Protein Functionality in Foods - American Chemical Society

5

Solubility and Viscosity JEROME L. SHEN Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 19, 2015 | http://pubs.acs.org Publication Date: March 6, 1981 | doi: 10.1021/bk-1981-0147.ch005

Ralston Purina, 900 Checkerboard Square, St. Louis, MO 63188

The manner in which proteins behave in a given food system or food application, i.e. its functionality, is a manifestation of the fundamental physicochemical properties of the proteins under the given conditions. Food systems are generally very complex, involving water, several types of protein as meat or soy, fats, salts, flavor and color compounds. Further, the functional properties of the food systems, generally, and that of soy proteins, particularly, are sensitive to past processing history, methods of preparation, and conditions of measurement. Because of this, it has been difficult to standardize functional testing, generalize the findings, and use the measured results from one system to predict the behavior in another system. Measurements that can have general applicability must be based upon the fundamental physiochemical properties of the components. Further, in order to predict functional behavior of the protein, it is necessary to determine the particular physicochemical states and interactions of the protein that result in the desired functionality. Solubility and viscosity are two experimentally measurable properties that can yield information about the functional behavior as well as the physicochemical nature of the proteins. In this chapter, the application of these two measurements to isolated soy proteins and how these measurements can explain the basic physicochemical nature of isolated soy proteins are discussed. Solubility The requirements for the thermodynamic definition of solubility are 1) well defined initial solid and final solution states, and 2) establishment of equilibrium between these two states. Under these conditions, the solubility at a given temperature and pressure is the concentration of the sample in solution. At a given temperature and pressure, the solid state is the stable crystalline state; and the solution state is 0097-6156/81/0147-0089$05.25/0 © 1981 American Chemical Society

In Protein Functionality in Foods; Cherry, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

90

PROTEIN FUNCTIONALITY

IN FOODS

f i x e d w i t h regard to such v a r i a b l e s as degree of p r o t e i n assoc i a t i o n , d i s s o c i a t i o n and p r o t e i n conformation. Defined i n t h i s manner, the thermodynamic s o l u b i l i t y i s independent of p a t h . As long as the i n i t i a l and f i n a l s t a t e s are unchanged, the s o l u b i l i t y w i l l not depend upon how the f i n a l s t a t e i s achieved. For example, i n the f o l l o w i n g r e a c t i o n

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 19, 2015 | http://pubs.acs.org Publication Date: March 6, 1981 | doi: 10.1021/bk-1981-0147.ch005

