the nonpolar molecule might p e n e t r a t e i n t o  i n t e r i o r o f t h e p r o t e i n and  lodge.  i n a) seems more l o g i c a l b e c a u s e changes.  On  If  f o r hydrocarbon or amphiphilic molecules.  d i f f e r e n t modes o f i n t e r a c t i o n b e t w e e n a p r o t e i n and  o r b)  remain  ( T a n f o r d , 1972).  l o n g hydrophobic patches are formed,  W e t l a u f e r and L o v r i e n  molecule:  complete  I n m o s t n a t i v e p r o t e i n s , some h y d r o p h o b i c g r o u p s  at the molecular surface or i n crevices  sufficiently  the  An  the r e l a t i v e l y  nonpolar indicated  occur w i t h v e r y minor  t h e o t h e r hand, i f b) i s t h e c a s e , i t would  s u b s t a n t i a l m o l e c u l a r rearrangement  penetration;  i n t e r a c t i o n of the s o r t  i t might  to the  structural  require  t o accomodate the n o n p o l a r  molecule  or  m o l e c u l e s , s i n c e the p r e - e x i s t e n c e of h o l e s i n the p r o t e i n  i s h i g h l y u n l i k e l y on e n e r g e t i c  interior  grounds.  Since the d r i v i n g force behind the hydrophobic i n t e r a c t i o n i s thought t o be e n t r o p i c  (Kauzmann, 1 9 5 9 ) , t h e s e i n t e r a c t i o n s s h o u l d be  stronger at h i g h e r temperatures because the term T A S i n A F = AH - T A S becomes i n c r e a s i n g l y u n f a v o r a b l e t o w a r d s d i s s o c i a t i o n o f n o n p o l a r b i n d i n g s . A t t h e same t i m e m o s t o f t h e o t h e r b o n d s a r e w e a k e n e d , s u c h t h a t phobic bonding would predominate. f a v o u r e d by h i g h i o n i c affect  strength  (von H i p p e l and  Schleich,  promoting  > P0,~ 4 1969a).  Non  ionic  ions  solutes like  ~ > and 4  dioxane  ( T a n f o r d , 1973).  CNS~  and  Urea, guanidine by  t h e s o l u b i l i z a t i o n o f h y d r o p h o b i c r e s i d u e s i n the aqueous phase  actually  Detergents minimize hydrophobic  The  interactions  f o r m c h e m i c a l b r i d g e s b e t w e e n h y d r o p h o b i c and  a d d i t i o n of a c i d or a l k a l i  groups, which are buried w i t h i n regions of the p r o t e i n molecule. a t t r a c t w a t e r m o l e c u l e s and h y d r o p h o b i c a s s o c i a t i o n s and 5.  Certain  This effect i s  > C l " > B r " > I ~ > CIO  p o r t i o n s of p o l y p e p t i d e s , thereby f a c i l i t a t i n g 1974).  l o w pH.  also  some s u r f a c t a n t s d i s r u p t h y d r o p h o b i c i n t e r a c t i o n s ,  ( G o r d o n and W a r r e n , 1 9 6 8 ) . a n d may  and  type of bonding.  a l c o h o l s weaken h y d r o p h o b i c i n t e r a c t i o n s h y d r o c h l o r i d e and  i n t e r a c t i o n s are  ( H j e r t e n , 1975)  the establishment of t h i s  d e c r e a s e d i n o r d e r o f SO. 4  Hydrophobic  hydro-  denaturation (Steinhardt,  i o n i z e s weakly  the i n t e r i o r ,  a c i d i c or b a s i c  supposedly i n hydrophobic  Upon i o n i z a t i o n ,  these charged  form h y d r a t i o n s h e l l s , which cause u n f o l d i n g  hydrophilic  (Perutz,  i n turn  groups disrupt  1974).  Determination of hydrophobicity:  The  i m p o r t a n c e o f h y d r o p h o b i c i n t e r a c t i o n s has a t t r a c t e d  the  attention  of  number  of  investigators  scale"  for  has  liquids  by  side  Phe,  or  Leu  the  (NPS)  (1964)  This  was  only  that  a l l nonpolar the  estimated that  a simple  outer using  highly  In  case  in  of  terms  of  frequencies  for  (Ve)  by  and  organic  nature  of  residues.  (Vi)  acids of  are present  the molecule.  s p e c i f i c volumes proteins  inside  are  of  The  amino  likely  acid  and  total  Trp,  proteins of  l i e ,  by  a globular on  the  volume  amino  (Vt)  that  acids  was  Fisher  because  protein.  assumption  a l l polar  residues.  nonspherical  phases.  calculated  nonpolar  a r i t h m e t i c c a l c u l a t i o n and based  amino  He  proteins.  for  in  proteins  of  i n t e r n a l volume  hydro-  solubilities  residues.  series  as  "hydrophobic  their  aqueous  nonpolar  a  A  small molecules,  the hydrophobic  defined  chemistry.  establish  calculated polarity values  surface  polar  the  protein  f r a c t i o n of  and V a l were  e x t e r n a l volume  on  of  attempted to  estimated  dividing  are  area  t h e i r p a r t i t i o n between  (1954)  Fisher  the  determined mainly  chain  Pro,  have  in  proteins.  determining  nonpolar Tyr,  of  been  Waugh simply  researchers  series  phobicity organic  many  suggested sort  of  Ve shape more  gives space  them a h i g h e r would  Klotz acids  outside  studies  show  exposed  to  accessible  proceed  one  that  to  available  (1970)  and  the  diffraction.  be  ones  almost  solvent,  of —  for  disagreed  apolar in  ratio  the  inside  the  a substantial molecules  For  some o f  them,  further:  to  sphere  of  of  polar  view  the that  the molecule.  a l l proteins  solvent  a  excess  with  the  step  than  in  ionic  number  of  the  chains  of  Hence  residues. polar  Although  examined  amino  crystalographic  are  groups  information is extent  same V t .  regards  apolar  a l l proteins  sufficient  estimate  side  the  fully seem  by  to  X-ray  available  exposure  of  be  each  to of  the  major groups o f apolar  Tanford an  residues  (Klotz,  (1962) c o n s i d e r e d  s o l u b i l i t y o f f r e e amino a c i d s a s  index f o rt h e i r hydrophobicities.  required  1970).  He d e t e r m i n e d t h e f r e e  t o t r a n s f e r one m o l e c u l e o f a m i n o a c i d f r o m w a t e r t o e t h a n o l .  N e g a t i v e v a l u e s were obtained to e t h a n o l .  f o r t h e amino a c i d s w h i c h p r e f e r r e d  These a r e supposed t o be p o l a r  be  Tanford  t h e c a l c u l a t e d v a l u e s were independent o f t h e k i n d  solvents.  According to Tanford t h i s  ( 1 9 6 2 ) showed  of  organic  f r e e e n e r g y f o r each amino a c i d c a n  r e g a r d e d a s t h e sum o f two t e r m s : o n e f o r t h e b a c k b o n e H-^N- c J  and  the other f o rthe side chain.  that  f o rglycine.  leads  With s o l u b i l i t y data f o rg l y c i n e , subtraction chain  itself.  f r e e e n e r g i e s c a l c u l a t e d by T a n f o r d were c a l l e d  These hydrophobicities  i s i n t e r e s t i n g to note that Tanford  ( 1 9 6 2 ) a n d Waugh  regard  g l y c i n e a s an amino a c i d o f l o w h y d r o p h o b i c c h a r a c t e r ,  Fisher  (1964) c o n s i d e r s  p o l a r by F i s h e r Tanford  residue  Also,  (1954)  whereas  t y r o s i n e was a s s u m e d  n o n p o l a r b y Waugh  (1954)  (1962).  (1967) c a l c u l a t e d an "average h y d r o p h o b i c " i n d e x  from  p e r c e n t a g e amino a c i d c o m p o s i t i o n o f t h e p r o t e i n and a f r e e  energy f o reach r e s i d u e phobicity  i t t o be n o n p o l a r .  ( b e c a u s e o f i t s OH g r o u p ) w h i l e  Bigelow the  therefore  D u n n i l l i n 1965. It  and  - COO H  The backbone s t r u c t u r e i s s i m i l a r t o  t o a t r a n s f e r f r e e energy f o r the s i d e  transfer by  water  s i d e c h a i n s and presumably  occur i n the outer s h e l l of the p r o t e i n molecule. that  energy  determined by Tanford  d i v i d e d b y t h e number o f r e s i d u e s  (1962).  Total  hydro-  i n the molecule y i e l d e d the  "average h y d r o p h o b i c i t y " .  B i g e l o w (1967)  suggested t h a t low m o l e c u l a r  weight p r o t e i n s have the l a r g e s t spread i n a l l o w e d h y d r o p h o b i c i t i e s while p r o t e i n s of high molecular weight range of  above mentioned  researchers determined  w i t h t h e i s o l a t e d p r o t e i n m o l e c u l e and  not r e p r e s e n t the a b i l i t y  for. of  of the p r o t e i n were not  t h e y c a n t a k e p a r t i n , and n o t o n l y on  s t r u c t u r e and amino a c i d determines  flexibility  Hydrophobic  There  bonds accounted  their molecular that  a s w e l l a s on i t s a m i n o a c i d c o m p o s i t i o n .  l i k e propane,  b u t a n e , and p e n t a n e  to study the nonpolar r e g i o n s of p r o t e i n s  carbon coated agaroses. chromatography  ( W i s h n i a and  C u a t r e c a s a s and A n f i n s e n ( 1 9 7 1 ) by a t t a c h i n g h y d r o c a r b o n  d i s t a n c e s from g e l m a t r i x backbone.  Agarose  have sometimes  ( W i s h n i a , 1962).  T h i s p r o p e r t y has been employed i n chromatography  The  Thomas,  w i t h the hydrodeveloped  ligands at varying  i s t h e most w i d e l y used  i n s o l u b l e m a t r i x f o r the p r e p a r a t i o n of the a f f i n i t y Sepharose,  degree  i s a need f o r a method  alkanes i n t e r a c t d i r e c t l y w i t h the hydrophobic areas  affinity  v a l u e s do  chromatography:  Hydrocarbons been used  compositions.  on  the e f f e c t i v e h y d r o p h o b i c i t y of a p r o t e i n which accounts f o r  c o n f o r m a t i o n and  1966).  The  f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s m o s t l y d e p e n d on t h e  interactions  6.  components.  of the p r o t e i n t o form hydrophobic  c o n f o r m a t i o n and f l e x i b i l i t y  The  hydrophobicities  t h e measurements were based  number and p r o p e r t i e s o f t h e a m i n o a c i d  because  narrow  hydrophobicities.  The  the  tend to occur i n q u i t e a  adsorbents.  a "beaded" form of agarose, w i t h i t s h i g h l y porous  structure,  is very suitable  f o r the preparation of substituted gels.  The  p o l y s a c c h a r i d e m a t r i x i s a c t i v a t e d b y t h e CNBr m e t h o d a n d t h e h y d r o - • c a r b o n l i g a n d s a r e s u b s e q u e n t l y c o u p l e d t o i t v i a t h e f r e e amino These h y d r o c a r b o n c o a t e d agaroses o r a l k y l - S e p h a r o s e s were h y d r o p h o b i c g e l s b y S h a l t i e l ert a l . ( 1 9 7 3 b ) a n d w e r e u s e d  group.  called extensively  f o r p u r i f i c a t i o n p u r p o s e s b y many w o r k e r s  ( C u a t r e c a s a s and A n f i n s e n ,  1970;  1975).  Cuatrecasas et a l . ,  Hofstee  (1974)  1968; J e n i s s e n ,  e s t i m a t e d t h e r e l a t i v e degree  of substitution  f r o m t h e d y e b i n d i n g c a p a c i t i e s o f t h e r e s i n a n d showed t h a t t h e d e g r e e of s u b s t i t u t i o n d e c r e a s e d upon s t o r a g e .  This i n a c t i v a t i o n took place  g r a d u a l l y e v e n when t h e g e l was r e f r i g e r a t e d .  F o r i n s t a n c e , i n one c a s e  a d e c r e a s e o f a s much a s 85 p e r c e n t o f t h e p o n c e a u b i n d i n g c a p a c i t y h a d o c c u r r e d d u r i n g a p e r i o d o f almost f i v e months. d e c r e a s e i n 40 days  s t o r a g e was o b s e r v e d  the adsorbents w i t h  t h e h i g h e s t degree  stable  I n another case, a 40%  ( H o f s t e e , 1974).  Basically,  o f s u b s t i t u t i o n were t h e l e a s t  ones.  The  adjustment  o f t h e column h y d r o p h o b i c i t y so as t o adsorb  but n o t denature t h e p r o t e i n s has been approached  i n two w a y s : 1) E r - e l  e t a l . ( 1 9 7 2 ) , H o f s t e e (1973b) and S h a l t i e l and E r - e l  (1973)  used  a  s e r i e s o f a l k y l - S e p h a r o s e s , o r ej-aminoalkyl-Sepharoses o f v a r i o u s a l k y l chain l e n g t h s , from which  t h e a p p r o p r i a t e o n e was f o u n d b y t r i a l ;  m a n i p u l a t i n g pH o n s t r o n g l y h y d r o p h o b i c a l k y l - S e p h a r o s e g e l , Y o n introduced electrostatic  2) b y (1974)  repulsion energies to control the binding  f o r c e s and a d s o r p t i o n p r o p e r t i e s o f t h e g e l m a t e r i a l .  S h a l t i e l e_t a l . ( 1 9 7 3 b ) s u g g e s t e d t h a t t h e r e t e n t i o n p o w e r of a l k y l - a g a r o s e s i s d e r i v e d m a i n l y from l i p o p h i l i c h y d r o p h o b i c p a c k e t s o r r e g i o n s i n t h e p r o t e i n and groups on t h e  r e t e n t i o n and  i n t e r a c t i o n s c o n t r i b u t e predominantly to the  s h o u l d be a w a r e t h a t i o n i c i n t e r a c t i o n s a l s o  the e l u t i o n p a t t e r n of these columns coated Sepharoses  seem u n l i k e l y  phobic i n t e r a c t i o n .  ( H o f s t e e , 1973a).  van der Waals, important roles  sulfate.  By p r e p a r i n g  t o o b t a i n a bed m a t e r i a l t h a t e x h i b i t e d o n l y h y d r o The  amino groups  and h y d r o g e n  bonding  of the l i g a n d s r e t a i n t h e i r ( H j e r t e n , 1973).  c o n t a i n s two  In the c r o s s l i n k e d  removed b u t t h e c a r b o x y l group  f o r c e s g e n e r a l l y p l a y more o r  agar, s u l f a t e groups i s present.  and  are almost completely  Even h i g h l y p u r i f i e d  (Porath et a l . ,  may  o f a c t i v a t i o n w i t h CNBr w h i c h  i n t r o d u c e d as a r e s u l t  less  alkyl-  types of charged groups, c a r b o x y l  c o n t a i n some n e g a t i v e c h a r g e s be  basic  Electrostatic,  i n c h r o m a t o g r a p h i c b e h a v i o u r o f p r o t e i n s on  Agar  affect  t h r o u g h t h e CNBr a c t i v a t i o n p r o c e d u r e , i t  p r o p e r t i e s a f t e r b i n d i n g to Sepharose  Sepharoses.  long chain a l k y l  d i s c r i m i n a t i o n power o f t h e a l k y l - S e p h a r o s e s . However, a t  t h e same t i m e , one  would  between  agarose.  Hydrophobic  hydrocarbon  interactions  1971)  and p o s i t i v e  agaroses charges  are  subsequently coupled with alkylamine.  It significant  i s evident that this  amounts o f i o n i c g r o u p s , m a i n l y c a t i o n i c  Hjerten,  1973).  effect.  The  limits  type of hydrophobic adsorbents  ( H o f s t e e , 1973b;  T h e r e f o r e , they e x h i b i t a superimposed  presence of these i o n i c  groups  contain  electrostatic  c o m p l i c a t e s and  t h e u s e f u l n e s s o f t h e s e s o r b e n t s when a s p e c i f i c  potentially  interaction i s  desired  ( H o f s t e e , 1974).  The  simultaneous presence of  i o n i c and h y d r o p h o b i c g r o u p s has b e e n f o u n d t o c a u s e nonspecific protein binding.  