Food Protein Deterioration - American Chemical Society

13 Effects of Ions on Protein Conformation and Functionality SRINIVASAN DAMODARAN and JOHN E. KINSELLA

Downloaded by CORNELL UNIV on October 10, 2016 | http://pubs.acs.org Publication Date: December 13, 1982 | doi: 10.1021/bk-1982-0206.ch013

Cornell University, Institute of Food Science, Ithaca, NY 14853

The unique three-dimensional structure of a protein molecule is the resultant of various attractive and repulsive interactions of the protein chain within itself and with the surrounding sol vent. The various types of interactions and forces which con tribute to the overall stability of the native structure of a protein may be broadly categorized as hydrogen bonding, electro static and hydrophobic interactions. For the protein to assume a particular ordered structure, the favorable free energy change from the above interactions in the ordered form should be more than the unfavorable free energy increase resulting from the configurational entropy of the chain. In other words, the favorable change in the free energy for the stability of the folded native protein may be expressed as Equation 1: ΔG = ΔG + ΔG + ΔG n

h

e

ф

TΔS

congifuraoitn

where ΔG , ΔG and ΔG are the free energy contribution from hydrogen bonding, electrostatic and hydrophobic interactions, respectively, and ΔS is the configurational entropy of the pro tein chain and Τ is the temperature. For many proteins the net free energy favoring the formation and stability of the native structure is only about 10-20 Kcal/ mole (1). Hence any perturbation leading to decrease in the free energy of stabilization even by few kilocalories would profoundly affect the structural stability of the native protein and hence its function. Such changes may be brought about by agents like heat, organic solvents and chaotropic salts. The major emphasis of this chapter will be to discuss the effect of ionic solutes on protein conformation and function, and to elucidate the mecha nisms of these interactions. Before venturing into this i t may be appropriate to understand the nature and magnitude of some of the major forces responsible for the stability of protein struc ture, viz. hydrogen bonds, electrostatic and hydrophobic inter actions . h

e

ф

0097-6156/82/0206-O327$09.00/0 © 1982 American Chemical Society Cherry; Food Protein Deterioration ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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328 Major Forces i n P r o t e i n Structure

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Hydrogen bonds. The existence and importance of hydrogen bonds i n p r o t e i n s i s w e l l documented. In f a c t , the α-helical and the 3-sheet s t r u c t u r e s i n p r o t e i n s are based on the formation of hydrogen bonds between the peptide groups. However, there has been considerable disagreement about the degree of s t a b i l i z a t i o n they provide to the p r o t e i n s t r u c t u r e . The reason f o r t h i s i s the f a c t that i n aqueous s o l u t i o n s , the solvent i t s e l f can com pete with both the acceptor and donor groups i n p r o t e i n s f o r hydrogen bond formation. In other words, i f the formation of hydrogen bonds i n aqueous s o l u t i o n i s expressed as Equation 2: protein-Η···Η 0 + Η 0··«H-protein 2

2

p r o t e i n - p r o t e i n + Η^0··«Η^Ο then, i n order to form i n t r a p e p t i d e hydrogen bonds the change i n the free energy of the above r e a c t i o n should favor the forward r e a c t i o n . With N-methylacetamide as a model compound, which i s comparable to groups i n p r o t e i n s , i t has been shown that the change i n the free energy f o r the formation of hydrogen bonds between N-me thy lace tamide molecules i s +0.75 Kcal/mole (2). But the data f o r h e l i x - c o i l t r a n s i t i o n i n s y n t h e t i c polypeptides shows that the f r e e energy c o n t r i b u t e d by the hydrogen bonds f o r the s t a b i l i t y of p r o t e i n s i s only about -0.15 Kcal/mole residue (3). One can, t h e r e f o r e , assume that the energy contributed by hydrogen bonds to the o v e r a l l s t a b i l i t y of p r o t e i n s t r u c t u r e i s very s m a l l . I t should be borne i n mind that the hydrogen bond i s p r i m a r i l y i o n i c i n character due to the d i p o l e moment of the pep t i d e group. Although the hydrogen bonds give s t a b i l i t y to the ah e l i c a l s t r u c t u r e , the d r i v i n g force f o r the formation of hydro gen bonds i s the hydrophobic i n t e r a c t i o n between the s i d e c h a i n groups ( 4 ) . Conversely, any p e r t u r b a t i o n which causes d e s t a b i l i z a t i o n of these hydrophobic i n t e r a c t i o n s i n the p r o t e i n would a l s o d e s t a b i l i z e the hydrogen bonds i n t h i s v i c i n i t y . 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 . The presence of charged groups l i k e glutamic, a s p a r t i c , l y s i n e and a r g i n i n e residues i n p r o t e i n s form the b a s i s of 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 . But l i t t l e i s known about the thermodynamic c o n t r i b u t i o n they provide f o r the o v e r a l l s t a b i l i t y of the p r o t e i n s t r u c t u r e . Near the i s o e l e c t r i c pH the e l e c t r o s t a t i c i n t e r a c t i o n between the o p p o s i t e l y charged groups may be s i g n i f i c a n t and may provide some s t a b i l i z a t i o n energy to the p r o t e i n s t r u c t u r e (5). Above or below the i s o e l e c t r i c pH range the p r o t e i n w i l l have a net negative or p o s i t i v e charge and hence r e p u l s i v e i n t e r a c t i o n s w i l l e x i s t w i t h i n the p r o t e i n . But binding of counter ions may decrease these r e p u l s i v e f o r c e s and provide s t a b i l i t y to the p r o t e i n s t r u c t u r e . Since most of the charged groups are present on the surface of the p r o t e i n , the charge r e p u l s i o n or a t t r a c t i o n between these groups may be minimal