A (25°C, lAtm)? Crystal

A (25°C, lAtm, pH 7, I-.1M) Solution

i t makes no d i f f e r e n c e whether the f i n a l e q u i l i b r i u m i s approached by d i s s o l v i n g more and more c r y s t a l l i n e A u n t i l the s o l u t i o n i s s a t u r a t e d , by s t a r t i n g with a saturated s o l u t i o n of A at 35°C and c o o l i n g to 25°C, or by d i s s o l v i n g A i n water and then adding s a l t u n t i l 0.1M i o n i c strength i s reached. As long as e q u i l i b r i u m i s e s t a b l i s h e d , the s o l u b i l i t y w i l l be unchanged. This type of behavior when p l o t t e d i n percent s o l u b l e p r o t e i n versus amount of s o l i d p r o t e i n added i s i l l u s t r a t e d i n Figure 1. As s o l i d A i s added to the s o l v e n t , 100 percent of the added p r o t e i n w i l l remain i n s o l u t i o n u n t i l a s a t u r a t i o n l i m i t i s reached. A f t e r t h a t , the percent i n s o l u t i o n w i l l drop off and approach zero a s y m p t o t i c a l l y as the amount of added p r o t e i n a p p r o a c h e s * i n f i n i t y . In c o n t r a s t , Figure 2 shows t h a t the percentage of p r o t e i n i n s o l u t i o n f o r soy i s o l a t e s remains constant as the amount of added p r o t e i n i s increased (1_). In other words, the amount of p r o t e i n i n s o l u t i o n increases l i n e a r l y w i t h i n c r e a s i n g amounts of added p r o t e i n . This behavior i s observed f o r a l l the i s o l a t e s we have studied up to the highest c o n c e n t r a t i o n of 18 percent. Thus, soy i s o l a t e s behave as i f they are composed of a completely s o l u b l e f r a c t i o n (A) and a completely i n s o l u b l e f r a c t i o n (B). Upon the a d d i t i o n of s o l v e n t , the s o l u b l e f r a c t i o n (A) d i s s o l v e s completely w h i l e the i n s o l u b l e f r a c t i o n (B) remains unchanged. There i s no e q u i l i b r i u m e s t a b l i s h e d between A and B such t h a t , i f B i s separated from A and r e s l u r r i e d i n a d d i t i o n a l amounts of s o l v e n t , no a d d i t i o n a l p r o t e i n w i l l go i n t o s o l u t i o n . (More p r e c i s e l y , no evidence of microscopic r e v e r s i b i l i t y was found on the time s c a l e of the experiment, i . e . 2 h r s . at 25°C. A c t u a l l y , f o r a l l the i s o l a t e s s t u d i e d , the amount of p r o t e i n i n s o l u t i o n at 25°C reaches a maximum plateau a f t e r 100 minutes i n a shaker b a t h . Thus, at l e a s t s t e a d y - s t a t e c o n d i t i o n s are reached i n our experiments. It i s not known whether much longer e q u i l i b r a t i o n times, days, w i l l change the amount of s o l u b l e p r o t e i n . ) Thus, a key requirement f o r thermodynamic s o l u b i l i t y i s not met. F u r t h e r , n e i t h e r the i n i t i a l s o l i d s t a t e nor the f i n a l s o l u t i o n s t a t e are w e l l - d e f i n e d . The i n i t i a l s t a t e i s a heterogeneous amorphous s o l i d . The d i s t r i b u t i o n of the f r a c t i o n s A and B depends upon the previous h i s t o r y of the sample, such as manner of e x t r a c t i o n , p r e c i p i t a t i o n , solvent



5.

SHEN

Solubility

and

Figure 1.

Solubility

Soy

91

Viscosity

behavior of a pure crystalline

protein

Proteins

100 *

75 t_

= 301 D 0

23 h

2 4 6 8 10 P r o t « i n a d d t d (g/lOOg) Cereal Chemistry Figure 2.

Solubility

behavior of soy protein isolates (1)



92


IN FOODS

treatment, heat treatment, and d r y i n g . The amount and the nature of the p r o t e i n i n s o l u t i o n depends upon how the sample i s put i n t o s o l u t i o n ( s l u r r y and blending methods, Figure 3 ) , upon how the i n s o l u b l e f r a c t i o n i s removed ( c e n t r i f u g a t i o n c o n d i t i o n s , Figure 4 ) , and upon the path by which the f i n a l s t a t e i s reached. For example, i n the f o l l o w i n g experiment, the s o l u b i l i t y i s very much dependent upon the path f o r reaching the f i n a l s t a t e . Percent 20ml H2O 5ml 1M NaCl ^ Soluble „ ^ 82.7 1 Hr, 25°C 2 H r s , 25°C Protein slurry K).2M NaCl 25°C 20ml 0.2M NaCl " 1 h r , 25°C

_5ml 0.2M NaC 1 h r , 25°C

36.2

I n t e r p r e t a t i o n of Soy P r o t e i n S o l u b i l i t y Experiments This observed behavior of soy p r o t e i n s complicates the d e f i n i t i o n of soy p r o t e i n s o l u b i l i t y , the comparison of s o l u b i l i t y d a t a , and the i n t e r p r e t a t i o n of s o l u b i l i t y experiments. Because the thermodynamic c r i t e r i a are not met, p r o t e i n s o l u b i l i t y becomes an o p e r a t i o n a l l y defined q u a n t i t y that depends upon the experimental methods of measurement. A number of d i f f e r e n t o p e r a t i o n a l d e f i n i t i o n s have been used t o measure p r o t e i n s o l u b i l i t y . Each has i t s own advantages and d i s a d v a n tages, and l i m i t e d u t i l i t y . This p l u r a l i t y , though often d e s i r a b l e , makes i t d i f f i c u l t t o compare experimental r e s u l t s . I n t e r p r e t a t i o n of s o l u b i l i t y data i n terms of the f o r c e s and the i n t e r a c t i o n s at the molecular l e v e l i s an even more d i f f i c u l t problem. Because thermodynamic c r i t e r i a are not met, s t r a i g h t f o r w a r d , thermodynamic analyses cannot be a p p l i e d . F u r t h e r , s t r i c t comparison of the r e s u l t s f o r soy p r o t e i n s w i t h the r e s u l t s of systems that meet the thermodynamic c r i t e r i a cannot be j u s t i f i e d . However, i t may be v a l i d t o draw some q u a l i t a t i v e i n s i g h t s i n t o the nature of soy p r o t e i n by making comparisons under favorable circumstances. One such f a v o r a b l e case would be the f o l l o w i n g h y p o t h e t i c a l mechanism Reaction I Reaction II