Hofstee,  substantial  These g e l s e x h i b i t b o t h  and h y d r o p h o b i c p r o p e r t i e s a t l o w i o n i c s t r e n g t h  extraneous  electrostatic  (Hjerten,  1 9 7 3 a ) and m a i n l y h y d r o p h o b i c i n t e r a c t i o n s a t h i g h  strength.  An  i d e a l uncharged  g e l with a hydrophobic ligand  1973; ionic should  give hydrophobic i n t e r a c t i o n s at a l l i o n i c strengths (Hjerten eta l . , 1974;  Rosengren  chromatography"  e_t a l . , 1 9 7 5 ) .  However, the term  "hydrophobic  has u n f o r t u n a t e l y been used f o r a l l e x p e r i m e n t a l  p r o c e d u r e s i n w h i c h an a d s o r b e n t w i t h a n o n p o l a r l i g a n d i s employed, i r r e s p e c t i v e of whether  7.  Uncharged  (1974) u s e d h y d r a z i d e s t o p r e p a r e u n c h a r g e d  Whereas  alkyl-substituted  Sepharose,  performed  ionic  the b i n d i n g of these p r o t e i n s to the r e p o r t e d t o b e weak.  on h y d r a z i d e d e r i v a t i v e s o f  (1975  the c o u p l i n g of f u n c t i o n a l groups  Sepharose.  These  workers  i n t e r a c t i o n i s i n v o l v e d when c h r o m a t o g r a p h y  N i s h i k a w a and B a i l o n in  agarose  a - l a c t a l b u m i n and o v a l b u m i n bound s t r o n g l y t o t h e  h y d r a z i d e - s u b s t i t u t e d r e s i n was c o n c l u d e d t h a t no  uncharged.  resins:  J o s t et_ a l _ . derivatives.  these l i g a n d s are charged or  is  Sepharose.  ) employed a c y l - h y d r a z i d e m o i e t i e s t o cyanogen b r o m i d e - a c t i v a t e d  T h e y s u g g e s t e d t h a t r e a g e n t s such as c a p r y l y l h y d r a z i d e  p r o v i d e s o r b e n t s t h a t were e s s e n t i a l l y  f r e e o f c h a r g e a t pH  7.0.  H j e r t e n e_t a l . ( 1 9 7 4 ) p r e s e n t e d a m e t h o d f o r a t t a c h i n g o r a r y l m o i e t i e s w i t h o u t i n t r o d u c i n g charged groups  alkyl  o n t o an a g a r o s e m a t r i x .  24  The an  gel  was  aprotic  ionic  prepared solvent.  groups. of  were  structure  based  is  generally  of  a protein that  of  gels were  on  for  claimed  use  their  agarose w i t h  in  the  believed  represents  free  energy  that a  the p a r t i c u l a r  state  of  this  of  minimum  state  is  the  individual  contacts  involved  in  the  equilibrium conformation.  easily  organic  potential  structural  types  of  etc.  substitution, Rosengren the  the  of  the  relatively  bond,  immediately affect  i t  further  is  types  protein  ambient The  spacer  and  of  of  such  as  It  apparent  that  that  of  be  temperature,  classified  are  the  ionic  as  bond  there of  native  can  salts,  can be  one  sum  the  electrostatic  exclusively  and  residue-solvent  However,  apparent  very  above  species  can  interactions.  chromatography,  and  the  the  substituent,  adsorbent,  temperature amount  energy,  algebraic  and  Perturbants  1973).  "native"  stable.  perturbants  which  hydrophobicity and  of  interaction  et^ a l _ . , 1975) .  length  is  three  hydrophobic  the  only  effect  i t  and  a l l  residue-residue  hydrophobic  perturbants  i n t e r a c t i o n between  strength,  is  (Tanford,  However,  affect  In  and  the  interactions,  potentially  the  by  hydrogen bond,  destabilizers. few  most'proteins  solvents,  of  and  free  the  of  destabilized  in  hydrophobicities.  energies  of  t o be- d e v o i d  separation  free  conformation  epoxides  factors:  It  furthermore  reaction  introduced  proteins  Influential  8.  the  The p r e p a r e d  They  purification  the  through  of  arm m a i n l y  many  namely  the  ligand  determine  the  degree  (Hjerten et  the  factors  a l . ,  bound  the  affect  ionic  of  the  1974; to  the  matrix  hydrophobicity  of  the g e l . alkyl  Tanford  (1973)  assumed t h a t t h e f i r s t  c h a i n a t t a c h e d t o a p o l a r group  give only a small contribution to  the h y d r o p h o b i c i t y o f t h e molecule. p r o p y l agaroses have minor  Salt effect:  I n f a c t , m e t h y l , e t h y l and even  o r no h y d r o p h o b i c  ( H j e r t e n et_ a l . ,  h a v e f o u n d , f o r many s y s t e m s , in free solution differs n e u t r a l s a l t s used. intensify  the adsorptioni n  decreases w i t h lowering i o n i c  1974).  von H i p p e l and S c h l e i c h  and C l  properties.  C o n t r a r y t o i o n exchange chromatography,  hydrophobic i n t e r a c t i o n chromatography strength  two c a r b o n a t o m s i n a n  (1969b) and H a t e f i and H a n s t e i n  t h a t t h e degree  significantly  Basically,  of hydrophobic  Phosphate  interactions  according to the type of the  s a l t i n g - o u t a g e n t s s u c h a s PO^  these k i n d s o f bonds w h i l e B r  a n d SCN  known a s s a l t i n g - i n a g e n t s d i m i n i s h t h e s e i n t e r a c t i o n s 1977).  , SO^  which' a r e  ( P a h l m a n _et a l . ,  and s u l f a t e a n i o n s a r e s t r u c t u r e f o r m i n g i o n s ,  a r e known t o d e c r e a s e t h e s o l u b i l i t y bonds between n o n p o l a r m o l e c u l e s  o f p r o t e i n and s t a b i l i z e  (von H i p p e l and S c h l e i c h ,  Ammonium s u l f a t e a n d p o t a s s i u m p h o s p h a t e  hydrophobic character.  (1973)  to purify  1969a).  b u f f e r s w i t h d e c r e a s i n g concenby  p r o t e i n s on t h e b a s i s o f t h e i r  R e c e n t l y P a h l m a n e_t a l . ( 1 9 7 7 )  d e c y l - S e p h a r o s e s o f t h e s e s e r i e s t o measure t h e degree i n t e r a c t i o n s based  which  hydrophobic  t r a t i o n g r a d i e n t have been a p p l i e d i n h y d r o p h o b i c chromatography Rimerman and H a t f i e l d  (1969)  used p e n t y l - a n d of hydrophobic  o n t h e amount o f t h e p r o t e i n b o u n d t o t h e a d s o r b e n t .  These workers m a i n l y s t u d i e d  the effect of different  s a l t s on t h e a d s o r p t i o n  o f t h r e e p r o t e i n s , n a m e l y o v a l b u m i n , BSA a n d p h y c o e r y t h r i n . T h e y t h a t 3 M sodium bromide  and 3 M sodium  found  t h i o c y a n a t e changed t h e c o n f o r m a t i o n  o f BSA  and  o v a l b u m i n , w h i c h i n m o s t c a s e s was  in protein-adsorbent conformational  interaction.  i n hydrophobic binding  S a l t s indeed and,  p l a y an  s a l t s are  of n e u t r a l s a l t s  capable of a l t e r i n g  polypeptides  conformational  stability,  and  t o be  t h e s t r u c t u r e and  sodium c h l o r i d e  chromatography.  p r o m o t e d by  high The  p r o p e r t i e s of  t o t h e i r e f f e c t s on  role  the  neutral proteins  solubility,  a s s o c i a t i o n - d i s s o c i a t i o n e q u i l i b r i a or  of t r a n s f o r m a t i o n a l r e a c t i o n s . specific  decrease  no  (Rosengren e t a l , , 1975).  i n s o l u t i o n due  a  extremely important  therefore, i n hydrophobic  hydrophobic i n t e r a c t i o n i s considered  concentrations  and  These w o r k e r s r e p o r t e d  change i n p r o t e i n i n the p r e s e n c e o f 3 M  or 1 M sodium s u l f a t e .  The  a c c o m p a n i e d by  I t i s c l e a r t h a t the v a r i o u s  p r e d i c t a b l e e f f e c t s on  m a c r o m o l e c u l a r s t r u c t u r e s and  the  stability  complexes.  b e e n s t u d i e d e x t e n s i v e l y by v o n  Hippel  rates  ions  have  of a wide v a r i e t y of  T h e i r e f f e c t s on p r o t e i n s  and  Schleich  (1969b).  When  have the  e f f e c t s of d i f f e r e n t s a l t s i n promoting hydrophobic i n t e r a c t i o n or a f f e c t i n g water s t r u c t u r e are comparing the c a n be ionic  made, f o r e x a m p l e , a t c o n s t a n t strength or constant  conformation  and  polyvalent  salt  space charge  Comparison  concentration,  constant  density.  affect fluorescence  p r o p e r t i e s of p r o t e i n s .  s o d i u m c h l o r i d e s h a v e b e e n shown t o be  concentrated  ions.  of monovalent c a t i o n s have been found to  d e s t a b i l i z a t i o n of macromolecules the  compared, the problem a r i s e s of  e f f e c t s o f m o n o v a l e n t and  Chlorides  and  t o be  responsible  (von H i p p e l  and  salt  s o l u t i o n s , changes i n the  could a l s o take place  ( S t e i n b e r g et_ a l . , 1 9 6 0 ) .  change Potassium  f o r causing  S c h l e i c h , 1969b).  s i z e of the At  low  salt  the In  protein concentrations,  electrostatic these  interactions  i n t e r a c t i o n s would  addition  of  increases  ammonium  the  into  be  or  for  In  similar  the  free  energy  Aggregation  of  (salting-out).  This  opposite  the  residue-solvent  interaction. binding  of  In  these  involved  in  been  the  The  1975).  nonpolar  (Kauzmann,  to  be  Pahlman  anions  change  and  anions  do  Temperature van  der  enthalpy in  the  effect:  Waals  forces  decrease.  opposite  play  a  by  et the  The  al.  in  the  the  results  urea  which  agents  enhance  compounds suggested  to  be  Sepharose Memoli  1973;  structure  of  water  salting-out  of  nonpolar  out  their  of  favours  protein  Hatfield,  of  proteins  has  conformational  (1977) r e p o r t e d  conformation  surface  residue-residue  salting  changes  now  e f f e c t of  and  in  to  stable  amphiphilic  on  by  solvent  relative  native  effect is  ions  strength  the  salting-out  with  role  to  salting-in  Rimer-man  solutions.  thiocyanate sulfate  proteins  e f f e c t of  accompanied  1959).  while  salting-out  1973;  groups must  from aqueous  shown  The  ionic  balance  r e l a t i v e to  sorbent,  et a l . ,  to  chromatography,  i n t e r a c t i o n of  (Porath  and D o e l l g a s t ,  compounds  to  bindings.  the  derivatives  around  hydrophobic  proteins  diminish  contact  concentrations  some p r e v i o u s l y  interactions.  stabilizes  the  contacts  residue-residue  effect is  salt  additives  converting  which  high  Increasing  residue-residue  interactions,  species  dominant.  absent.  sulfate  s t a b i l i t y of  residue-solvent residues  are  that  proteins  bromide while  structure and  chloride  not.  Some a d s o r p t i v e are  weakened  Hydrophobic  manner,  and  in  forces  by  a rise  bonding, gel  such in  being  as  hydrogen  bonding  temperature because an  chromatography  entropy  effect,  a r e t e n t i o n of  or of  the  behaves the  28 s o l u t e should occur w i t h increase i n temperature.  Hjerten  (1973) s t u d i e d  behaviour of the hydrophobic temperature d i d not d i s t u r b  t h e e f f e c t o f t e m p e r a t u r e on t h e  columns.  He f o u n d t h a t a d e c r e a s e i n  the adsorption of proteins  to the hydrocarbon  c o a t e d Sepharose w h i l e d e s o r p t i o n o f h y d r o p h o b i c p r o t e i n s from t h e epoxy d e r i v a t i v e s o f S e p h a r o s e 4B was a c h i e v e d u p o n a d e c r e a s e i n t e m p e r a t u r e f r o m 25°C t o 0°C ( H j e r t e n , since electrostatic intensify  1973).  elements  This finding  i s not surprising,  are present i n alkyl-Sepharoses which  i n t e r a c t i o n s a t lower temperatures.  Pure hydrophobic  on t h e o t h e r h a n d i s d e s t a b i l i z e d when t h e t e m p e r a t u r e The  e f f e c t o f t e m p e r a t u r e on h y d r o p h o b i c b o n d i n g  t h a t t h e s e k i n d s o f bonds a r e e n t r o p y  9.  Detergent  Another in  bonding  i s decreased.  i s due t o t h e f a c t  effects.  effects:  i n t e r e s t i n g phenomenon w h i c h h a s p r o v e d  t o be u s e f u l  t h e s t u d y o f membrane s t r u c t u r e i s t h e e f f e c t o f d e t e r g e n t s o n p r o t e i n s .  High detergent concentration d i s s o c i a t e s membranes a n d l i p o p r o t e i n s  the l i p i d s  ( E n g e l m a n et_ a l . ,  hydrophobic proteins s p e c i f i c a l l y  1967).  from the p r o t e i n s i n Detergents bind to  and t h e m a j o r d r i v i n g  force f o r this  a s s o c i a t i o n i s hydrophobic i n t e r a c t i o n s .  I n p r o t e i n - d e t e r g e n t systems,  the i n t e r a c t i o n  i s enhanced by i n c r e a s i n g  the hydrophobic chain length of  the detergent.  The s t a n d a r d f r e e e n e r g y  been found Blauer,  to decrease with  1974).  change f o r t h e s e p r o c e s s e s has  t h e a d d i t i o n o f - C ^ - r e s i d u e s (Yonath and  B i n d i n g o f T r i t o n X-100 ( n o n i o n i c d e t e r g e n t ) t o h y d r o p h o b i c  p r o t e i n s t a k e s p l a c e i n i t s monomeric form w h i l e t h e b u l k o f d e t e r g e n t remains  as m i c e l l e s .  H e l e n i u s and Simons  (1972) s t u d i e d , w i t h t h e a i d  of r a d i o a c t i v e d e t e r g e n t , i n t e r a c t i o n s of p r o t e i n s w i t h and d e s o x y c h o l a t e . detergent, while  Lipophilic  proteins  was  10.  no  These workers c o n c l u d e d t h a t  bonding  involved.  Affinity  partition:  Albertsson separation  (1971) p r e s e n t e d a p a r t i t i o n method f o r t h e  and p u r i f i c a t i o n  for  separation  the  two-phase system.  of macromolecules  i s the s e l e c t i v e  distribution  the p a r t i t i o n  concentration  and p r o t e i n s .  The  coefficient,  K.  the p a r t i t i o n  Ideally,  takes place  i s characterized  K i s independent  of  and a l s o i n d e p e n d e n t o f t h e v o l u m e r a t i o o f t h e p h a s e s .  i s m a i n l y a f u n c t i o n o f t h e p r o p e r t i e s o f t h e two p h a s e s ,  partitioned  s u b s t a n c e and  Distribution  the  temperature.  of the macromolecules  and p a r t i c l e s  is  i n f l u e n c e d by f a c t o r s s u c h as m o l e c u l a r w e i g h t o f t h e p o l y m e r s , tration  of the polymers, i o n i c  charge of the p a r t i c l e s ,  c o m p o s i t i o n of the phase  affinity  s u b s t a n c e s d i s s o l v e i n the phase The  partition  system they p a r t i t i o n  coefficient  a n d p r o b a b l y on t h e k i n d  o f p r o t e i n added.  