Cherry; Food Protein Deterioration ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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because of the high d i e l e c t r i c constant of the surrounding s o l vent. But the magnitude of such i n t e r a c t i o n s may become s i g n i f i cant i f these charges are p a r t i a l l y or completely b u r i e d i n s i d e the p r o t e i n where the d i e l e c t r i c constant i s lower. In such cases, each of the b u r i e d charges w i l l d e s t a b i l i z e the n a t i v e p r o t e i n s t r u c t u r e to the order of 20 Kcal/mole ( 6 ) . In the p r e s ence of an o p p o s i t e l y charged residue i n the i n t e r i o r of the prot e i n , a s a l t bridge may be formed which w i l l provide c o n s i d e r a b l e energy f o r s t a b i l i z i n g p r o t e i n s t r u c t u r e . Hydrophobic i n t e r a c t i o n s . I t i s recognized that the major force r e s p o n s i b l e f o r the s t a b i l i t y of p r o t e i n s t r u c t u r e i s the hydrophobic i n t e r a c t i o n between the nonpolar s i d e c h a i n s i n prot e i n s . The d r i v i n g force f o r such i n t e r a c t i o n s a r i s e not from the inherent a t t r a c t i o n o f nonpolar s i d e c h a i n s f o r each other, but i n the e n e r g e t i c a l l y unfavorable e f f e c t they have on the s t r u c t u r e of the water molecules around them. The b a s i s f o r t h i s fundamental concept i s the thermodynamic data concerning the t r a n s f e r o f v a r i o u s nonpolar compounds from apolar s o l v e n t s to water (7-10)· When a hydrocarbon i s t r a n s f e r r e d from a nonpolar solvent to water, the changes i n volume and enthalpy are negative and there i s a very large negative excess entropy change over the entropy of i d e a l mixing. T h i s leads to a l a r g e p o s i t i v e change i n f r e e energy and hence, low s o l u b i l i t y (11). These abnormal changes are considered to be the r e s u l t of an o r d e r i n g o f water molecules around the hydrocarbon chain forming c l a t h r a t e or cagel i k e s t r u c t u r e s . The above mentioned n o n - i d e a l thermodynamic behavior of hydrocarbon s o l u t i o n s i s due to the formation of these c l a t h r a t e s t r u c t u r e s and to the o r i e n t a t i o n a l s p e c i f i c i t y of the water molecules i n these c l a t h r a t e s t r u c t u r e s compared to that i n the f r e e bulk water i n the absence of nonpolar s o l u t e s (12). The requirements on the o r i e n t a t i o n a l s p e c i f i c i t y of water molecules f o r the formation of c l a t h r a t e - l i k e s t r u c t u r e s around hydrocarbons i s more s p e c i f i c (Figure 1; 12). F i g u r e 1A r e p r e sents the water p a i r geometry i n i c e or i d e a l i z e d bulk water. This i s the g e o m e t r i c a l l y p o s s i b l e o r i e n t a t i o n of two hydrogen bonded water molecules which permit the maximum entropy p o s s i b l e . The s t r u c t u r e shown i n Figure IB represents the water-water o r i e n t a t i o n i n c l a t h r a t e hydrates. The d i f f e r e n c e between these two o r i e n t a t i o n s i s that i n the second c o n f i g u r a t i o n one of the water molecules i s r o t a t e d through a t e t r a h e d r a l angle on the hydrogen bond. T h i s s t r u c t u r e w i l l have a higher energy s t a t e because of the closeness of the hydrogen atoms and the lone p a i r s of e l e c t r o n s which w i l l enhance the r e p u l s i v e i n t e r a c t i o n (12, 13). This would e x p l a i n the p o s i t i v e standard f r e e energy change observed f o r the t r a n s f e r o f hydrocarbons i n t o aqueous s o l u t i o n . Furthermore, such o r d e r i n g of the water molecules around the hydrocarbon would r e s t r i c t t h e i r freedom of r o t a t i o n a l and t r a n s l a t i o n a l motions and thus decrease the entropy of the system (14)· Such a s t a t e would be h i g h l y unstable and would tend to go back



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Figure 1. Mutual orientation of two water molecules in ice (A) (and liquid water), and in clathrate hydrates (B). Dots indicate the direction of lone electron pair orbitals, small circles refer to hydrogen atoms and big circles refer to oxygen atoms. Configuration Β is obtained from A by rotating one water molecule through a tetrahedral angle about the axis of the hydrogen bond, (Reproduced, with permis sion, from Ref. 12, Copyright 1975, Academic Press Inc. (London) Ltd.)



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to the o r i g i n a l s t a t e o f higher entropy. To do so i t i s o b l i g a tory f o r the system to expel the hydrocarbon from the s o l u t i o n . This i s p a r t i a l l y accomplished by f o r c i n g two hydrophobic molecules together, which allows formation o f a s i n g l e sphere of hyd r a t i o n around them with r e l e a s e o f some o f the water molecules to t h e i r e n t r o p i c a l l y f a v o r a b l e s t a t e (Figure 2 ) . Such grouping of hydrophobic molecules i n aqueous s o l u t i o n i s known as hydrophobic i n t e r a c t i o n . In other words, i t i s the i n t e r a c t i o n between the hydrophobic molecules f o r c e d by the thermodynamically unfavorable s t r u c t u r a l s t a t e of water i n the presence of these molecules. Since p r o t e i n s c o n t a i n such nonpolar r e s i d u e s , the hydrophobic i n t e r a c t i o n between these r e s i d u e s would r e s u l t i n the formation of hydrophobic regions which would impose a p a r t i c u l a r three dimensional conformation on the p r o t e i n . From the s o l u b i l i t y measurements of amino a c i d s i n water and i n e t h a n o l , Nozaki and Tanford (15) c a l c u l a t e d the f r e e energy o f t r a n s f e r of amino a c i d r e s i d u e s from water to e t h a n o l . Assuming that the t r a n s f e r from water t o ethanol i s comparable to t r a n s f e r of hydrophobic r e s i d u e s i n p r o t e i n from water environment to a nonpolar r e g i o n i n the p r o t e i n the data obtained by Nozaki and Tanford (15) suggest that on an average the hydrophobic f r e e energy c o n t r i b u t i o n f o r the p r o t e i n f o l d i n g and s t a b i l i t y by each hydrophobic r e s i d u e i s about -2 Kcal/mole r e s i d u e . Since many p r o t e i n s c o n t a i n about 4 0 to 60% content o f hydrophobic amino a c i d r e s i d u e s , i t may be deduced that the major f o r c e r e s p o n s i b l e f o r the s t a b i l i t y of the p r o t e i n conformation i s the hydrophobic interaction. As a c o r o l l a r y , i f the entropy d r i v e n hydrophobic f r e e energy i s r e s p o n s i b l e f o r the n a t i v e s t r u c t u r e of the p r o t e i n , then i t should be p o s s i b l e to manipulate the s t r u c t u r e o f the p r o t e i n by d e c r e a s i n g t h i s hydrophobic f r e e energy by changing the s o l vent c o n d i t i o n s . Such changes can be induced by c h a o t r o p i c i o n s . Before attempting t o study the i o n i c e f f e c t s on hydrophobic i n t e r a c t i o n , and hence on the p r o t e i n conformation and f u n c t i o n , i t i s p e r t i n e n t to understand the e f f e c t s of ions on the s t r u c ture of water. The type of i n t e r a c t i o n between an i o n and water i n s o l u t i o n i s the strong i o n - d i p o l e compared to the weak d i p o l e induced d i p o l e i n t e r a c t i o n i n hydrocarbon s o l u t i o n s . Such i o n d i p o l e i n t e r a c t i o n would c e r t a i n l y d i s r u p t the r e g u l a r t e t r a h e d r a l l y hydrogen bonded s t r u c t u r e of water while concomitantly imposing a new k i n d o f order around each i o n . A d e t a i l e d d i s cussion on the s t r u c t u r e of water w i l l not be presented s i n c e t h i s i s done elsewhere (13, 16). The o r i e n t a t i o n of water molecules around an i o n depends on the type o f i o n ; i . e . c a t i o n o r anion. In the h y d r a t i o n s h e l l o f a c a t i o n the hydrogen atoms of water are d i r e c t e d out, whereas with the anion they are d i r e c t e d i n (Figure 3)· T h i s q u a l i t a t i v e d i f f e r e n c e i n the o r i e n t a t i o n o f the water molecules around ions i t s e l f profoundly a f f e c t s the p o l a r i t y o f the bulk water (17) and i n t h i s respect the anions decrease the p o l a r i t y o f water to a



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Free water Figure 2,

Figure 3.

Scheme of water structure induced association of hydrocarbons.

Scheme of orientation of water molecules in the hydration spheres of cations and anions.