nA«=*A A —* B

n

n

in which s o l u b l e monomers A r e v e r s i b l y form aggregates (A ) followed by the i r r e v e r s i b l e conversion of the aggregate (A ) i n t o the i n s o l u b l e p r e c i p i t a t e ( B ) . The aggregates are l i k e the i n s o l u b l e p r e c i p i t a t e s i n a l l respects except f o r the f a c t o r s that prevent B from being r e v e r s i b l y r e s o l u b i l i z e d . If n

n


SHEN

Solubility

and

Viscosity


°c

I

I

I

I

1

2

3

i 4

! 5 6

I

I

I

7

8

I 9

I

I

I

10 11

Blending Speed (rpm x 10 ) 3

Cereal Chemistry Figure 3. The effect of blending on solubility (I). Samples A, B,D, and E are soy protein isolates. Sample C is a commercial sodium caseinate. T is the temperature of the slurry after blending.

J 5

J

J

I

I

1

6 7 8 9 Log Centrifugal Force (g) x Time (sec)

Cereal Chemistry Figure

4. The effect of centrifugation on solubility (I). Samples A, B, D, and E are soy protein isolates. Sample C is a commercial sodium caseinate.


94


IN FOODS

r e a c t i o n (I) i s f a s t e r than r e a c t i o n ( I I ) , then a s t e a d y - s t a t e c o n d i t i o n w i l l r e s u l t i n which the concentration of A remains r e l a t i v e l y c o n s t a n t . The r a t e of formation of B w i l l be p r o p o r t i o n a l to the steady s t a t e concentration of A , and the amount of B formed i n a given time span w i l l be a measure of the steady s t a t e c o n c e n t r a t i o n of A . Since steady s t a t e e q u i l i b r i u m c o n d i t i o n s are reached, thermodynamic treatment of e q u i l i b r i u m II i s v a l i d . n

n

n


Aggregation

and S o l u b i l i t y

There i s much i n d i r e c t evidence t h a t the above h y p o t h e t i c a l mechanism f i t s the behavior of the soy p r o t e i n s . Wolf and Tamura ( 2 J , w h i l e studying the heat denaturation of n a t i v e U S soy p r o t e i n , found that s o l u b l e aggregates are formed p r i o r to the formation of i n s o l u b l e p r e c i p i t a t e s . They proposed the f o l l o w i n g mechanism to e x p l a i n t h e i r r e s u l t s . 11S

* subunits A

+

C subunits] B

s o l u b l e aggregates i n s o l u b l e aggregates Catsimpoolas et a l . (3) and Hermansson (4) have found t h a t the r e a c t i o n leading to formation of the s o l u b l e aggregates i s reversible. R e c e n t l y , Takagi et a l . (_5) found that hydrophobic i n t e r a c t i o n s are p r i m a r i l y r e s p o n s i b l e f o r the formation of the s o l u b l e aggregates which are subsequently i r r e v e r s i b l y i n s o l u b i l i z e d through i n t e r m o l e c u l a r d i s u l f i d e bond interchange. We have used t u r b i d i t y to monitor the heat induced aggregat i o n of an u n f r a c t i o n a t e d , a c i d p r e c i p i t a t e d , spray d r i e d , soy p r o t e i n i s o l a t e as a f u n c t i o n of i o n i c strength and pH. The t u r b i d i t y data (Figures 5, 6, 7) when c o r r e l a t e d w i t h the corresponding s o l u b i l i t y data at 25°C show t h a t solvent c o n d i t i o n s f a v o r i n g aggregation a l s o favor i n s o l u b i l i z a t i o n . This i s i n d i r e c t evidence that the r e l a t i v e amounts of i n s o l u b l e p r e c i p i t a t e formed as a f u n c t i o n of changing s o l v e n t composition are measures of the r e l a t i v e e f f e c t s of these solvents on the steady s t a t e e q u i l i b r i u m between the s o l u b l e p r o t e i n and s o l u b l e aggregates. Our data i s i n general q u a l i t a t i v e agreement with those compiled by Hermansson who used a s l i g h t l y d i f f e r e n t procedure. Salt