d e p e n d s on s i z e ,  of groups exposed  concen-  system, net  o f t h e compounds t o t h e two  p h a s e v o l u m e r a t i o and t h e q u a n t i t y  manner.  basis  o f s u b s t a n c e s between  For s o l u b l e substances, d i s t r i b u t i o n  m a i n l y b e t w e e n t h e two b u l k p h a s e s , a n d  It  or  p r i m a r i l y h y d r o p h o b i c and h y d r o p h o b i c r e g i o n s on t h e p r o t e i n  m o l e c u l e s were  by  X-100  bound l a r g e amounts o f t h e  t h e h y d r o p h i l i c p r o t e i n s bound l i t t l e  d e s o x y c h o l a t e o r T r i t o n X-100.  Triton  phases,  When s o l u b l e in a  reproducible  charge, conformation  to the phases.  The  size  effect  i s such  that small molecules  the phases w h i l e l a r g e molecules  tend  concentrate  w h i c h d e p e n d s on f a c t o r s o t h e r t h a n  Shanbhag and hydrophobic Albertsson an and  Axelsson  to p a r t i t i o n evenly i n one  of the  phases,  size.  (1975) e s t i m a t e d  the extent  i n t e r a c t i o n s u s i n g t h e m o d i f i e d p a r t i t i o n method (1971).  The  m e t h o d was  between  b a s e d on p a r t i t i o n  of of  of p r o t e i n s i n  aqueous two-phase s y s t e m c o n t a i n i n g d e x t r a n , p o l y e t h y l e n e polyethylene glycol  palmitate.  The  p a r t i t i o n was  c o n d i t i o n s where c o n t r i b u t i o n s from e l e c t r o s t a t i c eliminated.  With  this  the degree of t h e i r workers suggested  technique  measured  interactions  could rank p r o t e i n s based  i n t e r a c t i o n s w i t h the p a l m i t a t e group.  t h a t the a f f i n i t y of p r o t e i n s toward  ester of polyethylene g l y c o l these p r o t e i n s .  they  glycol  can determine  under were on  These  the p a l m i t a t e  the h y d r o p h o b i c i t i e s of  31 MATERIALS AND METHODS  Materials: Albumin Bovine No.A-7511 e s s e n t i a l l y fatty acid free, cytochrome C No. C-4381 from Candida krusei-type VII, conalbumin No. C-0755 type I from chicken egg white,a -chyraotrypsin No. C-4129 from bovine pancreas type I I - s a l t free, y-globulin BG-II-bovine Cohn f r a c t i o n II approximately 9 9 % Y , B -lactoglobulin No. L-6879 from milk (contains B-lactoglobulins A and B and approximately 2% NaCl), Trypsin, pancreatic type II crude, were purchased from Sigma Chemical Company, St. Louis, Mo.  Ovalbumin 17268, l o t # 24427-A was from ICN  Pharmaceuticals Inc., Cleveland, Ohio.  Lysozyme, s a l t free 11.800,  was purchased from Worthington Biochemical Corporation, Freehold, N.J. Myoglobin 47592 (horse s k e l e t a l muscle), B grade 98% pure, were obtained from Calbiochem, Sah Diego, C a l i f .  Cyanogen bromide and 4-phenyl-  butylamine (PBA) were purchased from A l d r i c h Chemical Company.Inc., Milwaukee, Wis..  Ethylene g l y c o l was "Baker analysed" from J.T. Baker  Chemical Co., P h i l l i p s b u r g , N.J.  Sephadex G-150, Sepharose 4B and  3 Dextran T70 (M^ = 70 x 10 ) were purchased from Pharmacia Fine Chemicals, Sweden.  Polyethylene g l y c o l was obtained from Applied Science  Laboratories Inc., State College, Penna.  Palmitoyl chloride, boron  t r i f l u o r i d e ethylether, hexylalcohol and the n-alkylamines were from Eastman Organic Chemicals, Rochester, N.Y. Coleman and B e l l , Norwood, Ohio.  Octanol was from Matheson,  Epichlorohydrin, dioxane, corn o i l ,  ethylene diamine and o l e i c acid were from Fisher S c i e n t i f i c Company, F a i r Lawn, N.J. Seattle, WA  Butyl.alcohol was from American S c i e n t i f i c & Chemicals,  98199.  Biobeads SM-2  (20-50 mesh) was purchased from  Bio-rad  Laboratories,  was o b t a i n e d  Richmond,  Calif.  f r o m Pope S c i e n t i f i c  Triethylamine  P o l y v i n y l p y r r o l i d o n e K-30 No. 203  I n c . , Menomonee F a l l s , W i s .  AC-9421 was p u r c h a s e d f r o m A n a c h e m i a  C h e m i c a l s L t d . , Mo.  Methods:  1.  A c t i v a t i o n o f S e p h a r o s e 4B w i t h  v e n t i l a t e d h o o d 5 0 g o f S e p h a r o s e 4B was w a t e r and t o t h i s m i x t u r e 5 g o f f i n e l y added a l l a t once. pH a t 1 1 .  When t h e r e  was no f u r t h e r c h a n g e  m i x t u r e was q u i c k l y t r a n s f e r r e d washed w i t h  bicarbonate  i n pH ( p r o t o n  t o a Buchner f u n n e l  t o S e p h a r o s e 4B:  ( p H 9.5) c o n t a i n i n g  concentrated hydrochloric  release),  and e x t e n s i v e l y  (pH 9.5) u n d e r  suction  10 m l a m i n e was a d j u s t e d  a c i d and added  t o pH 10 w i t h  t o the g e l i n t h e Buchner a glass  rod, transferred to a  a m a g n e t i c s t i r r i n g b a r and g e n t l y  stirred  a t 4°C f o r  T h e g e l was t h e n t h o r o u g h l y w a s h e d w i t h b u f f e r b e f o r e  During  the procedure f o r coupling  t h e pH t o 1 0 . 0 .  This  packing.  octylamine to the activated  a p r e c i p i t a t e formed a f t e r t h e a d d i t i o n o f h y d r o c h l o r i c  adjust  of i c e  F i f t y m l o f c o l d 0.1 M s o d i u m  The g e l was i m m e d i a t e l y m i x e d w i t h  beaker containing  gel,  c y a n o g e n b r o m i d e was  1970).  C o u p l i n g amines  20 h r s .  50 m l  t o t h e s u s p e n s i o n and t h e r e a c t i o n  0.1 M s o d i u m b i c a r b o n a t e b u f f e r  (Cuatrecasas,  funnel.  divided  stirred with  S i x N s o d i u m h y d r o x i d e was u s e d t o m a i n t a i n t h e  amount o f i c e was r a p i d l y a d d e d  2.  gently  In a well  T e m p e r a t u r e was m a i n t a i n e d a t 20°C b y a d d i n g p i e c e s  as needed. a large  cyanogen bromide:  acid to  p r e c i p i t a t e was f o r m e d e v e n i n t h e p r e s e n c e  of d i o x a n e o r d i m e t h y l formamide, w h i c h were employed  t o keep  t h e amine  in solution.  When f o r m i c a c i d was  the c o u p l i n g r e a c t i o n took p l a c e  3.  M  successfully.  Coupling of 4-phenylbutylamine  S e p h a r o s e 4B was 0.1  used i n s t e a d of h y d r o c h l o r i c a c i d  immediately  sodium b i c a r b o n a t e  (50:50),  and  6 ml  The  f o l l o w e d by  0.1  (50:50).  prepared  a c t i v a t e d S e p h a r o s e 4B g e l was  W a s h i n g was  A suspension  ml  1.5  s o a p , 50 m l  water  t h e n 0.05  M  oleic  (2).  to  the  The s u b s t i t u t e d  1973) .  acid  i n 48 m l w a t e r  solution while  was  stirring  o f a m i n o e t h y l a m i n o - S e p h a r o s e 4B  and  g 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 w e r e a d d e d and  mixture by  this  dimethyl-  c o u p l i n g of ethylenediamine  s a p o n i f i e d a t pH w i t h 6 N s o d i u m h y d r o x i d e To  hours  Aminoethylamino-  according to procedure  o f 28.5  of  continued with cold  to Sepharose:  through  V/V  with  f o r 20  and  s t i r r e d a t 37°C f o r 3 d a y s .  a c e t y l a t i o n w i t h a c e t i c anhydride  g e l was  5. carried  then  Unreacted a t pH  7,  amino g r o u p s were 0°C,  t h o r o u g h l y washed w i t h w a t e r b e f o r e  f o r one  i n a column of  150  mm  l e n g t h and  hour.  the  blocked The  packing.  C h r o m a t o g r a p h y on a l k y l a m i n o - S e p h a r o s e s : out  of  8.0).  a g e d f o r 5 d a y s ( P e t e r s e_t a l _ . ,  vigorously.  stirred  M s o d i u m b i c a r b o n a t e b u f f e r (pH 9.5)  Coupling of o l e i c a c i d  S e p h a r o s e 4B was  a d j u s t e d t o 9.5  t h e n w a s h e d w i t h 100 m l  T r i s - h y d r o c h l o r i c a c i d b u f f e r (pH  4.  Activated  dimethylformamide:water  t h e m i x t u r e was  s u b s t i t u t e d g e l was  f o r m a m i d e : w a t e r V/V  20 m l  A f t e r t h e pH was  concentrated hydrochloric acid, a t 4°C.  to Sepharose:  t r a n s f e r r e d t o a b e a k e r c o n t a i n i n g 10 m l  (pH 9 . 5 ) ,  o f PBA.  (PBA)  10 mm  Chromatography diameter  was  packed w i t h  34 20 m l  alkylamino-Sepharose.  Tris-hydrochloric ethylene  acid,  pH  The  p r o t e i n s were e l u t e d  8.0.  Sodium c h l o r i d e  g l y c o l were s u c c e s s i v e l y  were c o l l e c t e d absorbances  w i t h an  added  I s c o M o d e l 326  were measured  a t 280  nm  w i t h 0.05  ( 0 . 1 M)  to the b u f f e r .  Fraction  and  M  50%  One-ml  Collector.  w i t h a n U n i c a m SP  fractions  The  800  Spectrophotometer.  6.  Synthesis of g l y c i d y l  glycidyl with  e t h e r s were p r e p a r e d by  epichlorhydrin  etherate  as  a catalyst flask,  funnel  a stirring  and  in  bath.  the  temperature  two  hour  The  and  C  C  H. 2  H  -  temperature was  filtered  then f i l t e r e d  -  0  -  and  then l e f t  added  2 ml  of  added  overnight. a t 20-25°C,  dropwise  -  CH„ I  dropwise  While  within  a two  1 Whatman f i l t e r  (CH-) / n  -  55°C  CH  a  o f 50  g  hour paper.  dried  3  over  stirring  a solution  vacuum d i s t i l l e d .  -  to  and m a i n t a i n i n g  the general formula:  CH. i.  borontri-  t h e m i x t u r e warmed  i n a separatory funnel,  a g a i n and  ethyl  separatory  intensively  t h r o u g h No.  washed w i t h water  CH.  added  alcohols  a three neck  g of a l c o h o l ,  the mixture  m i x t u r e was  r e p r e s e n t e d by  To  g e p i c h l o r h y d r i n was  i n 50 m l w a t e r  magnesium s u l f a t e , c a n be  e t h e r ) was  37  octyl-  (BF^)  thermometer, condensor,  stirring  m i x t u r e was  and  of b o r o n t r i f l u o r i d e  containing  m a i n t a i n i n g the  hydroxide The  The  hexyl-  of the r e s p e c t i v e  et. a l . - , 1963) .  with  c o n s t a n t , 46.6  f i l t r a t e was  ethers  bar,  While  period.  constantly  period.  equipped  (1.5% i n d i e t h y l  a water  sodium  (Ulbrich  Butyl-,  reaction  i n the presence  distilling  fluoride  ethers:  The  with glycidyl  7.  Treatment of Sepharose 4B:  Sepharose 4B (50 g)was washed  successively with a series of solvents of decreasing p o l a r i t y to exchange the water i n the gel with dioxane.  The washing procedure  suggested by Hjerten et_ a l . (1974) was performed as follows: 1 - Once with 50 ml water-dioxane  (4:1)  2 - Once with 50 ml water-dioxane  (3:2)  3 - Once with 50 ml water-dioxane  (2:3)  4 - Once with 50 ml water-dioxane  (1:4)  5 - Seven times with 50 ml dioxane.  After exchanging the water i n the Sepharose with dioxane, the  coupling with the g l y c i d y l ether was carried out.  8.  Coupling g l y c i d y l ethers to Sepharose:  The coupling procedure  introduced by Ellingboe et. a l . (1970) and modified by Hjerten et a l . (1974) was used to attach g l y c i d y l ethers to the polysaccharide matrix. F i f t y ml of gel was transferred to a round-bottomed equipped with a s t i r r e r .  reaction vessel  F i f t y ml dioxane and 1 ml of a 48% solution  of b o r o n t r i f l u o r i d e etherate i n diethyl ether were then added.  The  mixture was gently s t i r r e d f o r 5 minutes, then a mixture of one ml g l y c i d y l ether and 5 ml dioxane was added dropwise from a separatory funnel within a 40 minute period while s t i r r i n g constantly at room temperature.  Afterwards, the gel was washed with the same ratios of  water-dioxane mixtures but i n the reverse order and f i n a l l y was washed with 1 l i t e r of water. following formula:  The alkylepoxy derivatives produced have the  0 S e p h ) - OH + CH  'CH - C H  2  - OR  Seph)-0  where R r e p r e s e n t s  the a l k y l  group.  - CH  These  2  OH - CH - C H  g e l s were  - OR  2  called  alkyl-  epoxy-Sepharoses.  9.  Determination o f the d r yweight  Two m l g e l s u s p e n s i o n , w e l l h o m o g e n i z e d a glass  filter  washings The  funnel.  g e l suspensions:  by s t i r r i n g ,  were  The g e l s were d e h y d r a t e d t h r o u g h  w i t h a c e t o n e and f i n a l l y  d r y w e i g h t s were  determined.  10.  column  Hydrophobic  o f Sepharose  dried  Pharmacia  Fine  were used  f o r hydrophobic chromatography.  Sepharoses  were p a c k e d  0.002 M s o d i u m  buffer  G l a s s columns  a t 90°C.  from  16 ± 0.02mm d i a m e t e r , 200mm l e n g t h )  i n t h e column.  phosphate  successive  to constant weight  chromatography:  Chemicals (K16(26),  transferred to  Thirty  They  g of alkylepoxy-  were e q u i l i b r a t e d  (pH 6.9) c o n t a i n i n g  2 M  with  sodium 2  chloride.  P r o t e i n s were e l u t e d  temperature.  The e l u t i o n  280 nm u s i n g  a LKB Bromma  collected. each  application  their  R.C.  profiles  o f 3 ml/hr/cm  were m o n i t o r e d  8300 UVICORD  II.  a t room  continuously at  One m l f r a c t i o n s  T h e g e l was w a s h e d w i t h t h r e e b e d v o l u m e s  were  of buffer before  o f sample.  Hydrophobic as  a t a flow rate  retention  adsorption  of proteins  coefficients.  Ve - V t g dry g e l  This  v a l u e was c a l c u l a t e d  w h e r e Ve = e l u t i o n Vt  t o t h e s e columns  = total  was e x p r e s  from  volume  volume  g d r y g e l = gram d r y g e l p a c k e d  i n t h e column.  11. was  G e l chromatography w i t h d e t e r g e n t s :  p a c k e d w i t h S e p h a d e x G-150.  A column  (2 x 35 cm)  The b u f f e r e m p l o y e d was 0.025 M  s o d i u m p h o s p h a t e , pH 7.2, c o n t a i n i n g 0.05% T r i t o n X - 1 0 0 . s a m p l e s (lOmg) w e r e a p p l i e d t o t h e c o l u m n . was  maintained  collected  throughout the a n a l y s i s .  Protein  A f l o w r a t e o f 2ml/hr/cm  F r a c t i o n s o f 1.5 m l w e r e  i n t u b e s w i t h a n I s c o M o d e l 326 f r a c t i o n c o l l e c t o r .  elution profile monitoring  2  f o rthe protein-detergent  The  c o m p l e x was o b t a i n e d b y  t h e a b s o r b a n c e a t 280 nm w i t h a U n i c a m SP 800 S p e c t r o -  photometer.  12.  Removal o f t h e T r i t o n X-100:  A c o l u m n ( 1 x 8 cm) c o n t a i n i n g  5g o f m o i s t SM-2 B i o b e a d s was e q u i l i b r a t e d w i t h (pH  7.2).  The p r o t e i n - d e t e r g e n t  was  concentrated  pyrrolidone  lOmM p o t a s s i u m p h o s p h a t e  c o m p l e x c o l l e c t e d f r o m S e p h a d e x G-150  by d i a l y s i s a g a i n s t 20% aqueous s o l u t i o n s o f p o l y v i n y l -  (PVP) t h e n a p p l i e d t o t h e B i o b e a d column.  