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greater extent than c a t i o n s . The degree to which the water s t r u c t u r e i s a f f e c t e d depends on the s i z e and charge d e n s i t y of the anion. In general f o r anions, the extent of s t r u c t u r e breaking e f f e c t on water p r o g r e s s i v e l y f o l l o w s the order F~* < CHLCOO"" < S0 < C I " < Br"" < I"" < N0 ~ < ClO^-SCN"" < C l CCOO"; t h i s s e r i e s i s known as the l y o t r o p i c o r c h a o t r o p i c s e r i e s (18). One of the thermodynamic m a n i f e s t a t i o n s o f the e f f e c t o f ions on water s t r u c t u r e i s the p a r t i a l molar entropy (Table I ) . For monovalent i o n s , a p r o g r e s s i v e i n c r e a s e i n entropy accompanies with i n c r e a s i n g i o n i c r a d i u s , suggesting that the entropy gain may a r i s e p r i m a r i l y from the s t e r i c hindrance to the format i o n o f the water framework. =


4

3

Table I . Anion

E f f e c t s of anions on water s t r u c t u r e . * S.0(M)

a

S^ h y d r a t i o n

1

F~~ ciBr~ I~ SCN"

-2.3 13.5 19.7 25.3 36.0

P a r t i a l molar entropy of aqueous ions; of h y d r a t i o n . *Taken from H a t e f i and Hanstein (18).

-31.2 -18.2 -14.4 -10.1 - 8.1 p a r t i a l molar entropy

I t may a l s o be noted that with decreasing entropy of h y d r a t i o n , the p a r t i a l molar entropy o f the s o l u t i o n a l s o decreases. T h i s shows that the entropy of i o n i c s o l u t i o n s i s c l o s e l y r e l a t e d to the o r i e n t a t i o n and to magnitude of water molecules i n the hydrogen sphere o f the i o n s . Small ions with greater charge d e n s i t y on the surface tend to have the l e a s t e f f e c t on the entropy of bulk water, whereas large ions with lower charge d e n s i t y on the surface tend to break the hydrogen bonded s t r u c t u r e of water. Reviews on the e f f e c t of ions on the p h y s i c a l p r o p e r t i e s of water were presented by von Hippel and S c h l e i c h (16) and Dandliker and De Saussure (19). I t i s reasonable a t t h i s p o i n t to question what e f f e c t the chaotropic ions would have on the hydrophobic i n t e r a c t i o n s i n p r o t e i n s . Since the hydrophobic i n t e r a c t i o n i s i n t i m a t e l y r e l a t e d to the s t r u c t u r e of water, c h a o t r o p i c ions should have profound e f f e c t s on the s t r e n g t h o f the hydrophobic i n t e r a c t i o n . As explained i n the previous s e c t i o n s , the conformation of a p r o t e i n i n aqueous s o l u t i o n i s mainly induced by the c h a r a c t e r i s t i c s of the s o l v e n t such as water. Although the grouping of the nonpolar s i d e c h a i n s i n t o a p r o t e i n ' s hydrophobic r e g i o n i n v o l v e s a decrease i n the conformational entropy, the high gain i n the e n t r o py of the s o l v e n t i n the process o v e r r i d e s the decrease i n the


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c o n f o r m a t i o n a l e n t r o p y and t h u s t h e r m o d y n a m i c a l l y s t a b i l i z e s t h e f o l d e d n a t i v e s t r u c t u r e ( 5 ) . I f t h e h y d r o g e n bonded s t r u c t u r e o f w a t e r i s a l t e r e d by c h a o t r o p i c i o n s , t h e d r i v i n g f o r c e f o r t h e h y d r o p h o b i c i n t e r a c t i o n s w i t h i n and between p r o t e i n m o l e c u l e s ( a s s o c i a t i o n and p o l y m e r i z a t i o n r e a c t i o n s ) w i l l d e c r e a s e a n d hence would d e s t a b i l i z e t h e i r s t r u c t u r e s . E f f e c t o f s a l t s on t h e s o l u b i l i t y o f h y d r o c a r b o n s a n d m o d e l peptides. One p o s s i b l e a p p r o a c h t o u n d e r s t a n d i n g t h e e f f e c t o f s a l t s on p r o t e i n c o n f o r m a t i o n and f u n c t i o n i s t o s t u d y t h e s o l u b i l i t y o f a p p r o p r i a t e m o d e l compounds i n s a l t s o l u t i o n s . The c l a s s i c a l s t u d y was on t h e s o l u b i l i t y o f a m o d e l p e p t i d e , a c e t y l t e t r a g l y c y l e t h y l e s t e r (ATGEE), i n s a l t s o l u t i o n s ( 2 0 ) , T h i s s t u d y showed t h a t t h e l o g a r i t h m o f t h e a c t i v i t y c o e f f i c i e n t ( l o g S /S) o f ATGEE i n s a l t s o l u t i o n s e x h i b i t e d a l i n e a r r e l a tionship with the s a l t concentration. T h a t i s E q u a t i o n 3: 0

Log

S /S 0

=

K C S

S

where S i s t h e s o l u b i l i t y o f ATGEE i n w a t e r , S i s t h e s o l u b i l i t y i n a s a l t s o l u t i o n , C i s t h e m o l a r c o n c e n t r a t i o n o f t h e added s a l t and K i s t h e s a l t i n g - o u t c o n s t a n t . Here i t s h o u l d be men t i o n e d t h a t a n i n c r e a s e i n t h e s o l u b i l i t y o f ATGEE i n a s a l t s o l u t i o n corresponds t o a decrease i n the a c t i v i t y c o e f f i c i e n t (S /S). I o n s s u c h a s I " * , C 1 0 ^ " , SCN~ a n d C I 3 C C O O " e x h i b i t e d n e g a t i v e K v a l u e s , w h i l e S 0 ^ , ^PO^" a n d c i t r a t e h a d p o s i t i v e K values. C h l o r i d e a n d b r o m i d e e x h i b i t e d n e i t h e r s a l t i n g - i n nor s a l t i n g - o u t p r o p e r t i e s ; i . e . , t h e i r K v a l u e s were a l m o s t z e r o . I n a s i m i l a r a p p r o a c h S c h r i e r a n d S c h r i e r (21) s t u d i e d t h e s o l u b i l i t i e s o f N - m e t h y l a c e t a m i d e and N - m e t h y l p r o p i o n a m i d e i n various s a l t s o l u t i o n s . While theorder of effectiveness of v a r i ous n e u t r a l s a l t s a s a c t i v i t y c o e f f i c i e n t a f f e c t o r s were t h e same as t h a t o f ATGEE, t h e l e v e l a t w h i c h Κ c h a n g e s f r o m a p o s i t i v e t o a n e g a t i v e v a l u e f o r t h e s a l t s was d i f f e r e n t . F u r t h e r m o r e , i n c r e a s e i n t h e c h a i n l e n g t h o f t h e amide i n c r e a s e d t h e s a l t i n g out c o e f f i c i e n t i n d i c a t i n g t h a t t h e e f f e c t o f i o n s on t h e s o l u b i l i t y b e h a v i o r o f t h e s e m o d e l compounds was m a i n l y on t h e n o n p o l a r m o i e t i e s i n t h e s e m o d e l compounds. The s a l t i n g - o u t c o n s t a n t f o r N - m e t h y l a c e t a m i d e was 0.099 i n N a C l a n d -0.023 i n NaSCN. The change i n t h e f r e e e n e r g y f o r t r a n s f e r o f one mole o f N-methy l a c e t a m i d e f r o m w a t e r t o 1 M s a l t s o l u t i o n c a n be c a l c u l a t e d from the K v a l u e s by E q u a t i o n 4 ( 2 2 ) : Q

g

g

Q

g

=

g

g

g

AG

= 2.3 RTK C tr s s where C i s t h e m o l a r c o n c e n t r a t i o n o f t h e s a l t , R i s t h e g a s c o n s t a n t a n d Τ i s t h e t e m p e r a t u r e . A t 25°C, t h e f r e e e n e r g y o f t r a n s f e r o f N-methylacetamide from water t o 1M NaCl i s u n f a v o r a b l e t o t h e m a g n i t u d e o f a b o u t +133 c a l / m o l w h e r e a s t h e t r a n s f e r t o 1 M NaSCN i s f a v o r a b l e t o t h e o r d e r o f -30.9 c a l / m o l e . The e f f e c t s o f n e u t r a l s a l t s o n s o l u b i l i t y o f many a p o l a r g