Effects

Neutral s a l t s are known to exert s t r i k i n g e f f e c t s on the s o l u b i l i t y , the a s s o c i a t i o n - d i s a s s o c i a t i o n e q u i l i b r i u m , the



5.

SHEN

Figure

Solubility

5.

and

95

Viscosity

The effect of ionic strength on solubility bidity ( ) at pH 2

(S) and the heat-induced

T


tur-


96

PROTEIN FUNCTIONALITY I N FOODS

Figure

7.

The effect of ionic strength on solubility bidity (T) at pH 7

Table I.

(S) and the heat-induced

Relative Effectiveness of Various Ions in Stabilizinq the "Native" Form of Collaqen and Ribonuclease r~6) and the Predicted Relative Order using the Molal Surface Tension Increment (7) -

Heli -Nativ Salting-ou? Collagengelatin:

SO

2-

Coil DenaturedSalting-in

I i n s a l t i n g out e f f i c a c y . In the case of the SOzp a n i o n , there i s the i n i t i a l steep s a l t i n g o u t . But there are plateaus between 0.1 and 0.3M s a l t c o n c e n t r a t i o n instead of the minimum s o l u b i l i t i e s found i n the cases of the monovalent a n i o n s . A f t e r the p l a t e a u , the s o l u b i l i t i e s decrease g r a d u a l l y 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 tion. Q u a n t i t a t i v e l y , these two i s o l a t e s d i f f e r i n t h e i r i n i t i a l s o l u b i l i t i e s i n water and i n the magnitude of the s a l t effects. For a given s a l t , DPI i s on the average s a l t e d out twice as much as NPI. Our data agree q u a l i t a t i v e l y w i t h those of Hermansson (4) except f o r the lack of a minimum i n the Na2S04 c u r v e .


-

E f f e c t of S a l t on Other P r o t e i n s and Model

Polypeptides

For comparison w i t h these d a t a , the types of s a l t e f f e c t s t h a t have been observed f o r model p r o t e i n and polypeptide systems t h a t achieve true thermodynamic e q u i l i b r i u m i n s o l u t i o n can be summarized i n t o three c l a s s e s : Class I: P r o t e i n s o l u b i l i t y i s f i r s t increased ( s a l t i n g in) and then decreased ( s a l t i n g out) as 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 . Thus, there i s a maximum peak i n the solubility profile. Examples of p r o t e i n s that show t h i s behavior are carboxylhemoglobin and f i b r i n o g e n (7). Class I I : P r o t e i n s o l u b i l i t y i s e i t h e r monotonically increased or decreased by 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 . The model polypeptide a c e t y l t e t r a g l y c i n e e t h y l e s t e r (ATGEE) behaves i n t h i s manner. C i t r a t e s , s u l f a t e s , phosphates, a c e t a t e s , and c h l o r i d e s s a l t out ATGEE; whereas phenol, p e r c h l o r a t e s , t o s y l a t e s , t r i c h l o r o a c e t a t e s , t h i o c y a n a t e s , i o d i d e s , and bromides s a l t i n (8). The heat denaturation of ribonuclease a l s o f i t s t h i s cTass. Class I I I : Protein s o l u b i l i t y and i s then increased ( s a l t i n g c o n c e n t r a t i o n . Except f o r the other p r o t e i n s to my knowledge s o l u b i l i t y behavior. However,

i s decreased ( s a l t i n g out) in) by i n c r e a s i n g s a l t soy i s o l a t e s , there are no t h a t e x h i b i t t h i s type of i n the r e l a t e d p o l y m e r i z a -


100


IN FOODS

t i o n and denaturation r e a c t i o n s , there are examples of t h i s type of behavior. Unpolymerized G - a c t i n i s caused to p o l y merize to F - a c t i n by 0.1M N a l , but F - a c t i n i s depolymerized by 0.5M Nal (9). The u n f o l d i n g of the h e l i c a l s t r u c t u r e of DNA a l s o f i t s t h i s c l a s s ( 6 ) .