Following  e l u t i o n o f p r o t e i n , t h e b e a d s w e r e w a s h e d w i t h 5 0 % e t h a n o l p l u s 10% ethylene  g l y c o l i n lOmM p o t a s s i u m p h o s p h a t e b u f f e r  (pH 7.2) t o remove  T r i t o n X-100 bound t o t h e b e a d s .  13. was  Q u a n t i t a t i o n o f T r i t o n X-100:  used.  Ammonium c o b a l t o t h i o c y a n a t e was p r e p a r e d  o f ammonium t h i o c y a n a t e water.  ethylene  appropriate  (1973)  b y d i s s o l v i n g 17.8 g  a n d 2.8g o f c o b a l t n i t r a t e h e x a h y d r a t e i n  The r e a g e n t was d i l u t e d  0.5mg/ml s t a n d a r d 10%  The m e t h o d o f G a r e w a l  t o 100ml w i t h d i s t i l l e d w a t e r .  s o l u t i o n o f T r i t o n X-100 i n 5 0 % e t h a n o l  g l y c o l was p r e p a r e d  to construct  amount o f T r i t o n X-100 s t a n d a r d  the standard  A  containing curve.  An  s o l u t i o n was t r a n s f e r r e d t o  a test  t u b e a n d t h e v o l u m e was made t o 1 m l w i t h 5 0 % e t h a n o l .  Concentration of the detergent  r a n g e d f r o m 50 t o 5 0 0 ygm.  The c o n t e n t s  o f t h e t u b e s w e r e m i x e d , t h e n 0.50 m l o f t h e ammonium c o b a l t o t h i o c y a n a t e reagent  was a d d e d a n d t h e m i x t u r e was a l l o w e d  a t room t e m p e r a t u r e .  to stand  forfive  minutes  1.5 m l o f e t h y l e n e d i c h l o r i d e was t h e n a d d e d a n d  the contents were mixed f o r 2 min w i t h a v o r t e x shaker. w e r e t h e n c e n t r i f u g e d a t 1500 x G a n d t h e b o t t o m p h a s e  The s a m p l e s (ethylene  d i c h l o r i d e ) was r e m o v e d w i t h a P a s t e u r p i p e t t e f o r t h e s p e c t r o p h o t o m e t r i c analysis.  The  d i f f e r e n c e i n a b s o r b a n c e a t 622 nm  w i t h a U n i c a m SP 800  The collected  a n d 687 nm was  Spectrophotometer.  T r i t o n X-100 f r a c t i o n e l u t e d f r o m t h e B i o b e a d  i n a 5 ml v o l u m e t r i c f l a s k .  e t h a n o l a n d 1 m l o f t h i s s o l u t i o n was u s e d .  out  as d e s c r i b e d i n p r e p a r a t i o n o f samples f o r t h e s t a n d a r d  protein  Hydrophobic p a r t i t i o n :  c o l u m n was  I t was made up t o v o l u m e w i t h  50%  14.  recorded  T h e a n a l y s i s was  carried  curve.  To a n a p p r o p r i a t e amount o f t h e  (5-15 m g ) , i n 10 m l c e n t r i f u g e t u b e s  (conical, plain,  Pyrex  b r a n d ) , b u f f e r ( 1 0 0 mM K^SO^ a n d 2 mM K P h o s p h a t e , pH 7.1) was a d d e d t o make up t h e t o t a l w e i g h t  o f 2 0 0 mg.  shaking w i t h the v o r t e x shaker.  The p r o t e i n was s o l u b i l i z e d b y  T h e n 1.6 g o f 4 0 % p o l y e t h y l e n e  o r i t s p a l m i t a t e e s t e r a n d 3.2 g o f 2 0 % d e x t r a n T70 w e r e a d d e d . contents  o f e a c h t u b e were m i x e d f o r 2 m i n u t e s on t h e v o r t e x  A f t e r w a r m i n g a t 40°C i n a w a t e r b a t h  f o r 10 m i n u t e s ,  glycol The  shaker.  the tubes  were  39  c e n t r i f u g e d i n a c l i n i c a l c e n t r i f u g e a t 1500x G f o r 10 m i n u t e s .  An  a l i q u o t of 2g was withdrawn from each phase and d i l u t e d w i t h water for  the p o l y e t h y l e n e g l y c o l p a l m i t a t e phase w h i c h Was  a s o l u t i o n of 0.02%  SDS  i n 0.1 N sodium h y d r o x i d e .  made on a w e i g h t b a s i s .  The  c o n t a i n i n g no p r o t e i n handled  diluted with  A l l d i l u t i o n s were  absorbance of p r o t e i n was  280 nm a g a i n s t a s o l u t i o n of the c o r r e s p o n d i n g  except  measured a t  phase from a system  i n an i d e n t i c a l manner.  D i l u t i o n s from  p o l y e t h y l e n e g l y c o l p a l m i t a t e phases were warmed i n a 90°C water b a t h for  3 minutes b e f o r e the r e a d i n g was  taken.  The p a r t i t i o n c o e f f i c i e n t of p r o t e i n was  determined from  K = CU/CL where CU and CL a r e c o n c e n t r a t i o n s o f the p r o t e i n a t e q u i l i b r i u m i n the upper phase and lower phase r e s p e c t i v e l y . P a r t i t i o n c o e f f i c i e n t of a p r o t e i n i n polyethylene g l y c o l p a l m i t a t e and p o l y e t h y l e n e g l y c o l systems were d e s i g n a t e d as respectively. and was 15.  A l o g K was  A log K = log  - log  used as an i n d e x of h y d r o p h o b i c i t y of the p r o t e i n s . S y n t h e s i s of p o l y e t h y l e n e g l y c o l p a l m i t a t e :  p o l y e t h y l e n e g l y c o l was was  c a l c u l a t e d from  and 1^ ,  Two  hundred g  d i s s o l v e d i n 1200ml t o l u e n e and 200ml o f  d i s t i l l e d out of the s o l u t i o n t o remove t r a c e s of m o i s t u r e .  toluene Four g  t r i e t h y l a m i n e p u r i f i e d by d i s t i l l a t i o n over p h t h a l i c anhydride was Then, a s o l u t i o n of 5g p a l m i t o y l c h l o r i d e i n 50ml t o l u e n e was dropwise under c o n t i n u o u s  stirring.  15 minutes and then f i l t e r e d c o o l i n g t o 3°C  The m i x t u r e was  over Whatman No.  added.  added  gently refluxed for  1 f i l t e r paper.  Upon  the p o l y e t h y l e n e g l y c o l p a l m i t a t e p r e c i p i t a t e d and  was  c o l l e c t e d by vacuum f i l t r a t i o n .  The p r o d u c t  was  recrystalized  t w i c e f r o m a b s o l u t e e t h a n o l and has t h e f o l l o w i n g f o r m u l a : 0 II CH„ - ( C H J . . - C - 0 - CH  - CH„(-.0 - C H  0  - CHj  Polyethylene  16.  Interfacial  t e n s i o n measurements:  m e a s u r e d a t 25°C w i t h a F i s h e r S u r f a c e instrument according  was c a l i b r a t e d  r i n g was t h o r o u g h l y  washed  an a l c o h o l  A f t e r allowing the mixture to  e q u i l i b r a t e f o r 2 minutes, three consecutive intervals.  The  i m m e r s e d i n t h e 0.2% p r o t e i n s o l u t i o n s a n d  t h e c o r n o i l p h a s e was s l o w l y a d d e d .  2 minute  were  recommendation.  a n a l y s i s , the tensiometer  The r i n g was t h e n  tensions  Tensiomat Model 21.  w i t h benzene and then a c e t o n e f o l l o w e d by f l a m i n g over burner.  glycol  a t 49 d y n e s / c m w i t h t h e a i d o f 600mg w e i g h t  to the manufacturer's  Before  Interfacial  - OH  r e a d i n g s were  taken a t  RESULTS AND D I S C U S S I O N  1.  Chromatography on Sepharoses s u b s t i t u t e d w i t h  alkylamines:  Chromatography on t h e s e g e l s c l a s s i f i e d p r o t e i n s i n t o t h r e e d i f f e r e n t groups: a. and  P r o t e i n s w h i c h h a d no a f f i n i t y  towards t h e bed m a t e r i a l  w e r e e l u t e d w i t h i n t h e t o t a l b e d v o l u m e when e l u t e d w i t h 0.05 M  T r i s - H C l b u f f e r , pH 8.0. b. probably  P r o t e i n s w h i c h were bound t o t h e g e l m a t e r i a l , most  b y means o f e l e c t r o s t a t i c  protonated  matrix.  T h e s e m o l e c u l e s w e r e r e l e a s e d when t h e i o n i c  s t r e n g t h o f t h e e l u e n t was c.  a t t r a c t i o n s between p r o t e i n and  increased.  P r o t e i n s w h i c h were t i g h t l y bound t o t h e l o n g  h y d r o c a r b o n l i g a n d and were n o t d e s o r b e d even a t h i g h These m o l e c u l e s p r o b a b l y h y d r o p h o b i c and i o n i c until polarity the b u f f e r .  interacted with  associations.  reducing  strengths.  t h e l i g a n d b y means o f  These p r o t e i n s were n o t r e l e a s e d  agents such as e t h y l e n e  The e l u t i o n p a t t e r n s  ionic  chain of  g l y c o l were i n c l u d e d i n  o f some p r o t e i n s o n  alkylamino-  S e p h a r o s e s o f i n c r e a s i n g c h a i n l e n g t h a r e shown i n F i g . 1. suggests that lysozyme d i d n o t i n t e r a c t w i t h  It  the hydrophobic l i g a n d .  B o v i n e serum a l b u m i n , o v a l b u m i n and g - l a c t o g l o b u l i n were a d s o r b e d on the  g e lmaterial apparently  w e l l as by h y d r o p h o b i c  It  b y means o f e l e c t r o s t a t i c  i n t e r a c t i o n s as  bondings.  i s obvious that the b i n d i n g p r o p e r t i e s of these g e l  m a t e r i a l s r e q u i r e t h a t t h e p r o t e i n and t h e adsorbent a r e o p p o s i t e l y  42  F i g u r e 1B e h a v i o u r o f p r o t e i n s o n 1 5 x 1 cm c o l u m n s of b u t y l - and o c t y l a m i n o - S e p h a r o s e 4B. B u f f e r , 0.05 M T r i s h y d r o c h l o r i c a c i d (pH 8 . 0 ) . Arrows i n d i c a t e a d d i t i o n of 0.1 M s o d i u m c h l o r i d e a n d 50% e t h y l e n e g l y c o l (EG) in the buffer.  Absorbance B-lacto-  Y-globulin  (280  nm)  Ovalbumin  Lysozyme  Bovine  globulin  Serum  Albumin  O  't—•  O  t—'  °  i—'  o  charged. of  The  proteins  that  predominant  by  salt  groups  in  at  (butyl  the b u f f e r  least and  the  case  of  hydrophobic  gels,  by  c o n t r i b u t i o n of  serum  a l b u m i n was  columns.  This  column by  simply  A  protein  demonstrated  are  Positive and  attractive  or  butions  the  group  on  to  the  positively  made  was  c l e a r by with  readily  in  the  reversed  the  the  binding  observation  smaller by  hydro-  inclusion  of  the  interactions  p r o t e i n was ionic  in  the  responsible not  to  the  case  released ionic  desorbed,  strength.  bonds  of  from  e l u t e d by  On  the  strength  was  of  ionic  interactions  minor  other of  highly  hand,  a  bovine  hydrophobic  octylamino-Sepharose thus  suggesting  interaction.  decreasing  ionic  be  from even  the  highly  this  in  to  adsorption  strength, for  seemed  the  needed  Furthermore,  polarity  for  that  alone.  desorption  of  occur.  alkylamino-Sepharoses  activation  not  increasing bonds  is  adsorbents  hydrophobic  Interference  gels.  This  increasing  increase  to  the  hydrophobic  a l b u m i n was  concomitant  the  of  p r o t e i n was  the hydrophobic serum  electrostatic forces  1).  ovalbumin.  major  bovine  to  hexyl),  (Fig.  Involvement in  of  alkylamino- Sepharoses  the b i n d i n g ,  phobic  role  charges  relates are  mono a m i n e repulsive  matrix  charged  the method  introduced  forces the  can  exert  p r o t e i n due  to  of  the preparation of  Hence, great  hydrophobic  can prevent  chromatography  through  substitution.  formation of  gel  to  in  cyanogen the  hydrophobic  bond  these  bromide  electrostatic  positive  bonds.  on  The  or  negative  protonated  formation  electrostatic repulsions.  of  This  contri amino  45 repulsion leaving a l l  provides  large  them u n a b l e  eluted at  the  to  distances  interact.  first  stage  Chromatography (Fig.  1)  suggests  interfere was  with  attracted  differences proteins these  the to  in  like  gels,  that  both  gel  their  forces.  Increasing  second  stage  elution  globulin. was  These  added  addition more  to of  of  hydrophobic  chain eluted after  simply  including  Tris-HCl to and  this  i t .  50%  type  of  forces.  bovine  Under  Therefore,  No  of  However,  they  were  ionic  albumin,  ionic  appears to  reinforcing amount  a  0.1  large  effect of  the  the  of  the  the  case  of  bonds and  is  contribution a longer  alkyl  p r o t e i n was  Elution  took  chloride  strong  degree,  glycol after  charged  the  M sodium  that  the  3-lacto-  eluted  Sepharose has  strength.  at  hydrophobic  interactions,  and  and  In  to  electro-  ethylene  negatively  circumstances,  ovalbumin  by  ovalbumin 50%  is  to  the buffer  when  glycol i t  of  aided  repulsions  charged  and  released  protein  depends,  detectable  to be  a  hydrophobic  not  place in  binding  on  p r o t e i n was  4B  ovalbumin  negatively  strength  serum  and  due  M concentration.  these the  were  octylamino-Sepharoses  probably  of  seem  i n t e n s i f i e d when  ethylene  mutually  0.1  through  adsorbent  and  is  binding  not  this  thus  proteins  rejected while  desorption  to  increasing  buffer.  presumably  static  to by  like  is  was  This  ionic  caused  chloride  bonds  attached  the  buffer.  partly  charged  albumin, 3-lactoglobulin  Although  bound  In  original  dominant.  presumably  Lysozyme  were  proteins,  groups,  electrostatic attractions  proteins  sodium  apolar  become  the  positively  hexyl-  interactions  static  nonpolar  elution.  material.  serum  hydrophobic  of  of  charges.  bovine  The  on b u t y l - ,  binding.  the  between  only  the of  protein  cooperative and  electro-  released  from  its  complex w i t h  extensively  with  Y-globulin required of  the  or  bovine  increase  polarity  reducing  Elution  incapable proteins protein are  the of  that  the  possible  interactions  of  the  to  type gel  of  could  of also  washed  on  site  ligand.  with  the  size  of  octylamino-Sepharose  AB  well  that  as  the  that  presence  these  a complicated  and  occurred proteins  hydrophobicity  aggregation  interactions, of  on  mean  bonds; In  ionic  even  hydrophobic  the nature  i t  of  system  like  states  of  is  quite  pockets  may  upon are  these the  which  conceivable depend  of  the  ions  interactions  are  reinforced  cooperative be  of  With  when  present.  upon It  is  by  be  assumed  protein with this  similar  buffer.  gel, to  the  Sepharose to  the  occur phenyl  results  other  proteins  hydrophobic  chromatography  substituted  may  hydrophobic  Tris-HCl  e l e c t r o s t a t i c and  observed  (PBA)  PBA-Sepharose  interaction is  retained  complex  as  the  washed  forces.  hydrophobic  substituted  does not  hydrophobic  4-phenylbutylamine  proteins  the  of  3-lactoblobulin  conformation  and  that  ionic  and  hydrophobic  the  Formation  with  strength  a v a i l a b i l i t y and  quite  Disruption  strength  some e f f e c t .  pH  i n t e r a c t e d bed was  with  ionic  ovalbumin  dependent  strength,  buffer.  albumin  the  the  agents.  of  have  ionic  stronger  in  ionic  molecule,  strongly  serum  forming  must  when  the Tris-HCl  an  increasing  adsorbent  but  was  4B.  through group  suggested  performed  Binding  interaction on  the  that  alkylamino-Sepharoses. did  not  release  of  them  this This when  47  1.0  h  Effluent Figure (a) Tris M  2-  and  Behaviour of bovine H  chloride  and  50%  Arrows  (ml) albumin  4-phenylbutylamino-Sepharose  hydrochloride(P 8.0).  