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335

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s o l u t e s such as amides, benzene, purines and p y r i m i d i n e s , e t c . , i n aqueous s o l u t i o n s were reported (22, 23). These s t u d i e s with simple compounds r e v e a l that the n e u t r a l s a l t s a f f e c t both the p o l a r and apolar moieties i n these compounds and c e r t a i n ions e x h i b i t p o s i t i v e e f f e c t s while other ions have negative e f f e c t s on s o l u b i l i t y . In general the s a l t i n g - o u t e f f e c t of the anions and c a t i o n s f o l l o w the order =

S0, > C l ~ > Br" > NO ~ > CIO," > SCN~ > C l C C 0 0 " 4 3 4 3 + + + + 2+ 2+ 2+ NH > Κ ,Na > L i > Mg > Ca > Ba


o

Since p r o t e i n s have both p o l a r and apolar groups, more com parable to the above model compound, the s a l t i n g - i n or s a l t i n g out e f f e c t o f i o n s on these groups would have profound a f f e c t on the conformational s t a b i l i t y o f p r o t e i n s . Since the hydrophobic i n t e r a c t i o n between the apolar residues i s the major s t a b i l i z i n g force of the n a t i v e conformation o f the p r o t e i n , the ions which s a l t - o u t the hydrocarbons should enhance these hydrophobic i n t e r a c t i o n s i n the p r o t e i n and provide more s t a b i l i z i n g energy. In the presence of ions which e x h i b i t s a l t i n g - i n e f f e c t on hydro carbons , the hydrophobic i n t e r a c t i o n s i n the p r o t e i n would be de creased and hence d e s t a b i l i z e the p r o t e i n s t r u c t u r e . The simplest way to study the e f f e c t s o f ions on the confor mational s t a b i l i t y o f p r o t e i n s i s t o f o l l o w the change i n α-heli c a l content o f a p r o t e i n as a f u n c t i o n o f temperature a t d i f f e r ent s a l t concentrations. von Hippel and Wong (24) s t u d i e d the e f f e c t s of CaCl2 on the melting temperature o f c o l l a g e n at ρΗ 7.0. The melting temperature, Τ , which i s defined as the tem perature at which the t r a n s i t i o n from h e l i c a l conformation to random c o i l i s 50% complete, decreased l i n e a r l y with increase i n CaCl2 c o n c e n t r a t i o n . In a s i m i l a r approach von H i p p e l and Wong (25) s t u d i e d the temperature t r a n s i t i o n o f ribonuclease as a f u n c t i o n o f s a l t c o n c e n t r a t i o n . The p l o t s o f T versus s a l t con c e n t r a t i o n f o r many s a l t s e x h i b i t e d l i n e a r r e l a t i o n s h i p s . In general the r e l a t i o n s h i p between T and the s a l t concentration may be f u n c t i o n a l l y expressed as Equation 5 (16): m

m

Τ

m

= Τ ° + ΚC m ss

where T ° i s the t r a n s i t i o n temperature i n the absence o f s a l t , C i s the molar c o n c e n t r a t i o n o f the s a l t and K i s the molar e f f e c t i v e n e s s of the s a l t i n p e r t u r b i n g T . In the study with r i b o n u c l e a s e , PO^", S0^ and C l ~ e x h i b i t e d p o s i t i v e K values i n d i c a t i n g that these ions s t a b i l i z e d the p r o t e i n . Br and SCN" e x h i b i t e d negative K values i n d i c a t i n g that they d e s t a b i l i z e d the p r o t e i n s t r u c t u r e . The r e l a t i v e e f f e c t i v e n e s s o f both anions and c a t i o n s i n p e r t u r b i n g the s t a b i l i t y o f r i b o n u c l e a s e followed the same chaotropic or l y o t r o p i c order observed with model com pounds. One can assume that the observed e f f e c t s o f ions on the m

g

s

m

=

g

g



336

FOOD P R O T E I N

DETERIORATION

s t a b i l i t y o f p r o t e i n s c a n n o t be due t o s i m p l e n o n s p e c i f i c i n t e r a c t i o n s w i t h t h e i r charged groups. As mentioned i n t h e p r e v i o u s s e c t i o n , t h e f r e e e n e r g y c o n t r i b u t e d by t h e 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 t o t h e o v e r a l l s t a b i l i t y o f p r o t e i n i s v e r y s m a l l and hence cannot a c c o u n t f o r t h e magnitude o f t h e s a l t e f f e c t on p r o t e i n c o n f o r m a t i o n . I n most p r o t e i n s t h e 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 a r e e s s e n t i a l l y s u p p r e s s e d a t about 0.1-0.15 M s a l t c o n c e n t r a t i o n (26). H e n c e , t h e l y o t r o p i c e f f e c t o f i o n s o n t h e p r o t e i n conformation, which i s observed a t r e l a t i v e l y higher conc e n t r a t i o n s , may be due t o t h e i r e f f e c t on t h e h y d r o p h o b i c i n t e r a c t i o n s mediated v i a changes i n t h e w a t e r s t r u c t u r e . I f t h e c o n f o r m a t i o n a l changes i n p r o t e i n i n t h e presence o f v a r i o u s i o n s a r e due t o s t a b i l i z a t i o n o r d e s t a b i l i z a t i o n o f t h e h y d r o p h o b i c r e g i o n s t h a t a r e mediated v i a changes i n t h e w a t e r s t r u c t u r e , t h e n s u c h c h a n g e s w i l l be r e f l e c t e d i n t h e b i n d i n g a b i l i t y o f h y d r o p h o b i c l i g a n d s t o t h e p r o t e i n . Damodaran and K i n s e l l a (27) s t u d i e d t h e e f f e c t o f a n i o n s o n t h e b i n d i n g o f 2nonanone t o b o v i n e serum a l b u m i n . B i n d i n g o f 2-nonanone t o b o v i n e serum a l b u m i n e x h i b i t e d p o s i t i v e c o o p e r a t i v i t y w h i c h was i n t e r p r e t e d as b e i n g due t o i n i t i a l b i n d i n g i n d u c e d c o n f o r m a t i n a l changes i n t h e p r o t e i n w h i c h i n c r e a s e d t h e b i n d i n g a f f i n i t i e s a t t h e s u b s e q u e n t b i n d i n g s i t e s (27). I n o t h e r w o r d s , t h e p o s i t i v e c o o p e r a t i v i t y may be due t o s t a b i l i z a t i o n o f t h e h y d r o p h o b i c b i n d i n g s i t e s i n b o v i n e serum a l b u m i n upon i n i t i a l b i n d ing. As a c o r o l l a r y , i f t h e e x h i b i t i o n o f p o s i t i v e c o o p e r a t i v i t y i s due t o b i n d i n g i n d u c e d s t a b i l i z a t i o n o f t h e b i n d i n g s i t e s , then i n the presence o f s t r u c t u r e s t a b i l i z i n g o r d e s t a b i l i z i n g a g e n t s t h e d e g r e e o f s u c h s t a b i l i z a t i o n s h o u l d be i n c r e a s e d o r decreased, r e s p e c t i v e l y . The b i n d i n g i s o t h e r m s f o r t h e i n t e r a c t i o n o f 2-nonanone w i t h b o v i n e serum a l b u m i n i n t h e p r e s e n c e o f v a r i o u s a n i o n s i s shown i n F i g u r e 4. I n t h e p r e s e n c e o f C l " * , Br"", SCN" and C I 3 C C O O " , t h e c u r v e s a r e s i g m o i d a l , w h e r e a s i n t h e p r e s e n c e o f F " and S0^ , t h e y a r e h y p e r b o l i c . When t h e same d a t a a r e p r e s e n t e d i n t h e f o r m of Scatchard p l o t s , the b i n d i n g e x h i b i t e d p o s i t i v e c o o p e r a t i v i t y i n t h e p r e s e n c e o f a l l a n i o n s e x c e p t F~ ( F i g u r e 5 ) . The i n i t i a l p o s i t i v e slopes a t low molal r a t i o s o f binding v a r i e d d i s t i n c t l y w i t h the type o f a n i o n . The e f f e c t o f a n i o n s i n i n c r e a s i n g t h e i n i t i a l p o s i t i v e slope followed the c l a s s i c a l Hofmeister s e r i e s o r c h a o t r o p i c s e r i e s (18) ; i . e . , F" > S O ^ > CI"" > B r " > SCN" > CI3CCOO"". These i o n s , i n t h e same o r d e r , have b e e n known t o a f f e c t t h e w a t e r s t r u c t u r e (18) as w e l l as s t a b i l i t y o f g l o b u l a r p r o t e i n s (16). Apart from changing the s l o p e s o f t h e Scatchard p l o t s , these ions a l s o induced a s h i f t i n the p o s i t i o n o f the maximum i n t h e S c a t c h a r d p l o t away f r o m t h e o r d i n a t e . A s h i f t away f r o m t h e o r d i n a t e may r e f l e c t i n c r e a s e d c o o p e r a t i v i t y i n binding. I n t h i s r e s p e c t , t h e r e s u l t s show t h a t t h e b i n d i n g o f 2-nonanone t o b o v i n e serum a l b u m i n i s h i g h l y c o o p e r a t i v e i n t h e p r e s e n c e o f Cl^CCOO" and SCN" i n c o n t r a s t t o i t s b e h a v i o r i n t h e p r e s e n c e o f SO, and F". =