The Theory of Melander and Horvath (7,

10)

R e c e n t l y , Melander and Horvath (_7, 10) have proposed a s i n g l e theory to account f o r the e f f e c t s of n e u t r a l s a l t s on the e l e c t r o s t a t i c and the hydrophobic i n t e r a c t i o n s i n the s a l t i n g out and the chromatography of p r o t e i n s . In s i m p l i f i e d terms, the theory accounts f o r the s o l u b i l i t y of p r o t e i n s i n terms of two c o n t r i b u t i o n s , e l e c t r o s t a t i c and hydrophobic i n nature. l n (W/Wo) = ( o F i e c t r o s t a t i c ) / R T

+ (AF

e

c a v i t

y ) / R T + Const

where W i s the weight of p r o t e i n s o l u b l e i n 1 l i t e r s a l t s o l u t i o n of given c o n c e n t r a t i o n and Wo i s the weight of p r o t e i n s o l u b l e i n 1 l i t e r of H2O. The e l e c t r o s t a t i c term, A F ] t r o s t a t i c » "> * change i n e l e c t r o s t a t i c f r e e energy when the p r o t e i n goes from the c r y s t a l l i n e s t a t e to the s o l u t i o n s t a t e . Melander and Horvath have expressed t h i s term by combining the proper terms from the Debye-Huckel and Kirkwood t h e o r i e s (7). This term i s always p o s i t i v e , and i s r e s p o n s i b l e f o r s a l T i n g i n . The hydrophobic term, F c U y , i s the f r e e energy r e q u i r e d t o c r e a t e a c a v i t y i n the bulk solvent to house the hydrophobic groups that are exposed when a p r o t e i n goes from a c r y s t a l l i n e s t a t e to a s o l u t i o n s t a t e . e

e c

s

t

i e

a v

AF

c

a

v

i ty = -ft

o m 7s, - AA O m

where X i i s a term p r o p o r t i o n a l to the increase i n hydrophobic surface area (AA) as a p r o t e i n goes i n t o s o l u t i o n , a i s the molal surface tension increment and m i s the m o l a l i t y . The molal surface t e n s i o n increment, a , i s defined by the r e l a t i o n s h i p : y = y + o m where T i s the surface t e n s i o n of the s a l t s o l u t i o n a n d T o i s the surface t e n s i o n of water. For i n o r g a n i c n e u t r a l s a l t s , a i s p o s i t i v e . Thus, f o r these s a l t s , A F - j t y i s always negative i f hydrophobic surface area i s exposed upon s o l u b i l i z a t i o n (AA i s p o s i t i v e ) . This term accounts f o r the s a l t i n g out of p r o t e i n s . The combination of the hydrophobic s a l t i n g out and the e l e c t r o s t a t i c s a l t i n g i n terms e x p l a i n s very n i c e l y the C l a s s I type behavior. The data f o r carboxyhemoglobin and f i b r i n o g e n are w e l l f i t t e d by the theory (Figure 10). Also the order of decreasing molal surface t e n s i o n increment g e n e r a l l y f o l l o w s the l y o t r o p i c order (_7). 0

c a v



5.