sodium  volume serum  4B  on  Sepharose  (b). Buffer,  indicate  ethylene glycol  addition  i n the  0.05 of  buffer.  4B M 0.1  48  Fig.  2 shows t h e e l u t i o n p a t t e r n  P B A - S e p h a r o s e 4B c o m p a r e d t o u n s u b s t i t u t e d i n t e r a c t i o n o f PBA w i t h glycol  to release  bound s t r o n g l y ovalbumin the  to this  t o 0.1 M.  bound under  sodium  often  caused  ( F i g . 3 ) , the  o f d i f f e r e n t c o m p o s i t i o n i n t o two a f t e r increasing the  c h l o r i d e t o 0.1 M, w h e r e a s y - g l o b u l i n  ethylene g l y c o l to the buffer.  of p r o t e i n s .  while  When a m i x t u r e o f 3 - l a c t o g l o b u l i n  t h i s c o n d i t i o n and desorbed o n l y  found u n s u i t a b l e  a n d BSA w e r e  after the ionic strength of  3 - l a c t o g l o b u l i n was r e l e a s e d  with  strong  the presence o f ethylene  t o a PBA-Sepharose column  were e l u t e d by b u f f e r s  strength  The  g e l m a i n l y by hydrophobic i n t e r a c t i o n s  and 3 - l a c t o g l o b u l i n were r e l e a s e d  d i s t i n c t i v e peaks.  4B.  y-globulin  the hydrophobic p r o t e i n .  a n d y - g l o b u l i n was a p p l i e d  ionic  Sepharose  serum a l b u m i n r e q u i r e d  b u f f e r was i n c r e a s e d  proteins  o f b o v i n e serum a l b u m i n on  upon t h e a d d i t i o n o f 50%  H o w e v e r , PBA s u b s t i t u t e d S e p h a r o s e  f o r the determination  Inhomogeneity  remained  of the effective  of thebinding  inhomogeneous a d s o r p t i o n  sites  hydrophobicity  i n alkylamino-Sepharoses  and d e s o r p t i o n  of proteins;  Thus p r o t e i n m o l e c u l e s bound t o b o t h e l e c t r o s t a t i c and h y d r o p h o b i c W h i l e t h e m a i n p o r t i o n o f t h e p r o t e i n was d e s o r b e d strength, sites  some m o l e c u l e s r e m a i n e d  on t h e g e l .  bound, a p p a r e n t l y  These m o l e c u l e s were r e l e a s e d  at elevated  of  thebinding  hydrophobic  ionic  l a t e r when t h e  as t h e f r a c t i o n a l e l u t i o n o f t h e p r o t e i n s  main f a c t o r r e s p o n s i b l e  This  effect  ( F i g . 1 ) . The  f o r t h i s b e h a v i o u r seems t o b e  inhomogeneity  s i t e s on t h e adsorbent which had b o t h i o n i c and  s i t e s a v a i l a b l e f o r the protein molecule.  amount o f p r o t e i n  i sapplied  t o t h e column,  sites.  to the hydrophobic  h y d r o p h o b i c bonds were d e s t a b i l i z e d by e t h y l e n e g l y c o l . appeared  was  When a l a r g e  some o f t h e p r o t e i n  49  1.0 e c §0.8 U  c  XI  f-l in  < 0.4  ^NaCl  Ethylene Y  glycol  0.2  40  60  80 Effluent  100  120  140  160  volume(ml)  F i g u r e 3- h l u t i o n o f a m i x t u r e o f - l a c t o g l o b u l i n and y-globulin from phenylbutylamino_Scpharose 4B b y s u b s e q u e n t a d d i t i o n o f 0 1 M s o d i u m c h l o r i d e and 50% e t h y l e n e g l y c o l t o b u f f e r Buffer 0.05 M T r i s h y d r o c h l o r i d e ( P H 8 . 0 ) . ' 3  180  molecules the  bind  ionic  is  binding  causes  between  site  degree  of  Reducing on  the  groups  is  be  through  and  substances hydrophobic  upon  to be  in  minimizes  hydrophobic  hydrophobic  interactions,  Chromatography  phobic  g r o u p w h i c h was  with  o l e i c groups  in  system  such  was as  an  hydrophobic  In  protein  the  applied  that  the  of  to  possibility  to be  adsorption  emulsion  in  hydrocarbon  similar  In  of  that  be  a c i d was  protein-lipid and  similar  construct  be and  contribution  Interaction  4B.  oleic  chains  Oleic  protein  ideal  the bed  the  of  these  for  should  between  increase  to  an  favorable  the  Sepharose  molecule  on p r e p a r a t i o n  e l e c t r o s t a t i c a t t r a c t i o n s must  coupled  binding.  distance  Use  formation.  to  in  protein  bond  order  binding  protein  the  of  conditions  association  affinity  most  predominate  on o l e i c - S e p h a r o s e 4B:  thought  more  the  section  the  certain  bindings.  the  of  decreased,  of  size  to  the  lower  is  of  the  of  i f  of  ratio  to  than  provides  analysed.  2.  sites  the p o i n t  i n t e r a c t i o n chromatography,  dependent  and  amount  kinds  discussed  basically  interactions  to  larger  w i l l  hydrophobic  the  adsorbent  as  interact mainly  other  substitution  Sepharoses,  first  bind  proximity  contain  l i m i t e d number  arm,  weak  their  the  eliminate these  of  with  to  some m o l e c u l e s  should  protein/adsorbent  to  kinds  a  several  way  epoxy  of  the  type  alkyl  the  occupied  As  substituted  entirely  are  s p e c i f i c due  the  uncharged  which take  and  a v a i l a b i l i t y and  surface  would  of  the  gel  sites.  another  on  sites  The  binding  one  Lowering  to  sites  they  become  favorable  is  depends  interaction.  would  hydrophobic  residues.  present,  for  the  sites  interacting strong  to  minimized.  another of  protein  interactions  fatty  the major  hyd  acids  part  of  the  lipid  structure.  S e p h a r o s e was  However,  chromatography  unsuccessful  as  effective  hydrophobicity  of  i n t e r a c t i o n s between  strong  Bovine  serum  acted with ethylene  instead this  glycol  in  amines  0.1 M N a C l  to  substitution  and because prepared strong  hydrophobicity  not  must  series  washings were  latter  water  and by  up  100% d i o x a n e .  gel,  this  adding  Sepharose  r e a c t i o n was  cyanogen  of  ionic  in  the vain  due of  to  the  the  Fifty  to  the  elute  Coupling  neutral  coupling  longer  kind  carbon  interpercent  protein.  alcohols  adsorbents.  for  This  proteins.  ovalbumin  a c t i v a t i o n was  sites.  occurrence  applied  bromide the  the  column.  Sepharoses:  provided  introduced  be  alcohols.  4B, the  to  used  in  coupled  In  order  should  be  c a r r i e d out  increasing  Sepharose  from  to  bind  swollen for  in  In  omitted  reaction, of  gel  chains  After  this  4B i n  a nonpolar  100% d i o x a n e  in  the  were  the  exhibited were  of  back  to  of  washings  pure  water.  ether  starting to  from  to  solvents.  dioxane  environment.  glycidyl  synthesized  organic  adaptation process,  series  form of  a glycidyl  Sepharose by  concentrations  terminated, another  time  to  h e x y l - and o c t y l - g l y c i d y l e t h e r s  respective  coupled  were  e s p e c i a l l y when  Sepharose  to  was  a n d many  remained  substituted  Sepharose.  ethers.Butyl-,  of  oleic  determination of  This  gel  gel matrix  devoid  Alcohols  their  the  alkylepoxy-  procedure  were  to  on  the  amine was  gels  introduced  proteins.  t h e bed m a t e r i a l and  of  for  the  a l b u m i n , y - g l o b u l i n , 3 - l a c t o g l o b u l i n and  Chromatography  3.  of  a method  on  A  from  the  pure  solvent  glycidyl  ethers  When  the  coupling  were  applied  to  were  the  The prepared gels were washed extensively with water to remove any unbound residues.  The gels were packed i n 30 ml columns and  were equilibrated with 0.002 M sodium phosphate buffer (pH 6.9).  Hydrophobic chromatography  on alkylepoxy-Sepharoses provided  a technique to c l a s s i f y proteins based on their a f f i n i t y towards the hydrophobic g e l material.  Considering that the g e l contains only  hydrophobic s i t e s , the interaction must be apolar i n nature.  Those  molecules without any a f f i n i t y for the hydrophobic ligand would pass through the column, while those molecules having a binding a f f i n i t y are retarded.  The degree of retardation would depend on the binding constant  of the macromolecule.  A molecule with a weak binding a f f i n i t y might be  retarded enough to prevent i t s emergence at the void volume when the column i s eluted with the buffer.  A molecule with a s l i g h t l y greater  binding a f f i n i t y may require several void volumes before emerging from the  column, while a molecule with a very high binding a f f i n i t y may never  be eluted from the column. the  In the case of molecules t i g h t l y bound to  g e l , i t i s necessary to change the eluent composition to promote  desorption of those proteins.  The hydrophobic residues or side chains which are available on the surface of the protein molecule are able to form hydrophobic bonds with the carbon chain present on the Sepharose g e l . The nonpolar amino acids which are buried inside the protein molecule are unable to p a r t i c i p a t e i n the interactions. the  This relationship seems to be similar to  function of proteins which interact mildly during emulsification.  In a l l chromatography procedures there  i s a risk  s u b s t a n c e s t o be c h r o m a t o g r a p h e d u n d e r g o d e n a t u r a t i o n . exists  i n hydrophobic i n t e r a c t i o n  that the  This  risk  also  chromatography, but i s r e l a t i v e l y  s m a l l i f the bed m a t e r i a l i s chosen to give o n l y moderately i n t e r a c t i o n s with hydrophobic p r o t e i n s .  The e x t e n t  probably  o f p r o t e i n s i n an o i l / w a t e r  i s comparable w i t h d e n a t u r a t i o n  i n t e r f a c e d u r i n g e m u l s i f i c a t i o n . However, t h e s e are r e v e r s i b l e (Gonzalez  The  difficult  and M a c R i t c h i e ,  of  strong  kinds  denaturation  of denaturation  1970).  t a s k w i t h t h e h y d r o p h o b i c e p o x y g e l was t o  choose t h e l i g a n d w h i c h does n o t b i n d  to protein i r r e v e r s i b l y .  m o s t s u i t a b l e g e l i s t h e one w h i c h o n l y r e t a r d s t h e h y d r o p h o b i c and  The proteins  e v e n t u a l l y r e l e a s e s them s i m p l y b y f l o w o f t h e o r i g i n a l b u f f e r .  more h y d r o p h o b i c p r o t e i n s w e r e f o u n d  The  t o b e more r e s i s t a n t t o d e s o r p t i o n .  P r e p a r a t i o n of the gels w i t h v a r y i n g carbon chain lengths provided  a  series of adsorbents with d i f f e r e n t h y d r o p h o b i c i t i e s .  C h r o m a t o g r a p h y on binding with proteins.  o c t y l e p o x y - Sepharose r e s u l t e d i n s t r o n g  This very hydrophobic l i g a n d prevented  of p r o t e i n s upon w a s h i n g t h e g e l w i t h t h e b u f f e r .  elution  Most o f t h e samples  t i g h t l y b o u n d t o t h e g e l i n 0.002 M s o d i u m p h o s p h a t e ( P H 6.9 ) c o n t a i n i n g 2 M NaCl.  E l i m i n a t i o n o f sodium c h l o r i d e from t h e b u f f e r d i d n o t cause  d e s o r p t i o n o f t h e bound p r o t e i n f r o m was a c h i e v e d  octylepoxy-Sepharose.  o n l y by i n c l u d i n g e t h y l e n e  Naturally,  Elution  glycol i n the buffer.  such a s t r o n g b i n d i n g i s u n d e s i r a b l e  o c t y l e p o x y - Sepharose i s u n s u i t a b l e f o r d e t e r m i n a t i o n  and a c c o r d i n g l y  of the e f f e c t i v e  54 hydrophobicity.  L e s s h y d r o p h o b i c g e l s w e r e f o u n d more s u i t a b l e f o r  this objective.  However, the o c t y l e p o x y -  success  experiments.  i n other  b i t t e r peptides were r e p o r t e d  t o be  hydrophobic i n nature  c o l l e c t e d from the  It  g e l was  from a c a s e i n h y d r o l y s a t e .  bound to the o c t y l e p o x y was  The  S e p h a r o s e was effective The  which  H a t a ,1972) hydrolysate  column.  i s known t h a t t h e a d s o r p t i o n o f p r o t e i n s on  s u b s t i t u e n t s ( P a h l m a n e_t a l _ . , 1 9 7 7 ) .  p h o b i c i t y needed f o r a p p r o p r i a t e not  i n removing  the d e b i t t e r e d  g e l s i n c r e a s e s w i t h t h e d e g r e e o f s u b s t i t u t i o n and the  with  b i t t e r peptides  ( M a t o b a and  S e p h a r o s e g e l and  used  hydrophobic  hydrophobicity  However, the  critical  r e t a r d a t i o n o f p r o t e i n s on  only a f u n c t i o n of the h y d r o p h o b i c i t y  of  hydro-  the g e l i s  of the adsorbent but  also  d e p e n d s on  the s a l t environment i n which the p r o t e i n i s chromatographed  (Fig.  I t i s shown t h a t t h e r e t e n t i o n v o l u m e o f b o v i n e s e r u m a l b u m i n  4).  i n c r e a s e d when c h r o m a t o g r a p h y was higher  c o n c e n t r a t i o n of s a l t s .  p r o t e i n s as w e l l . b u f f e r has  c a r r i e d out  This  e f f e c t was  S a l t c o n c e n t r a t i o n up  been used w i t h o u t  any  i n the presence of observed f o r  denaturation  e f f e c t on  A d d i t i o n of s a l t s a l s o obviates  of involvement of i o n i c  forces.  S a l t s a r e w e l l known t o d e c r e a s e t h e However, the e f f e c t  any  nonpolar  at high  A p r o t e i n w h i c h p r e c i p i t a t e s at a low  concentration should  l i k e w i s e be  low  Similarly,  strength.  possibility  s o l u b i l i t y of  i s appreciable  the  protein  concentration only.  ionic  other  to 3 M sodium c h l o r i d e i n  ( P a h l m a n e_t a l . ,1977) .  substances i n water.  a  salt  salt  r e t a i n e d on h y d r o p h o b i c s u p p o r t s the p r o t e i n s which p r e c i p i t a t e at  at  a  high  55 salt  concentration  to a f f e c t  should  require r e l a t i v e l y higher  effective  ammonium s a l t s .  i n f l u e n c e d by  the  c o n c e n t r a t i o n of s a l t s . i s not  d r i v i n g f o r c e i s the entropy  the water surrounding  to  The  compared  c h l o r i d e anions  gain a r i s i n g  are  of p r o t e i n s .  underlying  c l e a r l y understood, but  The  be  principle presumably  from s t r u c t u r e changes i n  the i n t e r a c t i n g hydrophobic groups.  change i n the p r o t e i n m o l e c u l e o c c u r s 1 M  more  o f e i t h e r g e l , p r o t e i n , o r b o t h r e a c t a n t s must  of s a l t i n g _ o u t a d s o r p t i o n  mational  S u l f a t e and  s a l t i n g - o u t agents which reduce s o l u b i l i t y  hydrophobicity  up  s o d i u m c h l o r i d e w e r e f o u n d t o be  i n r e t a i n i n g p r o t e i n s on h y d r o p h o b i c a d s o r b e n t s as  with corresponding  the  strengths  their retention.  