=



13.

DAMODARAN A N D KINSELLA

Ion Effects on

Proteins

337

Figure 4. Binding isotherms for the interaction of 2-nonanone to bovine^ serum albumin (BSA) in the presence of various anions at 0.15 M ionic strength, ν is the number of moles of 2-nonanone bound per mole of BSA and [L] is the free 2-non anone concentration. (Reproduced from Ref. 27.)





13.

DAMODARAN A N D K I N S E L L A

Ion Effects

on

Proteins

339

The above changes i n the b i n d i n g behavior o f 2-nonanone to bovine serum albumin i n the presence of v a r i o u s anions may be i n t i m a t e l y r e l a t e d to changes i n the p r o t e i n s t r u c t u r e . I n other words, the i n i t i a l p o s i t i v e slopes (Figure 5 ) , which may be cons i d e r e d to be p r o p o r t i o n a l to the net s t a b i l i z a t i o n o f the hydrophobic s i t e s upon l i g a n d b i n d i n g , are the r e s u l t o f two opposing f o r c e s ; i . e . , the d e s t a b i l i z i n g e f f e c t o f anions and the s t a b i l i z i n g e f f e c t o f 2-nonanone upon b i n d i n g . Assuming that the s t a b i l i z a t i o n energy provided by 2-nonanone b i n d i n g i s constant, the i n c r e a s e i n the d e s t a b i l i z i n g energy provided by d i f f e r e n t anions would o b v i o u s l y decrease the i n i t i a l p o s i t i v e slope i n the order of the l y o t r o p i c s e r i e s . If the d e s t a b i l i z a t i o n / s t a b i l i z a t i o n o f the p r o t e i n s t r u c ture by anions i s v i a changes i n the water s t r u c t u r e , then one would expect a c o r r e l a t i o n between the changes i n the b i n d i n g parameters and the p a r t i a l molar entropy o f the s o l u t i o n s o f v a r ious anions. F i g u r e 6 shows the c o r r e l a t i o n between the f r e e energy of i n t e r a c t i o n , AG , (27), which i s c a l c u l a t e d from the i n i t i a l p o s i t i v e s l o p e s , t i e H i l l c o e f f i c i e n t (28) which i n d i cates the degree o f cooperative changes, and the p a r t i a l molar entropy o f s o l u t i o n . I t i s obvious that as the p a r t i a l molar entropy o f the s o l u t i o n i s increased (which i n d i c a t e s increased d i s o r d e r l i n e s s of the water s t r u c t u r e ) , the hydrophobic s i d e chains i n the b i n d i n g s i t e s would be d i s s o c i a t e d mainly by a decrease i n the hydrophobic i n t e r a c t i o n between them. In such an environment, the b i n d i n g induced s t a b i l i z a t i o n o f the b i n d i n g s i t e s w i l l be r e l a t i v e l y unfavorable. This i s r e f l e c t e d by an increase i n the f r e e energy change (more p o s i t i v e ) with i n c r e a s e i n the p a r t i a l molar entropy o f the s o l u t i o n . However, the coo p e r a t i v i t y o f 2-nonanone b i n d i n g to bovine serum albumin i n creases with i n c r e a s e i n the p a r t i a l molar entropy o f the s o l u t i o n as revealed from the H i l l c o e f f i c i e n t . In the presence o f F~ i o n which has s a l t i n g - o u t e f f e c t on hydrocarbons, i t i s p o s s i ble that the s t r u c t u r a l s t a t e o f the b i n d i n g s i t e s would be compact so that the i n i t i a l b i n d i n g o f 2-nonanone would not r e s u l t i n f u r t h e r s t a b i l i z a t i o n o f the b i n d i n g s i t e s . In the presence of CI"*, Br"" and SCN" which have p o s i t i v e p a r t i a l molar entropy values (Table I; 18), the b i n d i n g s i t e s may have some degree of freedom f o r f u r t h e r strengthening o f these regions upon 2-nonanone b i n d i n g . T h i s i s r e f l e c t e d i n the c o o p e r a t i v i t y o f b i n d i n g . I t may be pointed out that the above b i n d i n g s t u d i e s with the various ions were done a t 0.15 M i o n i c s t r e n g t h . The r e s u l t s show that even a t t h i s i o n i c s t r e n g t h the anions e x h i b i t l y o t r o pic e f f e c t s on the hydrophobic f o r c e s i n the p r o t e i n , and such e f f e c t s f o l l o w the same Hofmeister s e r i e s as one would expect a t much higher s a l t c o n c e n t r a t i o n s . This i s f u r t h e r confirmed from the s o l u b i l i t y s t u d i e s o f 2-nonanone a t 0 to 0.15 M concentration of NaCl and NaSCN (Figure 7). Even a t t h i s low c o n c e n t r a t i o n , NaCl tends to s a l t - o u t 2-nonanone whereas NaSCN tends to s a l t - i n the hydrocarbon. This i n d i c a t e s that even a t t h i s low


FOOD PROTEIN

340

so;

I

CI

i

-

DETERIORATION

SCN

ΒΓ

- ?.o

1

l. G


_

i.e

-

-

-

0

10

1

1

20

30

L.2

m

P A R T I A L MOLAR ENTROPY

Figure 6. Relationship between partial molar entropy of solutions of various anions and the free energy of interaction, aG (O - O), and the Hill coefficient (• - •). (Reproduced from Ref. 27.) int

C10^, I " > B r " > CI"". S i n c e a p p a r e n t l y no 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 are i n v o l v e d i n the p o l y m e r i z a t i o n o f s i c k l e hemoglobin, the s a l t i n g - i n e f f e c t by s a l t s c a n n o t be due t o e l e c t r o s t a t i c s h i e l d ing effects. Even i 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 a r e i n v o l v e d , a s o l u t i o n a b o u t 0.15 M i o n i c s t r e n g t h s h o u l d be s u f f i c i e n t t o s u p p r e s s t h e s e i n t e r a c t i o n s ( 2 6 ) . The o b s e r v e d s a l t i n g - i n e f f e c t e v e n a t c o n c e n t r a t i o n s a s h i g h as 1 M i n d i c a t e s t h a t t h e



13.