S o d i u m s u l f a t e and  known as  ionic  No  confor-  i n the presence  of  s o d i u m s u l f a t e o r 3 M s o d i u m c h l o r i d e ( P a h l m a n e_t a l .  1977).  Increasing salt highly  c o n c e n t r a t i o n makes t h e  Also,  s u b s t i t u t e d g e l s s h r i n k because of t h e i r h y d r o p h o b i c i t i e s .  r e a s o n has  been a t t r i b u t e d to the f a c t t h a t the water molecules  i n a c e r t a i n order  around the  numbers o f i o n s s e e k i n g  s o l v a t e d g e l a r e d i s t u r b e d by  hydration.  The  competition  between s o l v a t e d polymer chains  and  and  g e l analogous.  results  ionic  gel shrink.  i n s h r i n k i n g of the  strength, l i p o p h i l i c  T h i s may  be  compared w i t h  increase  in ionic  i o n s e n d s up  the  i n favour  great  W i t h an  of the  increase  the well-known s a l t i n g - o u t e f f e c t . t o 1.0  arranged  f o r water molecules  s o l u t e s are pushed towards the g e l  s t r e n g t h f r o m 0.5  The  ions in  matrix. An  M sodium s u l f a t e i n c r e a s e d  the r e t e n t i o n volume of b o v i n e serum a l b u m i n a p p r e c i a b l y  (Fig. 4).  56  Hexylforming of  and  interactions manipulated  chloride  and by  ionic  found  column.  thus  using  of  to be  Sepharoses  interactions anions.  the  The  ionic  suitable  and  of  the  caused  salt  induced  considerable  decrease  in  flow  rate.  chosen  so  as  not  The by  Our  time  determine  showed  suitable  to  on  their  r e t e n t i o n volume.  or  the  butylBy  r e t e n t i o n volume  or  obtained  ionic in  with  the  required  that  With  becomes  group  strength  salt  so  on  (Fig.  a volume  buffer  greater  5).  the chloride  proteins  the  gel  this,  in  and  the  a  the  column  concentration  was  of  hydrophobicity  the  gels  correct  the  that  of  gel  chain  is  not  proteins  proteins  the  matrix.  hydrophobic  hydrophobic  are  experiment  lengths  presented  based  not  interesting for  eluted  is  s u c h as  r e t e n t i o n volumes  the results.  proteins  time.  such as  ligand  the  determination  Sepharose  substances  choosing  the gel  carbon  shorter  was  in  large  reasonable  column  in  of  of  presence  proteins  select  these  hydrophobic of  a result  a highly  shorter  comparatively  Hydrophilic  to  the  Two M s o d i u m  effective hydrophobicity  Substituting  hexylepoxy  adjusting  were  the  is  the  used  of  of  hydrophobic  the  retention  The  in  After  shrinkage As  capable  the  in  were  denature  in  of  series.  time.  problem  chromatography  experience  impractical.  to  p r e c i p i t a t e or  difficult  hydrophobic  group.  from  to  proteins  proteins  mild  of  repacked  the  strengths.  presence  be  quite  identical conditions  a l l proteins  to  were  intensity  The  had  with  retention of  different  strength,  chromatography was  butylepoxy-  mild hydrophobic  sulfate  proper  and  glucose  Elution  than  the  showed  of  total  no  interaction  hydrophobic volume  of  proteins the  bed.  0.71  20  30  40 50 60 70 80 1 E f f l u e n t volume (ml) F i g u r e 5R e t e n t i o n volume o f b o v i n e serum a l b u m i n (BSA) and g l u c o s e on h e x y l e p o x y - S e p h a r o s e 4B. B u f f e r , 0.002 M s o d i u m p h o s p h a t e c o n t a i n i n g 1 M s o d i u m s u l p h a t e (pH 6 . 9 ) .  The  r e t e n t i o n volumes of the p r o t e i n s were used t o c a l c u l a t e the  retention coefficients.  The  r e s u l t s obtained  from hydrophobic  chromatography of the s e l e c t e d s e r i e s of p r o t e i n s showed t h a t b o v i n e serum albumin had  the g r e a t e s t a b i l i t y to form h y d r o p h o b i c i n t e r a c t i o n s .  I t has been suggested t h a t i n g l o b u l a r p r o t e i n s the of n o n p o l a r / p o l a r  ratio  amino a c i d s i n c r e a s e s w i t h i n c r e a s i n g m o l e c u l a r  w e i g h t s ( F i s h e r , 1964).  T h i s was  shown to be the r e s u l t of an  increase  i n i n t e r n a l volume which i s composed m o s t l y of a p o l a r amino a c i d s . However, a p p a r e n t l y  not a l l n o n p o l a r groups are a b l e to p a r t i c i p a t e i n  hydrophobic i n t e r a c t i o n s .  T h i s i d e a i s s u p p o r t e d by the f a c t t h a t  t h e r e i s no c o r r e l a t i o n between the e f f e c t i v e h y d r o p h o b i c i t y m o l e c u l a r w e i g h t s of the p r o t e i n s  (Fig. 6).  and  T h i s r e s u l t a l s o suggests  t h a t no m o l e c u l a r s i e v i n g e f f e c t has been i n v o l v e d i n r e t e n t i o n of p r o t e i n s on the h y d r o p h o b i c columns.  Thus, the r e t e n t i o n must be  the a  r e s u l t of h y d r o p h o b i c i n t e r a c t i o n s .  I t i s i n t e r e s t i n g to i n v e s t i g a t e whether the e f f e c t i v e hydrophobicity  i s the same parameter as the average  hydrophobicity  c a l c u l a t e d by B i g e l o w (1967) , based on the n o n p o l a r amino a c i d c o n t e n t of p r o t e i n s . two  F i g u r e 7 shows t h a t t h e r e i s no c o r r e l a t i o n between these  expressions  of h y d r o p h o b i c i t y  i n the e x p e r i m e n t a l  proteins.  apparent t h a t h y d r o p h o b i c chromatography does not determine the  It is total  c o n t e n t of the n o n p o l a r amino a c i d s i n the m o l e c u l e , but r a t h e r measures the a b i l i t y of p r o t e i n t o take p a r t i n h y d r o p h o b i c i n t e r a c t i o n s whether p o l a r or n o n p o l a r amino a c i d s are  involved.  ©  60  75,000  © 60,000  45,000  ©  •H CO  * 3  30,000  O  0  15,000  0  2  4  ©  6 8 10 12 14 16 18 R e t e n t i o n c o e f f i c i e n t on HS4B F i g u r e 6C o r r e l a t i o n between h y d r o p h o b i c i t y ( r e t e n t i o n c o e f f i c i e n t ) d e t e r m i n e d on a h e x y l e p o x y - S e p h a r o s e 4B c o l u m n and m o l e c u l a r w e i g h t of proteins, o: ovalbumin, a: a - l a c t a l b u m i n , c: c o n a l b u m i n , m: m y o g l o b u l i n , 8: B - l a c t o g l o b u l i n , 1: l y s o z y m e , b : bovine serum a l b u m i n a n d c*2: a-chymotrypsin.  61  1240  1210  *1180  o  ©  1150  rH  <D bfl •H CQ  1120  ©  4->  o  1090  ©  ©  OU  o  >>1060 bO <u  >  1030.  1000  970  I  0  © 2  4  6 8 10 12 14 16 18 R e t e n t i o n c o e f f i c i e n t on HS4B Figure 7 - C o r r e l a t i o n between e f f e c t i v e h y d r o p h o b i c i t y measured on a h e x y l e p o x y - S e p h a r o s e 4B(HS4B) c o l u m n and t h e a v e r a g e hydrop h o b i c i t y c a l c u l a t e d b y B i g e l o w ( 1 9 6 7 ) . a : a - l a c t a l b u m i n , o.: ovalbumin, c: c o n a l b u m i n , m: m y o g l o b i n , fS: ( 3 - l a c t o g l o b u l i n , b: b o v i n e s e r u m a l b u m i n , 1: lysozyme.  62 Chromatography classified appear is  to  proteins provide  expected  should  be  and F i g .  ligand. with  proteins  Thus h i g h l y  proteins  a  of  t h e more  is  not  less  Triton  X-100,  Triton  X-100  Sephadex  for  highly  a non-ionic to  are  t h e more  columns in  Fig.  apolar  interaction  considering  that  The more  hydrophobic  nonpolar  ligands.  two methods  was  8  more  a more  (Fig.  o b t a i n e d when  calculation.  are  able  to  is  more  The  the  for  two  least  results  determine a wide suitable  10).  range  analysis  butylepoxy-Sepharose  can  be  proteins.  was  A  comparison  and  Due  to  a hydrophobic  i n v o l v e d , more  the  in  the  than  that  was  attempted.  less  of  binding  i n which  molecules the  results  the presence  fact  process  detergent  proteins  of  o t h e r methods  c a r r i e d out  detergent.  hydrophobic  the  from the  the  This  both  greater  the  .97)  chromatography  the protein is  proteins  bind with  =  methods  II).  in  shows  showed  detergents:  G-150  and  towards  surprising  whereas  apolar  with  from hydrophobic on  which  mutual e f f e c t s .  excluded  I  Both  elution patterns  affinity  proteins  b o t h methods  Chromatography  Chromatography  of  hexylepoxy- Sepharose  for  Sepharoses  interactions  comparison  affinity  apolar proteins,  efficiently  of  the  Tables  c o r r e l a t i o n between  were  although  hydrophobicity  to  are  more  proteins  of  regions  of  (see  c o r r e l a t i o n c o e f f i c i e n t (r  that  obtained  results  a greater  This  significant  hydrophobic  4.  hexylepoxy-  their hydrophobicities.  hydrophobic  interactions  high  suggest  have  have  or  interesting effect  moiety.  should  T h e r e was  used  However,  9 r e v e a l e d an  hydrophobic  on  the mechanism  similar.  the hexyl  A very  based  comparable  since  hydrophobic  on b u t y l -  the  were  of apolar  thought  hydrophobic  ones.  c e 3 c x •H e rt •H  00  0.8  3  Ov  r-H Oj  •M O  rt  i—ii a  c  •H  • H  .—1  3  1/)  PH  >, (H  c  4-> O E  •rH  X) o r-H  i— O  CO  O  i  X) O «-H  CO  O  <->  o  rt  c  o  •H  in  i—1 3  £  X O  a  o C  N  O  -—t  > rt  l/l  H°  3  X  X  o  ;>-  0.7 . >  *V \> (  A  7  < V  0.6  T I  <  <  < <  •-0.5  < <  o  CO  •>  <  rsi  >  < <  0.4  <  rt X>  <  '0.3 X <  0.2  V  0.1  /  V  / I_3H.  30 Figure sodium  32  8 - R e t e n t i o n volume phosphate containing  34  36 Effluent  38 volume  40  42  44  46  (ml)  o f p r o t e i n s on b u t y l e p o x y - S e p h a r o s e 2M s o d i u m c h l o r i d e .  4B.  Buffer,  0.002  M  48  c  c  • H  e  3 Xi  i—i  in  c • H  6 3 43  &,  E 3  r-H  rt  O  rt r—(rt 1  X  C  rH  - H  o  .—1  OO  rt o  O  rt  j  ll  Ji  CO I  >-  ,  o  'I  i—i  r-H 1  • s  a  3 43 O  o  +-> 43 O O E X 00 4= O O X  • r-H  > O  3 43  • H  • H  >  j  <  > <  i l  Hi >M  > > > >  11  'ii  >  i i i / / /  / /' /  >  V  34  Figure  9  sodium  phosphate  -  36  W  ; \ \  \ 32  > >  \ \  40 42 Effluent  38  R e t e n t i o n volume containing  of  proteins  2M  sodium  . 44 volume (ml]  46  on h e x y l e p o x y - S e p h a r o s e  chloride.  50  48  4B  52  Buffer,  Table  I-  Retention  chromatography  on  coefficients  of proteins  butylepoxy-Sepharose  by  hydrophobic  column  Ovalbumin  a-lactalbumin  1.33 1.33 1.33 1.33  2.00, 1.33 1.33 2.00  2.66 2.66 2.66 2.00  2.66 2.66 2.66 4.00  4.66 4.00 4.00 5.00  5.33 4.66 4.66 4.66  Mean,  1.33  1.66  2.49  2.99  4.41  4.82  7.49  10.33  Standard deviation, Statistical,, difference  0.00  0.38  0.33  0.67  0.49  0.33  0.64  0.38  Source  of variation  Among, Within,  Determined  of  freedom  Myoglobin  Sum  7 -  the  D u n c a n ' s New  of  squares  4.9  31 by  B-lactoglobulin  y-globulin  Lysozyme  Bovine serum albumin  6.66 8.00 8.00 7.33  10.00 10.66 10.00 10.66  _** Sx  -Mean s q u a r e s  270.2  24  Total, *)  Degrees  a-chymotrypsin  determined  4B  0.20  0.225  265.3 Multiple  Range  f i c a n t l y d i f f e r e n t groups. **) S t a n d a r d e r r o r o f t h e t r e a t m e n t , S x = y W i t h i n  test; mean  a=0,05; square/n.  a , b', c ,  d  and  e  are  signi-  Table IIRetention c o e f f i c i e n t s of proteins c h r o m a t o g r a p h y on h e x y l e p o x y - S e p h a r o s e 4B.  Ovalbumin  a-lactalbumin  Conalbumin  determined by h y d r o p h o b i c  a-chymotrypsin  Myoglobin  column  3-lactoglobulin  y-globulin  Lysozyme  Bovine serum albumin  1.33  2.00 3.33 3.33  11.33  10.66  12.66  2.00  1.33 2.00 2.00  10.66 9.33  10.00 11.66  12.66 11.33  mean,  1.55  1.78  2.89  10.44  10.77  Standard deviation,  0.38  0.38  0.76  1.01  0.85  1.33  Statistical*  of  18.00  14.66 14.66  18.00 18.66  12.21  14.43  15.10  18.22  0.76  1.01  0.77  0.38  b-  variation  Among, Within, Total,  *)  16.00  15.33 14.66  a  difference,  Source  13.33 '  Degrees  8 18 26  of  freedom  Sum o f  squares  Mean  933.7 10.0 923.7  D e t e r m i n e d b y t h e D u n c a n ' s New M u l t i p l e R a n g e t e s t ; a = 0 . 0 5 ; a , ficantly different groups. * * ) S t a n d a r d e r r o r o f t h e t r e a t m e n t , S x = / W i t h i n mean s q u a r e / n .  squares  Sx  0.55  b,  c,  d,  **  0.43  e and  f  are  signi-  ©  ©  ©  ©  © © ©  0  2  4  6 Retention  8 10 coefficient  on  12 HS4B  14  16  Figure 10- C o r r e l a t i o n between r e t e n t i o n c o e f f i c i e n t s measured by two h y d r o p h o b i c chromatography t e c h n i q u e s . O r d i n a t e and a b s c i s s a a r e r e t e n t i o n c o e f f i c i e n t s o f p r o t e i n s on b u t y l - a n d hexylepoxy-Sepharose 4B, r e s p e c t i v e l y . r=0.86, s i g n i f i c a n t at a=0.05. b : b o v i n e s e r u m a l b u m i n , 1: l y s o z y m e , m:myoglobin, o : o v a l b u m i n , a: a-lactalbumin, a : a - c h y m o t r y p s i n , 8: 8l a c t o g l o b u l i n a n d y: y-gl°bulin.  18  68 Thus  the concentration  complex Based was  should  on t h i s  carried  micellar which  of Triton  be a good  criterion  assumption,  concentration  contained  chromatography  of Triton  detergent  was b o u n d  detergent  was r e c o v e r e d  against SM-2  from  by complexing w i t h  into  sensitive  f o r the assay  the ethylene  column.  As a r e s u l t  After solution,  of destabilization,  p r o t e i n s was n o t s p e c i f i c . 3 - The p o l y e t h y l e n e from i t s complex w i t h  The  o f 50% e t h a n o l  m e t h o d was u s e d  Ammonium these  oxide)  group  phase.  This blue  forms a  precipitate  The method  i s claimed  This  the Biobead  i s not adequately to separate  SM-2 b e d i n t e r f e r e s  4 - Complexity  t o be  bound t o sensitive.  the detergent with the  of the procedure f o r  and s e p a r a t i o n o f t h e p r o t e i n - d e t e r g e n t  i s then  c o u l d be  1 - T h e amount o f t h e d e t e r g e n t  2 - The method  blue  X-100 ( G a r e w a l ,  repeatability.  g l y c o l w h i c h was u s e d  reaction.  i n T r i t o n X-100  cobaltothiocyanate  groups.  showed p o o r  to determine the  The method i s  o f m i c r o g r a m amounts o f T r i t o n  However, o u r r e s u l t s  cobaltothiocyanate  pyrrolidone  spectrophotometrically.  