1

Ο

0.1

Ion Effects on

1

I

0.2

0.3

[SODIUM S A L T ] Figure 11.

Proteins

1

i

0.4

0.5

349

(M)

Salting-in effects of anions on the solubility of deoxysickle hemoglobin. (Reproduced from Ref. 36.)



350

FOOD P R O T E I N

DETERIORATION

s o l u b i l i z a t i o n o f s i c k l e h e m o g l o b i n was due t o d e c r e a s e d h y d r o p h o b i c i n t e r a c t i o n s between s i c k l e hemoblobin m o l e c u l e s . This i s incompatible w i t h the n o t i o n that the increase i n surface t e n s i o n o f the s o l u t i o n should always r e s u l t i n s a l t i n g - o u t o f the p r o t e i n (33). A l s o , i t i s g e n e r a l l y b e l i e v e d t h a t t h e e f f e c t of i o n s on the h y d r o p h o b i c i n t e r a c t i o n s o c c u r o n l y a t h i g h e r s a l t c o n c e n t r a t i o n s (16)· The d a t a on s i c k l e h e m o b l o b i n and o u r r e s u l t s on t h e s o l u b i l i t y ( F i g u r e 7 ) and b i n d i n g o f 2-nonanone t o b o v i n e serum a l b u m i n ( 2 7 ) i n d i c a t e t h a t t h e l y o t r o p i c e f f e c t o f i o n s on h y d r o p h o b i c i n t e r a c t i o n s c a n o c c u r a t r e l a t i v e l y l o w concentrations. The argument t h a t t h e d e c r e a s e i n e l e c t r o s t a t i c interactions at low s a l t c o n c e n t r a t i o n s i s r e s p o n s i b l e f o r the s a l t i n g - i n e f f e c t (34, 35) may be v a l i d f o r s i m p l e g l o b u l a r p r o t e i n s . B u t , complex p r o t e i n systems, l i k e p r o t e i n s h a v i n g m u l t i s u b u n i t s o r s y s t e m s h a v i n g more t h a n one f r a c t i o n , may behave d i f f e r e n t l y a t low s a l t c o n c e n t r a t i o n s . F o r example, as the s a l t c o n c e n t r a t i o n i s i n c r e a s e d t o a b o u t 0.2 M i o n i c s t r e n g t h , t h e s o l u b i l i t y o f s o y p r o t e i n d e c r e a s e s , and t h e n i n c r e a s e s a t h i g h e r c o n c e n t r a t i o n s ( F i g u r e 12; 38, 39). I n t h i s r e s p e c t b o t h N a C l and C a C l exhib i t e d t h e same e f f e c t s ( 3 9 ) . S i m i l a r r e s u l t s were o b t a i n e d w i t h s o l u b i l i t y o f wheat g l u t e n i n v a r i o u s s a l t s o l u t i o n s (40). T h u s , i f the e l e c t r o s t a t i c e f f e c t a t low s a l t c o n c e n t r a t i o n has s a l t i n g i n e f f e c t s , t h e n how d o e s t h e s o l u b i l i t y o f s o y p r o t e i n d e c r e a s e at low s a l t c o n c e n t r a t i o n . T h i s may be due t o c o m p l e x i n t e r a c t i o n s i n v o l v i n g 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 f o r c e s b e tween t h e s u b u n i t s and d i f f e r e n t p r o t e i n f r a c t i o n s ( g l y c i n i n and c o n g l y c i n i n ) i n soy. Although the e l e c t r o s t a t i c s h i e l d i n g of the c h a r g e d g r o u p s by s a l t s may i n c r e a s e p r o t e i n s o l u b i l i t y , t h e d e c r e a s e i n t h e e l e c t r o s t a t i c r e p u l s i o n b e t w e e n p r o t e i n s may e n hance t h e h y d r o p h o b i c i n t e r a c t i o n b e t w e e n t h e i r n o n p o l a r s u r f a c e s and l e a d t o f o r m a t i o n o f a g g r e g a t e s . This i s f u r t h e r supported by t h e f a c t t h a t t h e o b s e r v e d s o l u b i l i t y i s minimum a t a b o u t 0.Ι Ο.15 M i o n i c s t r e n g t h ( F i g u r e 1 2 ) , a t w h i c h t h e e l e c t r o s t a t i c i n t e r a c t i o n i n p r o t e i n s i s s u p p r e s s e d ( 2 6 ) . The d a t a a l s o s u p p o r t s o u r c o n t e n t i o n t h a t even a t low c o n c e n t r a t i o n s t h e i o n s h a v e l y o t r o p i c e f f e c t s on t h e h y d r o p h o b i c i n t e r a c t i o n s . I f t h e i o n i c strength i s s o l e l y responsible f o r the decrease i n the s o l u b i l i t y a t low s a l t c o n c e n t r a t i o n s , then the s o l u b i l i t y l e v e l a t 0.15 M i o n i c s t r e n g t h s h o u l d be t h e same i r r e s p e c t i v e o f t h e n a t u r e o f t h e i o n . B u t t h e d a t a ( F i g u r e 12) show t h a t t h e minimum s o l u b i l i t y l e v e l a t 0.15 M i o n i c s t r e n g t h v a r i e s w i t h t h e n a t u r e o f t h e a n i o n and t h e i n c r e a s e i n s o l u b i l i t y a t t h i s i o n i c s t r e n g t h a p p a r e n t l y f o l l o w s t h e l y o t r o p i c s e r i e s ; i . e . , Cl"*= B r " < N O 3 < I"". T h i s c l e a r l y i n d i c a t e s t h a t e v e n a t t h i s l o w c o n c e n t r a t i o n t h e a n i o n s e x e r t l y o t r o p i c e f f e c t s on t h e h y d r o p h o b i c i n t e r a c t i o n s . B u t , the magnitude o f t h i s l y o t r o p i c ( s o l u b i l i z i n g ) e f f e c t a t t h i s l o w s a l t c o n c e n t r a t i o n may be s m a l l e r t h a n t h e magnitude o f t h e f a v o r a b l e h y d r o p h o b i c i n t e r a c t i o n between mole c u l e s and h e n c e r e s u l t i n i n s o l u b i l i t y . As t h e s a l t c o n c e n t r a t i o n 2


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i s i n c r e a s e d , t h e m a g n i t u d e o f t h e s e h y d r o p h o b i c i n t e r a c t i o n s may be e i t h e r i n c r e a s e d o r d e c r e a s e d d e p e n d i n g on t h e l y o t r o p i c n a t u r e o f t h e i o n and t h u s r e s u l t i n d e c r e a s e d o r i n c r e a s e d s o l u b i l i t y , respectively. To s u p p o r t t h e above a r g u m e n t , we s t u d i e d t h e b i n d i n g o f 8 - a n i l i n o n a p h t h a l e n e - 1 - s u l f o n a t e (ANS) t o s o y p r o t e i n i n t h e presence of v a r i o u s s a l t s . The r a t i o n a l e i n t h i s method i s t h a t when ANS i s h y d r o p h o b i c a l l y bound t o s o y p r o t e i n , i t s quantum y i e l d of fluorescence i s increased. Thus by m o n i t o r i n g t h e f l u o r e s c e n c e o f ANS i n a s o y p r o t e i n - A N S s y s t e m , i t i s p o s s i b l e t o measure t h e c h a n g e s i n t h e h y d r o p h o b i c i n t e r a c t i o n a s a function of salt concentration. The e f f e c t s o f s a l t s on t h e f l u o r e s c e n c e i n t e n s i t y o f ANS bound t o s o y p r o t e i n i s shown i n F i g u r e 1 3 . The ANS c o n c e n t r a t i o n was 1,54 χ 1 0 " ^ M and t h e p r o t e i n c o n c e n t r a t i o n was 0,0027%. A t t h i s p r o t e i n c o n c e n t r a t i o n no p r e c i p i t a t i o n was o b s e r v e d i n s a l t s o l u t i o n s a t t h e c o n c e n t r a t i o n range r e p o r t e d here. In the presence o f a l l the s a l t s s t u d i e d , the fluorescence i n t e n s i t y of ANS i n c r e a s e d up t o a b o u t 0.15 M s a l t c o n c e n t r a t i o n . The i n crease i n the f l u o r e s c e n c e i n t e n s i t y a t t h i s low i o n i c s t r e n g t h i s due t o an i n c r e a s e i n t h e h y d r o p h o b i c i n t e r a c t i o n b e t w e e n ANS and s o y p r o t e i n . The f l u o r e s c e n c e i n t e n s i t y u n d e r g o e s a t r a n s i t i o n a t a b o u t 0.15 M s a l t c o n c e n t r a t i o n and above 0.15 M t h e f l u o r e s c e n c e i n t e n s i t y e i t h e r i n c r e a s e s or decreases depending on t h e l y o t r o p i c e f f e c t o f t h e i o n on h y d r o p h o b i c i n t e r a c t i o n s . I n t h i s r e s p e c t S 0 ^ and F~ f u r t h e r i n c r e a s e d t h e f l u o r e s c e n c e , i n d i c a t i n g i n c r e a s e d h y d r o p h o b i c i n t e r a c t i o n b e t w e e n ANS and s o y p r o t e i n , and Br"", I"* and SCN~ i o n s d e c r e a s e t h e f l u o r e s c e n c e intensity. These r e s u l t s c l e a r l y d e m o n s t r a t e t h a t t h e o b s e r v e d d e c r e a s e i n t h e s o l u b i l i t y o f s o y p r o t e i n ( F i g u r e 11) a t l o w s a l t c o n c e n t r a t i o n s i s due t o enhancement 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 between p r o t e i n m o l e c u l e s . I n o t h e r w o r d s , t h e s a l t i n g - i n and s a l t i n g - o u t p r o f i l e o f p r o t e i n s by s a l t s a t v a r i o u s c o n c e n t r a t i o n s i s the n e t r e s u l t o f the maximization or m i n i m i z a t i o n o f 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 f o r c e s , a t d i f f e r e n t s a l t c o n c e n trations. I t w o u l d a l s o depend on t h e r a t i o o f n o n p o l a r s u r f a c e t o c h a r g e d e n s i t y on t h e s u r f a c e o f t h e p r o t e i n . I f t h e p r o t e i n has l a r g e r n o n p o l a r s u r f a c e a r e a s t h e d e c r e a s e i n t h e e l e c t r o s t a t i c i n t e r a c t i o n a t l o w s a l t c o n c e n t r a t i o n s w o u l d promote h y drophobic aggregation of such p r o t e i n s r e s u l t i n g i n decreased solubility. At higher s a l t c o n c e n t r a t i o n s , f u r t h e r decrease or i n c r e a s e i n s o l u b i l i t y depends on t h e l y o t r o p i c n a t u r e o f t h e ion. I n soy p r o t e i n , S 0 ^ f u r t h e r decreases the s o l u b i l i t y whereas o t h e r anions i n c r e a s e the s o l u b i l i t y i n the o r d e r o f C I " < B r " < NO3" < I*" ( F i g u r e 1 2 ) . On t h e o t h e r h a n d , i f t h e h y d r o p h o b i c s u r f a c e a r e a on t h e p r o t e i n i s l e s s and t h e c h a r g e d e n s i t y i s h i g h , t h e p r o t e i n w i l l be s a l t e d - i n a t l o w e r s a l t c o n c e n t r a t i o n s and a t h i g h e r s a l t c o n c e n t r a t i o n s t h e s o l u b i l i t y w o u l d f o l l o w t h e l y o t r o p i c e f f e c t o f d i f f e r e n t i o n s . Such com p l e x i n t e r p l a y o f d i f f e r e n t f o r c e s may be t h e r e a s o n f o r t h e =

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Figure 13. Effect of sodium salts on the binding of 8-anilinonaphthalene-l-sulfo nate (ANS) to soybean protein in 30 mM tris-HCl buffer, pH 8.0. Protein and ANS concentrations were 0.04% and 1.5 χ 10 M , respectively. ANS binding was monitored as the increase in fluorescence intensity at 485 nm with excitation at 360 nm. 6


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354

observed d i f f e r e n c e s i n the s o l u b i l i t y b e h a v i o r of v a r i o u s p r o t e i n s i n s a l t s o l u t i o n s (36, 38, 4 0 , 41).