dichloride  to the f o l l o w i n g reasons:  cleavage  20% p o l y v i n y l  of the poly(ethylene  extracted  due  c o m p l e x was c o l l e c t e d .  glycol.  (octylphenoxypolyethoxyethanol).  1973).  containing  The f r a c t i o n o f t h e e f f l u e n t  ammonium c o b a l t o t h i o c y a n a t e  on t h e p r e s e n c e  precipitate  o f p r o t e i n s o n S e p h a d e x G-150  the bed by a p p l i c a t i o n  concentration o f the detergent based  of proteins.  t o t h e b e d m a t e r i a l and p r o t e i n e l u t e d .  10% e t h y l e n e  The  X-100.  the protein-detergent  was a p p l i e d t o a B i o b e a d  containing  of hydrophobicity  out i n the presence o f phosphate b u f f e r  c o n c e n t r a t i o n by d i a l y s i s it  X-100 i n t h e p r o t e i n - d e t e r g e n t  complex.  The  69 direct  c o b a l t o t h i o c y a n a t e r e a c t i o n a p p l i e d to the d e t e r g e n t - p r o t e i n  fraction  5.  e l u t e d f r o m S e p h a d e x G-150  Hydrophobic p a r t i t i o n :  revealed different  d i d not  The  hydrophobic  affect  determines  are expected  to  the p a r t i t i o n of p r o t e i n s .  nm.  The  coefficient  d e x t r a n and  t h e r a d i a t i o n w h i c h was at  p r o t e i n s (Table I I I ) .  of the p r o t e i n which  of groups exposed to the surroundings  Partition 280  situation.  p a r t i t i o n method  values of A l o g K f o r d i f f e r e n t  H y d r o p h o b i c i t y as w e l l as c o n f o r m a t i o n the n a t u r e  improve the  was  determined  polyethylene glycol e l i m i n a t e d by  o f t h e two  phases i s comparatively  t h e l o w p o l y m e r c o n c e n t r a t i o n and However, p o l y e t h y l e n e g l y c o l  solutions slightly  s u b t r a c t i n g the v a l u e s  the i d e n t i c a l c o n c e n t r a t i o n s of polymers.  polarity  p h o t o m e t r i c a l l y at  The  s m a l l , and  polymer i s lowered,  i s mainly  i s s l i g h t l y more h y d r o p h o b i c  particles  The  can  than  cause  to  polymers. dextran.  drastic  general trend i n  i s t h a t when t h e m o l e c u l a r  the a f f i n i t y  non-  due  the h y d r o p h i l i c nature of the  changes i n the d i s t r i b u t i o n of the p r o t e i n s .  o f one  measured  d i f f e r e n c e i n the  D e l i c a t e changes i n the phase c o m p o s i t i o n  p a r t i t i o n o f p r o t e i n s and  absorbed  of the p r o t e i n s towards  weight that  phase i s i n c r e a s e d .  Neither polyethylene glycol less,  nor  dextran are charged.  i o n s i n f l u e n c e d i s t r i b u t i o n o f p r o t e i n s w i t h i n t h e two  P r o t e i n s which are p o s i t i v e l y  or n e g a t i v e l y charged  t h e u p p e r o r l o w e r p h a s e , d e p e n d i n g on system.  the i o n i c  c a n be  phases.  pushed  composition  When a l a r g e amount o f s a l t i s a d d e d t o t h e  Neverthe-  of  toward  the  dextran-polyethylene  Table IIIHydrophobicity of proteins determined by the hydrophobic p a r t i t i o n technique. A l o g K was c a l c u l a t e d f r o m A l o g K = l o g k / k where , k is the p a r t i t i o n c o e f f i c i e n t of p r o t e i n s i n t h e p o l y e t h y l e n e g l y c o l p a l m i t a t e / d e x t r a n system and is the p a r t i t i o n coe f f i c i e n t of proteins i n the polyethylene glycol/dextran system. 2  Lysozyme  k  * —  i  k ,* 2  k /k  Alog  K  CytochromeC  1  Conalbumin  Ovalbumin  Trypsin  a-chymotrypsin  3-lactoglobulin  Myoglobin  0.115  0.164  0.195  0.877  0.123  0.156  0.010  (0.002)  (0.003)  (0.004)  0.501  0.047  0.001  (0.001)  (0.001)  (0.000)  0.305 t CO.010)  0.120 * (0.004)  (0.002)  0.391  0.116  0.074  (0.007)  (0.003)  (0.001)  (0.003) (0.004)(0.004)  0.094  0.105  0.423  (0.005)(0.003)(0.005)  Bovine serum albumin  0.77  1.03  1.55  1.75  1.85  2.07  2.46  3.39  8.93  -0.10  0.01  0.19  0.24  0.26  0.31  0.39  0.53  0.95  *) Means o f 3 t o 4 d e t e r m i n a t i o n s . * * ) S t a n d a r d d e v i a t i o n s o f t h e means  in  parenthesis.  g l y c o l system, a l l p r o t e i n s tend phase.  S o d i u m c h l o r i d e c a n be  push p r o t e i n s to the s t r e n g t h w o u l d be  to p a r t i t i o n i n favour  added a t c o n c e n t r a t i o n s  top phase.  The  of the up  top  to 5 M  main e f f e c t of i n c r e a s i n g the  the i n c r e a s e i n the K v a l u e .  o f the p r o t e i n s f o r the upper p h a s e , w h i c h has  The  increased  a less polar  t r i v a l e n t anions  a s w e l l as Rb  character be  t h e " s a l t i n g - i n " phenomenon.  > K > Cs > Na  ionic  affinity  t h a n t h e b o t t o m p h a s e , w i t h i n c r e a s i n g s a l t c o n c e n t r a t i o n may c o m p a r e d w i t h t h e e f f e c t known a s  to  Di-  > L i c a t i o n s enhance  and  this  phenomenon ( A l b e r t s s o n , 1 9 7 1 ) .  The attraction I t has by  p a r t i t i o n o f a p r o t e i n depends b o t h on  to the  two  p h a s e s and  on  i t s net  b e e n s u g g e s t e d t h a t t h e dependence on  i n c l u d i n g an  appropriate  p o t a s s i u m s u l f a t e was s o l e l y on  salt  i n the  the b a s i s of the g e n e r a l  charge (Johansson, the  system.  u s e d i n t h e b u f f e r and  i t s relative  latter  c o u l d be  I n the p r e s e n t  solvation ability  of the  i n the h y d r o p h o b i c i t y  o f one  charge e f f e c t s were b a l a n c e d . identical  composition  but  two  were c o n c e n t r a t e d u p p e r p h a s e and  dextran  of p r o t e i n were used to c a n c e l  the o p p o s i t e i n the lower  group w h i c h i s bound to p o l y e t h y l e n e the  differing  determined under c o n d i t i o n s  phase  polymers employed to o b t a i n the b i p h a s i c into  phases.  but  c o n t r i b u t i o n o f e f f e c t s s u c h as p o l y m e r c o n c e n t r a t i o n and  The  determined  phase, the other e f f e c t s i n c l u d i n g  Blanks  devoid  removed work  t h e p a r t i t i o n was  M o r e o v e r , by making b o t h systems i d e n t i c a l i n c o m p o s i t i o n only  1974).  phases: polyethylene phase.  the viscosity.  system  glycol in  With the s t r o n g  the  hydrophobic  g l y c o l , a consequent i n c r e a s e  s o l v a t i n g capacity f o r hydrophobic molecules occurs  of  i n the  in  phase  72 rich  in  this  polymer.  concentrated  in  The dissolve low  in  the  Therefore  phase  palmitate  the  buffer  c o n c e n t r a t i o n was  between was  polyethylene  sufficient  to  containing  ester and  very  produce  a fine  glycol  distribution separation  analysis were  of  were  denaturation  1500 K  x  used  G was  value  of  of  protein but in  the  used  to  (partition  the  study.  0.2%  in  SDS  palmitate  tubes this  facilitate  same  the  0.1  most  the  the  separation  and  palmitate-protein  fairly  used  samples  solution  to  for  even  30  sec  phases  in  the  viscosity  of  the  required  to  caused  achieve  a  delay  spectrophotometric  difficulties, solutions  probably  makes  N NaOH was The  two  was  of  interactions  viscosity  these  concentration  measurements.  the  in  solutions  to  in  the  the  samples  test  shaker.  spectrophotometric  to  high  be  difficult  shaking  the  shaking  was  strong  the  eliminate  the  interaction.  of  closer  Centrifugal the  constant  over  to  dilute  the  the of  Although a range  used  in  the  of this  polyethylene  t h e n warmed necessary  to  force  phases.  p r o t e i n was  was  mechanical  even  of  were  This  caused  systems  emulsification process.  phases.  viscosity  a l l  c o e f f i c i e n t ) was  quantity  to  of  This  dilution  a vortex  shaking  protein  glycol  after  due  to  extensive  eliminate  and  mixed w i t h  due  created problems  To  aqueous  w i l l  ligand.  glycol  Although  the m a t e r i a l s .  and  weight  Severe  condition  of  samples.  taken by  tubes  palmitate  of  dispersion  system,  proteins  hydrophobic  possibly  glycol molecules.  polyethylene  phase  the  polyethylene  high,  glycol/dextran  in  of  hydrophobic  the v i s c o s i t y  polyethylene  uniform  more  to  up  before  the  decrease  the  t u r b i d i t y produced  possibly  due  The higher than  in  and  observed  polyethylene  thus by  Considerable  were  proteins  showed  resulting  great  A effective  been  showed  amino tend  acid to  throughout nine  toward  K value  the  residues,  in  by  the  the  with  and  large  the  of  a  the  third  of  palmitate this  lower  the  of  glycol  hydrophobic  group,  increased,  a f f i n i t y was  and  caused  palmitate as  only  ligand,  hydrophobic  the  from  is  are once  and  partition  and  However,  for  protein,  chromatography ligand  chain  hydrophilic  proportion  and  yielded  terminal primarily  end,  the are  side  129  chains  distributed first  thirty-  hydrophobic  hydrophilic,  hydrophobic  The  containing  randomly of  which  methods,  p a r t i t i o n method.  than being  again  the  This  palmitate  and  between  12).  polypeptide  sizeable  residues  sequence  11  hydrophobic  amino  found  hydrophobic  hydrophobic  rather  starting  terminal  values  Since  excluded.  the hydrophobic  clusters  forty  was  single  A  next  by  hydrophobic  polypeptide.  the  appreciably  polyethylene  designated  (Figs.  lysozyme  consists  residues,  was  distribution pattern  c o r r e l a t i o n was  methods  hydrophobic  residues,  occur  the  protein  determined  analyses  molecule  and  containing  Because  positive  chromatography  affinity  in  compared.  towards K.  system  III).  significant  a negative A log lysozyme  affinity  the  the  protein hydrophobicity  (Table  found  no  systems  i n t e r a c t i o n between  these c o r r e l a t i o n had  in  p a l m i t a t e were  hydrophobicities  hydrophobic  the  in  resulting  differences  larger A log  K represents  coefficient  protein  thus  when  glycol  in  hydrophobic  Alog  of  the bottom phase,  unity.  proteins  concentration  and  the  (Bernhard,  1968)  74  ©  10  /  / /  / 0 / 0  0.1  0.2  /  /  /  /  /  /  /  /  /  ©  /  0.3  0.4 0.5 Hydrophobic  0.6 0.7 coefficient  0.8  0.9  1.0  Figure 11C o r r e l a t i o n between h y d r o p h o b i c i t i e s measured by hydrophobic c h r o m a t o g r a p h y o n b u t y l e p o x y - S e p h a r o s e 4B a n d h y d r o p h o b i c p a r t i t i o n . O r d i n a t e and a b s c i s s a a r e r e t e n t i o n c o e f f i c i e n t on b u t y l e p o x y - S e p h a r o s e 4B (BS4B) and h y d r o p h o b i c c o e f f i c i e n t d e t e r m i n e d b y h y d r o p h o b i c p a r t i t i o n method r e s p e c t i v e l y . r = 0 . 9 5 , s i g n i f i c a n t a t a= 0 . 0 5 . b : bovine serum a l b u m i n , m:myoglobin, o: o v a l b u m i n , a: a - c h y m o t r y p s i n and 6:6lactoglobulin.  75  ©  18  / 16  to  /  c 14 o c <u o 12  • -H 4-1 4-1  <U O ° 10  ©  c o  /  />  /  /  /  0) • 0)  © 0  0.2  0.4 Hydrophobic  0.6  0.8  coefficient  F i g u r e 12C o r r e l a t i o n between h y d r o p h o b i c i t i e s measured by h y d r o p h o b i c c h r o m a t o g r a p h y on h e x y l e p o x y - S e p h a r o s e 4B and h y d r o p h o b i c p a r t i t i o n . O r d i n a t e and a b s c i s s a a r e r e t e n t i o n c o e f f i c i e n t on hexylepoxy-Sepharose 4B (HS4B) a n d h y d r o p h o b i c c o e f f i c i e n t d e t e r m i n e d b y t h e h y d r o p h o b i c p a r t i t i o n m e t h o d , r e s p e c t i v e l y . r= 0 . 8 7 , s i g n i f i c a n t a t a = 0 . 0 S . b: b o v i n e serum a l b u m i n , c: c o n a l b u m i n , o: o v a l b u m i n , m: myoglobin, a: a - c h y m o t r y p s i n a n d 8: 3-lactoglobulin.  Lysozyme arrangement able  to  of  nonpolar  may  with  palmitate  the  not  The proteins  high  are  polyethylene interface  have  soluble  weight  only  capacity  significant  role  of  with  The  the  With  to  consider,  of  the  the  in  at  respect  or  such  patches  These  an which  hydrophobic  orientation  to  are  sites,  interact  the  high of  tend is  interface.  of  this  suspended  the  and  the  weights  phases  lower of  cause  to  dextran  in  not  Many  at  the  play  and  the  the in  phases; a  may  Particles  interface.  adsorption  at  fact  "phases"  phases.  proteins  the  any  interface  particles.  not  or  distribute  the  proteins  that  i n t e r f a c e has  does  are  is  the method.  the  The  greater there  in  dissolved  proteins  l i q u i d phases  inter  two  in  proteins  molecular the  dissolve  protein  the p a r t i c l e s ,  the  used  soluble  the  food  systems  Although  to  p a r t i t i o n method  molecules of  quantity  between  of  not  These  formed  upper,  either  do  amount  insoluble  particles,  proteins  proteins  large  themselves size  the phase  adsorption.  distribute  interface.  groups.  size  since  hydrophobic  the hydrophobic  in  a small  a relatively  larger  butyl  suitable  phases.  a precipitate usually certain  the  and  provides  protein  ligand.  glycol  and  a hydrophobic  residues  l i m i t a t i o n of  molecular  adsorb  as  interact with hexyl  however,  a l l  appears  The  and to  three  large  the  radius  insolubility  locate  at  of  the  interface.  Sometimes then  the  particles  gravitational  force  gravitational are is  pulled  forces  down by  a f f e c t e d by  the  the  overcome former.  size  and  interaction The  density  forces;  influence of  the  of  particles  as  well  as  proteins  density  of  the  w e r e e x a m i n e d by  Y-globulin  and  glycol  was  added.  result  of  This  The  of  the  i s not  Biuret  protein  This  was  hydrophobic  the  and  were p r e c i p i t a t e d  when  i n s o l u b i l i z a t i o n phenomenon was  results with t u r b i d i t y of  applicable  to  the  the  in  the  g l y c o l and  In  partitioned  measurement o f  separated  for  of  the  t o be  the  proteins.  showed  spectrophotometric  partition coefficient.  the  phases.  case  polyethylene thought  phases  food  the  some s o l u b i l i z e d f o o d p r o t e i n s  Lowry methods were a p p l i e d  concentration  c a s e when v e g e t a b l e  p a r t i t i o n method.  i n t e r a c t i o n between p o l y e t h y l e n e  because  analysis  the  i n s u l i n , proteins  Preliminary that  phases.  determination  of  However,' d e x t r a n made  a  2+ complex w i t h formed  a  method  of  Cu  most  Layne  is  free  hydroxide and  trichloroacetic acid  phase of  t o be  respectively. and  phobicities partition  avoid  polymers. f o r washing  TCA-precipitated protein  positive  determined  the  Biuret  g l y c o l nor  and  p r o b l e m s w o u l d be is precipitated  and  diethyl  ether  g l y c o l and  i s then d i s s o l v e d  i s measured  according  Lowry  dextran  HCIO^, i t i s p r e d i c t e d  away p o l y e t h y l e n e protein  glycol  by  is that  as TCA  and  are dextran  i n 0.5  M  sodium  to  the  Biuret  found between  the  hydro-  1957). w h i c h were  hydrophobic  ( F i g . 11)  effective  and  protein  Chloroform  correlations by  i n the  polyethylene  foregoing  the  Polyethylene  F o l i n reagent  (TCA)  the  concentration  (Layne,  technique  determining  to  separation,  the  used  Lowry method The  of  formed.  Since neither  c o n v e n i e n t way After  suggested  upon a d d i t i o n  (1957).  by  follows: washed  a p r e c i p i t a t e was  precipitate  precipitated the  and  suggest  chromatography that  hydrophobicities.  and  hydrophobic  b o t h methods are  useful  in  Interfacial  6. taken  as  a representative  proteins,  since  these  interfacial  tension  measured  0.2%  The  in  results  showed  t h e more  should  be  on  o i l  more  of  to  proteins  hydrophobic  emulsion  is  are  This  could easily  and b r i n g  proteins  the  are  take  is  up  a  such  as  a logical  an  configuration with  the polar  nonpolar  phase  (oil).  denaturation  need not of two  such  be  as  the the  in  were IV).  the  This  15).  greater  o i l  of  (Table  effective  means  depression  that in  the  hydrophobic  proteins  and w a t e r m o l e c u l e s ,  denature  energy  down.  to  be  able  arrives  at  the boundary  to  Consequently  increase'the  interfacial  phase  systems.  phase As  resistance  films  that  against  has  i t  allows  the  free  energy  i n which  (water)  and  a consequence  occurs  It  low  and It  are  film  with  emphasized,  the mechanical  to  forces  two-  the  polar  side  arises. assume  indeed of  determines  with  the  interfacial  This that  to  to  groups  groups  mechanism  respect  strength  a  protein molecule  nonpolar  this  obvious  important  external  the  of  a protein  seems  been  of  i n t e r f a c e , o r i e n t a t i o n of  because  with  irreversible.  Alexander,1968), its  and  expected  expected  tensions  interface  interfacial  oil/water  result  interact  protein  the  with  properties  correlation with 14  was  capacity.  system  chains  o i l  tension  r e l a t e d - t o :- c h a n g e s  Interfacial  13,  be  When a p r o t e i n m o l e c u l e phase  surfactant  negative  (Figs.  Interfacial  closely  the p r o t e i n ,  interact  surface  for  solutions/corn  significant  tension.  able  parameter  (Kinsella,1976).  hydrophobic  interfacial  measurements:  properties  protein  hydrophobicities  the  tension  denaturation  the  the  (Pearson  properties  stability  of  and  the  protein  film  and  to  a great  extent  the  Table  IV-  Interfacial  Bovine serura albumin  tension  Ovalbumin  of  0.2%  protein  Lysozyme  y-globulin  solution/corn  Myoglobin  6 .0 6, .0 6 . .0  7.. 0 6 , .5  9 .0 9 .5 9 . .5  17 .5  18 .0 1 8 .0  18 .0  Mean,  6 . .0  6 . .6  9 . .3  1 7 . .6  Standard  0 . 00  0 . 29  0 . 28  6 .5  1 7 .5  o i l  8-lactoglobulin  interface.  Trypsin  Conalbumin  a-chymotrypsin  21 .5 21 .5 21 . 0  21 ,.5 2 1 . ,5  22.5 22.0  1 8 . .5  21 .5 2 0 .0 2 0 .0  22. 0  22.0  18. 1  2 0 . .5  2 1 , .3  21. 6  22.1  0 . 29  0 . 29  0 . 86  deviation,  0 . .29  0 . 29  0.29  Statistical difference,*  Source  of  variation  Degrees o f  Among, Within, Total,  *) **)  D e t e r m i n e d by t h e D u n c a n ' s different groups. Standard  error  of  the  freedom  ;  Sum  of  squares  8  1071  18 26  21 1092  New M u l t i p l e  treatment,  Sx=  Range  J Within  test; mean  Mean  of  squares  Sx**  1.2  a=0.05; square/n  a, .  b,  0.62  c  and  d  are  significantly  80  \  22  20  18  X 9  e  ©  \  u  e X T3 O •H to  c  \  • a>  12 rt  •H o  £io  ©  ID  ©  0  1  2  3 4 Retention  5 6 7 c o e f f i c i e n t on BS4B  8  9  10  F i g u r e 13- C o r r e l a t i o n between h y d r o p h o b i c i t y determined on b u t y l e p o x y Sepharose 4B (BS4B) and i n t e r f a c i a l t e n s i o n o f 0.2% p r o t e i n s o l u t i o n / c o r n o i l i n t e r f a c e . r= -0.95, s i g n i f i c a n t at a= 0.05. b:bovine serum a l b u m i n , 1: lysozyme, m: m y o g l o b i n , a: a - c h y m o t r y p s i n , 8: 3 - l a c t o g l o b u l i n and y: y - g l o b u l i n . ovalbumin i s shown by "*".  81  0  2  4  6 8 10 12 Retention coefficient  14  16  F i g u r e 14.C o r r e l a t i o n b e t w e e n h y d r o p h o b i c i t y d e t e r m i n e d on h e x y l e p o x y - S e p h a r o s e 4B (HS4B) a n d i n t e r f a c i a l t e n s i o n o f 0 . 2 % protein solution/corn o i l interface. r= - 0 . 7 7 , s i g n i f i c a n t a t a= 0 . 0 5 . b : b o v i n e s e r u m a l b u m i n , c : c o n a l b u m i n , 1: lysozyme, m: m y o g l o b i n , a : a - c h y m o t r y p s i n , 8 : 8 - l a c t o g l o b u l i n a n d y : yglobulin.  18  \  82  22  \  20  18  16  14  e 12  c C  \ © \ \  \  \  o  CO  \  10  O •rH +->  c  +J  O  rt <4H rH  <D •P  \  \  \  \  \ ©\  C  0  0.2  0.3  0.4  0.5 0.6 0.7 0.8 0.9 1.0 Hydrophobic c o e f f i c i e n t Figure 1 5 - C o r r e l a t i o n between h y d r o p h o b i c i t y (hydrophobic c o e f f i c i e n t ) o f p r o t e i n s determined by the hydrophbic p a r t i t i o n method and i n t e r f a c i a l t e n s i o n o f 0.2% p r o t e i n s o l u t i o n / c o r n o i l interface. r=-0.97, s i g n i f i c a n t a t a=0,05. b: bovine serum • albumin, c: c o n a l b u m i n , m: myoglobin, t : t r y p s i n , a: ac h y m o t r y p s i n a n d 8: B-lactoglobulin. O v a l b u m i n i s shown by " * "  stability mainly for  of  the  its  a  two phase  viscoelasticity  mechanical  property elastic  of  the  one,  position  of  but  interfacial  layer  factor  which for  One ovalbumin.  suggested were  not  presence  the  to be covered  the It  the o i l  in  was  complex and  to  be  decisive  shown  mechanical  that  a pure  the  viscous a  nor  a  pure  super-  elasticity.  these r h e o l o g i c a l bond  This  properties  formation  in  information of  protein  the  of  the  protein  film  agrees  with  as  outstanding  an  our  properties.  this  hypothesis had  been  was  found  i n t e r f a c i a l tension  the  contaminants  appears  c h a r a c t e r i z e d by  hydrophobicity  to  this  i t  f i l m w h i c h was  neither  one,  droplets.  the  also  hydrophobic  the  to  protein  general,  protein, which  due  Furthermore,  attributed  exception  depressed  of  a  surfactant  This  phobicity,  to  suggest  its  in  viscosity  (1976)  surrounds  results  is,  rather  both  of  strength.  film  Schut  which  system.  other  study. such  as  properties This  found  in  the  to  of  low  be  considerably. of  this  e f f e c t might  phospholipid.  protein be  case  of  hydroThis  is  which  attributed  to  the  SUMMARY AND  To and  the  investigate  surfactant  the  CONCLUSION  relationship  properties  of  between  proteins,  the  hydrophobicity  d i f f e r e n t methods  were  applied:  1.  Chromatography  substituted  gel  proteins  so  tightly  polarity  reducing  prohibited  2. Triton buffer. amount to  at  of  the  hydrophobicity  and  could  buffer.  be  to  the  in  analysis  for  by  with  Sepharoses  contained  positively  charged  repulsion  only  The retained  after  many  using  hydrophobicity  determination  G-150  in  the  on  or  detergent  and  the  of  proteins.  This  of  the  X-100.  of  Triton the  of  mixed  nitrogen  detergents:  sodium was  The  The  determined  suffered method  phosphate  eluted.  c o m p l e x was  Sepharoses:  Phenylbutylamine ring  complex  method  with n-alkylamines  aromatic  in  of  from  was  a  poor  found  hydrophobicity.  alkylamino  coupling  presence  included  the protein-detergent  substituted  Also, gel  done  Such a h i g h  utilized  Sephadex  determination  4B w a s  hydrophobic  static  on  bound  Chromatography  (C4-C6-C8).  4B:  i t s m i c e l l e c o n c e n t r a t i o n was  in  for  the  Sepharose  proteins.  hydrophobicity  unsuitable  high  elution  from being  detergent  repeatability  Sepharose  in  substituted  very  the  agents  of  Protein  express  3.  that  Chromatography X-100  oleic  demonstrated  the method  hydrophobicity  on  was  hydrophobic rendered  attraction forces  obtained. and  these  ionic gels  CNBr of  activated  different  to  chain  Sepharose  The  the  alkylamino  groups.  The  undesirable  w h i c h made, t h e s e  4B  lengths  gels  presence  due  to  of  electro  incapable  of  measuring  pure  Sepharoses  provided  interactions alkylamino proteins  are  dominant  3 was  contain  carried  alcohols,  out  the  reactions  showed  the  capability  the  Butyl-,  following of  and  of  tightly  hydrophobicity.  experiments  to  determination protein gels  with  were  ionic  of the  volumes  to  a)  obviate  of  with to  gel  any  determined However,  salt.  by  from  use  in of  to  calculate  ethers  from to  prepared.  for  determination  in  other  suitable  nonpolar  for  regions  non-ionic  suitable  mild hydrophobic  interactions.  of  amphiphilic  provided  The  interactions, retention  hydrophobicities.  found  between  hexylepoxy-Sepharose  hydrophobic  ethers  hydrophobicity,  found  of  adsorbents  hydrolysate.  2 M NaCl  ionic  glycidyl  successfully  these  for  the  glycidyl  unsuitable  were  of  and  between  Alkylepoxy-  of  were  high  casein  c o r r e l a t i o n was  butyl-  t h e more  its  used  conditions  used  positive  of  coupling  Interaction  The  possibility were  Preparation  found  was  available  stipulate  Significant  tography.  of  and was  peptides  groups  the proteins  phobicities  and  4B,  hexylepoxy-Sepharoses  alkyl  Moreover,  .  synthesis  because  hydrophobicity.  mediated  strength  b)  and  bitter  hydrophobic  discrimination  octylepoxy-Sepharoses  this  alkylamino  alkylepoxy-Sepharoses:  order:  proteins  However,  remove  Butyl-  and  with  a protein.  of  groups.  Sepharose  hexyl-  of  example).  on  hydrocarbon  Octylepoxy-Sepharose, interacted  an  chromatography  conditioning  Sepharose.  as  with  e l e c t r o s t a t i c or  in  only  in  chromatography  on w h e t h e r  presented  Hydrophobic  Sepharoses  However,  information  Sepharoses  (Fig.  4.  was  hydrophobicity.  proteins  showed  the  column greater  hydrochromaaffinity  toward  t h e h e x y l m o i e t y compared t o t h e b u t y l c o u n t e r p a r t .  No c o r r e l a t i o n was and  found between the e f f e c t i v e  the "average h y d r o p h o b i c i t i e s " of Bigelow  amino a c i d c o m p o s i t i o n o f p r o t e i n s . residues take part i n hydrophobic  No and  c o r r e l a t i o n was  hydrophobicities  (1967) c a l c u l a t e d  A p p a r e n t l y , o n l y exposed  from the  hydrophobic  interactions.  found between the e f f e c t i v e h y d r o p h o b i c i t y  the molecular weight of p r o t e i n .  This contradicts  the p r i m i t i v e  v i e w t h a t h i g h e r m o l e c u l a r w e i g h t g l o b u l a r p r o t e i n s a r e more hydrophobic. ratio  o f n o n p o l a r amino a c i d s w h i c h a r e n o t n e c e s s a r i l y  hydrophobic  5.  In f a c t , high molecular weight proteins contain a greater involved i n  interactions.  Hydrophobic  partition:  Determination of the e f f e c t i v e  h y d r o p h o b i c i t y was a l s o c a r r i e d o u t e m p l o y i n g o f them c o n t a i n e d a h y d r o p h o b i c l i g a n d .  a two p h a s e s y s t e m .  The p a r t i t i o n  c o e f f i c i e n t s of  p r o t e i n s were d e t e r m i n e d and t h e n t h e h y d r o p h o b i c c o e f f i c i e n t s calculated  f r o m them.  Positive  c o r r e l a t i o n was  found  solubilized  were  between  h y d r o p h o b i c i t i e s d e t e r m i n e d by h y d r o p h o b i c p a r t i t i o n and by chromatography.  One  hydrophobic  A p p l i c a t i o n o f t h e h y d r o p h o b i c p a r t i t i o n method  food p r o t e i n s needs f u r t h e r s t u d y t o s o l v e  to  insolubility  problems which occur i n the presence of the polymers.  6.  Interfacial  tensions:  The i n t e r f a c i a l t e n s i o n s o f v a r i o u s  p r o t e i n s o l u t i o n s / o i l showed a g o o d c o r r e l a t i o n w i t h t h e e x p e r i m e n t a l l y d e t e r m i n e d v a l u e s o f h y d r o p h o b i c i t y . The n e g a t i v e c o r r e l a t i o n w h i c h f o u n d between t h e i n t e r f a c i a l t e n s i o n s and t h e e f f e c t i v e  was  hydrophobicities  suggests more  that  more  hydrophobic  efficiently.  emulsifying  capacity  In their  These  short,  hydrophobicity  or  the  degree  of  the  partition  The with  their  This  implies  surfactant enable  us  of  proteins  through  proteins as  their  were  reduce  by  of  the  to  their to  the  expected  found  interaction  to  interfacial have  higher  interfacial  differ  tension  energy.  significantly  in  r e t e n t i o n on h y d r o p h o b i c  a hydrophobic  moiety  gels  (palmitate)  system.  of  i n t e r f a c i a l tensions t h e more  properties. to  are  reduction  measured  hydrophobicity  that  proteins  predict  its  apolar and  thus  hydrophobic  Information  proteins with  the  is  their  protein,  negatively surfactant  properties  the  the  on h y d r o p h o b i c i t y  functionality.  correlated  of  better  protein  would  BIBLIOGRAPHY  A l b e r t s s o n , P.A., " P a r t i t i o n o f C e l l P a r t i c l e s a n d M a c r o m o l e c u l e s " , S e c o n d E d i t i o n , W i l e y I n t e r s c i e n c e , New Y o r k (.1971). 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