Mechanisms o f S a l t E f f e c t on P r o t e i n

Structure

The e f f e c t s o f i o n s on p r o t e i n c o n f o r m a t i o n and f u n c t i o n may be d i v i d e d i n t o two c a t e g o r i e s : First, their electrostatic i n t e r a c t i o n w i t h t h e c h a r g e d g r o u p s and p o l a r g r o u p s ( e . g . , amide g r o u p s ) i n p r o t e i n s , and s e c o n d , t h e i r e f f e c t on h y d r o p h o b i c f o r c e s v i a i n f l u e n c e on t h e s t r u c t u r e o f w a t e r . From t h e f o r e g o i n g arguments t h e e l e c t r o s t a t i c s h i e l d i n g e f f e c t w h i c h r e a c h e s a maximum a t 0.15 M i o n i c s t r e n g t h may n o t have g r e a t i n f l u e n c e on t h e c o n f o r m a t i o n a l t r a n s i t i o n s i n p r o t e i n s t h a t a r e more p r o n o u n c e d a t h i g h s a l t c o n c e n t r a t i o n s . The s a l t may i n t e r a c t w i t h the p e p t i d e groups i n p r o t e i n s (42, 4 3 ) . Mandelkern e t a l . (44) have s u g g e s t e d t h a t s a l t i n t e r a c t i o n w i t h t h e p e p t i d e g r o u p s may a l t e r t h e p a r t i a l d o u b l e bond c h a r a c t e r o f t h e p e p t i d e bond. T h i s p e r m i t s f r e e r o t a t i o n o f the p e p t i d e backbone w h i c h m i g h t l e a d t o c o n f o r m a t i o n a l changes i n p r o t e i n s . A l t h o u g h the p o s s i b i l i t y o f s u c h i n t e r a c t i o n s b e t w e e n s a l t s and p e p t i d e g r o u p s e x i s t , i t has b e e n shown t h a t t h e h y d r a t i o n o f t h e i o n s g r e a t l y p r e v e n t s f o r m a t i o n o f s u c h c o m p l e x e s ( 4 5 ) . Hence i n aqueous s o l u t i o n s , c o n c e n t r a t i o n s a t which the ions induce c o n f o r m a t i o n a l changes i n p r o t e i n s , i t i s u n l i k e l y t h a t t h e f o r m a t i o n o f t h e c o m p l e x b e t w e e n i o n s and p e p t i d e g r o u p s i s t h e m a j o r c a u s e f o r t h e c o n f o r m a t i o n a l changes i n p r o t e i n s . Although i t i s thought t h a t s u c h a mechanism e x i s t s ( 4 3 ) , 46, 47) t h e r e a r e no d i r e c t evidence to support t h i s h y p o t h e s i s . The c o n f o r m a t i o n a l c h a n g e s i n p r o t e i n s i n t h e p r e s e n c e o f i o n s may be m a i n l y v i a i o n i c e f f e c t s on w a t e r s t r u c t u r e (16, 19). As m e n t i o n e d e a r l i e r , t h e n a t i v e s t r u c t u r e o f a p r o t e i n i s i m p o s e d by t h e s t a t e o f t h e w a t e r s t r u c t u r e b e c a u s e o f i t s t h e r m o dynamically unfavorable i n t e r a c t i o n with nonpolar sidechains. The c h a n g e s i n t h e h y d r o g e n bonded s t r u c t u r e o f b u l k w a t e r i n t h e p r e s e n c e o f s a l t s , due t o i o n - d i p o l e i n t e r a c t i o n , w o u l d a l t e r t h e d e g r e e o f h y d r a t i o n as w e l l as t h e o r i e n t a t i o n o f t h e w a t e r m o l e c u l e s around the n o n p o l a r s i d e c h a i n s . I n t h i s r e s p e c t , the i o n s w h i c h enhance t h e h y d r o g e n bonded s t r u c t u r e o f t h e b u l k w a t e r ( e . g . , F", S0^ ) p r e s u m a b l y h a v e t h e t e n d e n c y t o i n c r e a s e the degree of h y d r a t i o n of the n o n p o l a r groups i n p r o t e i n s . The i o n s w h i c h i n c r e a s e t h e e n t r o p y o f t h e b u l k w a t e r ( e . g . , B r " , I"", C10^~, SCN") tend to decrease the h y d r a t i o n o f the hydrophobic s i d e c h a i n s . As m e n t i o n e d e a r l i e r , t h e d r i v i n g f o r c e f o r t h e h y d r o p h o b i c i n t e r a c t i o n s between the n o n p o l a r m o l e c u l e s i s the e n t r o p i c a l l y u n f a v o r a b l e water-water o r i e n t a t i o n i n the h y d r a t i o n s p h e r e o f t h e h y d r o c a r b o n ( F i g u r e I B ) . Such r e l a t i v e o r i e n t a t i o n o f the water m o l e c u l e s around the hydrocarbon e x e r t s an i n t e r f a c i a l t e n s i o n o f a b o u t 51 e r g s / c m ^ o f w a t e r - h y d r o c a r b o n c o n t a c t a r e a (48). I n o t h e r w o r d s , s e p a r a t i o n o f 1 cm^ a r e a o f w a t e r - h y d r o c a r b o n i n t e r f a c e t o f o r m 1 cm^ o f w a t e r - w a t e r and =


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z

1 cm o f hydrocarbon-hydrocarbon i n t e r f a c e s w i l l be favored by about -102 e r g s . I t i s t h i s f r e e energy which i s r e s p o n s i b l e f o r the formation o f hydrophobic regions i n p r o t e i n s . The ions which s t a b i l i z e or d e s t a b i l i z e these hydrophobic regions may do so by i n c r e a s i n g o r d e c r e a s i n g the i n t e r f a c i a l tension, r e s p e c t i v e l y . I f we a s s i g n an i n t e r f a c i a l t e n s i o n o f 51 ergs/cm f o r the waterwater o r i e n t a t i o n i n the h y d r a t i o n s h e l l as shown i n Figure IB, (13), the ions which i n c r e a s e the i n t e r f a c i a l t e n s i o n above 51 ergs/cm^ may do so by a l t e r i n g the water-water geometry i n the h y d r a t i o n s h e l l i n such a way that the r e p u l s i o n between the lone p a i r o f e l e c t r o n s and the protons i s i n c r e a s e d . Conversely, the ions which decrease the i n t e r f a c i a l tension below 51 ergs/cm^ may do so by changing the water-water geometry such that the r e p u l s i v e i n t e r a c t i o n s between the lone p a i r o f e l e c t r o n s and the hydrogen atoms are decreased. However, such changes i n the water-water o r i e n t a t i o n i n the h y d r a t i o n s h e l l o f hydrocarbon by ions i s only through the changes i n the bulk water s t r u c t u r e , which can be d e f i n e d i n terms o f the p a r t i a l molar entropy. One can e x p e r i m e n t a l l y determine the decrease i n the i n t e r f a c i a l tension between water and hydrocarbon i n the presence of v a r i o u s i o n s . From these e s t i m a t i o n s and assuming that 51 ergs/cm corresponds to water-water o r i e n t a t i o n as shown i n Figure IB (12, 13), i t may be p o s s i b l e to c a l c u l a t e the r e l a t i v e water-water o r i e n t a t i o n around the hydrocarbon with a s t a t i s t i c a l - m e c h a n i c a l approach f o r v a r i o u s i n t e r f a c i a l tensions i n the presence of d i f f e r e n t i o n s . T h i s would provide an understanding of the r e l a t i o n s h i p s between the p a r t i a l molar entropy, i n t e r f a c i a l t e n s i o n and the hydrophobic e f f e c t and the i n f l u e n c e o f these e f f e c t s on p r o t e i n conformation and f u n c t i o n . Conclusion The primary e f f e c t of i o n s on p r o t e i n s t r u c t u r e a t low conc e n t r a t i o n i s t h e i r e l e c t r o s t a t i c i n t e r a c t i o n with the counter charges on the p r o t e i n . Such charge n e u t r a l i z a t i o n may induce d i s s o c i a t i o n o f the subunits i n o l i g o m e r i c p r o t e i n s which are h e l d together by 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 . At s u f f i c i e n t l y higher c o n c e n t r a t i o n s the ions e i t h e r weaken or strengthen the hydrophobic i n t e r a c t i o n s i n p r o t e i n s , depending on the nature o f the i o n . Such e f f e c t s on hydrophobic i n t e r a c t i o n s are mediated v i a changes i n the water s t r u c t u r e i n the presence of i o n s . However, some of the evidences suggest that even a t low concentrat i o n s (0.15 M) anions a f f e c t hydrophobic i n t e r a c t i o n s . The e f f e c t i v e n e s s of v a r i o u s anions i n d e s t a b i l i z i n g hydrophobic i n t e r a c t i o n s f o l l o w the Hofmeister s e r i e s . The changes i n e l e c t r o s t a t i c and hydrophobic f o r c e s i n p r o t e i n s by ions induce c o n f o r mational changes i n p r o t e i n s and hence a f f e c t t h e i r f u n c t i o n a l behavior. Systematic i n v e s t i g a t i o n of such e f f e c t s may provide b e t t e r understanding of the f a c t o r s a f f e c t i n g the f u n c t i o n a l p r o p e r t i e s of p r o t e i n s i n food systems.


356

FOOD P R O T E I N

DETERIORATION

Acknowledgment Support from the N a t i o n a l S c i e n c e F o u n d a t i o n Grant //CPE 80-18394 i s g r a t e f u l l y a c k n o w l e d g e d .

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RECEIVED June1,1982.


Food Protein Deterioration - American Chemical Society

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