SHEN

Solubility

and

Viscosity

101

The theory f a i l s to e x p l a i n the behavior of soy p r o t e i n s (Class I I I ) because the s a l t i n g i n e l e c t r o s t a t i c term i s more dominant at low s a l t c o n c e n t r a t i o n s . As s a l t concentrations i n c r e a s e , the c a v i t y term becomes more dominant. The o v e r a l l r e s u l t i s s a l t i n g i n followed by s a l t i n g o u t . The f a i l u r e of the theory i n d i c a t e s that soy p r o t e i n behavior i s more complex than Melander and Horvath's (_7, 10) model. In d e r i v i n g t h e i r simple theory, Melander and Horvath assumed that the changes i n hydrophobic surface area, i n the d i p o l e moment, and i n the net charge of the p r o t e i n upon s o l u b i l i z a t i o n are i n v a r i a n t w i t h respect to s a l t species or s a l t c o n c e n t r a t i o n . In other words, they assumed t h a t the s o l u b l e p r o t e i n s have the same thermodynamic s t a t e r e g a r d l e s s of s a l t species or s a l t c o n c e n t r a t i o n . The r e s u l t s f o r soy p r o t e i n s can be t r e a t e d i n the framework of the Melander and Horvath's theory i f i t i s expanded to allow the exposed surface a r e a , the d i p o l e moment, and the net p r o t e i n charge to be f u n c t i o n s of the s a l t species and the s a l t c o n c e n t r a t i o n . Thus, there are three d i f f e r e n t ways to account f o r t h i s soy p r o t e i n behavior as f o l l o w s : 1)

Exposed surface i s decreased by i n c r e a s i n g s a l t concent r a t i o n . The hydrophobic term i s p r o p o r t i o n a l t o AA and m. If AA decreases more r a p i d l y than the increase i n m, the s a l t i n g out term and, thereby, the degree of s a l t i n g out, w i l l decrease with i n c r e a s i n g s a l t concent r a t i o n . Since associated p r o t e i n s (dimers, t r i m e r s , e t c . ) w i l l have l e s s exposed hydrophobic surface area than monomers, i n c r e a s i n g amounts of associated p r o t e i n s with 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 w i l l r e s u l t i n decreasing A A v a l u e s . Soy p r o t e i n s are known t o a s s o c i a t e with 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 . At pH 7 . 6 , U S and 7S p r o t e i n s d i s s o c i a t e i n t o subunits at i o n i c strength below 0.1M and 0.5M, r e s p e c t i v e l y (11, 12). This i s , t h e r e f o r e , a p l a u s i b l e e x p l a n a t i o n o f the observed s o l u b i l i t y behavior. The sharp minimum at 0.1M s a l t c o n c e n t r a t i o n i n the case of the NPI might correspond to the cooperative a s s o c i a t i o n r e a c t i o n s i n v o l v i n g U S s u b u n i t s . Even though there has been no experimental demonstration, i t would not be unreasonable to p o s t u l a t e that random a s s o c i a t i o n r e a c t i o n s a l s o occur f o r denatured soy p r o t e i n s . The broader minimum between 0.1 and 0.2M s a l t c o n c e n t r a t i o n might be i n d i c a t i v e of l e s s cooperative a s s o c i a t i o n r e a c t i o n s .

2)

Dipole moment increases 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 . The d i p o l e moment of a p r o t e i n i s a f u n c t i o n of the t o t a l net charge of the p r o t e i n , the d i s t r i b u t i o n of the charges on the p r o t e i n , and the p r o t e i n c o n f o r mation. As s a l t c o n c e n t r a t i o n ( i o n i c strength) i s


102


IN FOODS


i n c r e a s e d , the solvent becomes i n c r e a s i n g l y b e t t e r f o r s o l v a t i n g charged s p e c i e s . Thus, i t i s p l a u s i b l e t h a t the p r o t e i n c o i l might expand. This would r e s u l t i n g r e a t e r separation of charged s i t e s on the p r o t e i n and a l a r g e r d i p o l e moment. The i n c r e a s i n g d i p o l e moment w i l l make the e l e c t r o s t a t i c s a l t i n g i n term more dominant as s a l t c o n c e n t r a t i o n i n c r e a s e s . I t i s d i f f i c u l t t o determine whether t h i s i s an important c o n t r i b u t i o n i n the cases studied here. 3)

Ion binding causes net charge to go through p o i n t of zero net charge. I f the net charge on the p r o t e i n i s reduced, the a i p o l e moment should a l s o be reduced. The d i p o l e moment should be minimum at zero net charge. It i s p o s s i b l e that there i s a s p e c i f i c binding of counter ions that causes the p r o t e i n to go through a p o i n t of zero net charge as 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 . If t h i s o c c u r s , the e l e c t r o s t a t i c s a l t i n g i n term w i l l decrease as s a l t c o n c e n t r a t i o n i s increased and reach a minimum at a point where the net charge i s zero. This w i l l r e s u l t i n a minimum i n the s o l u b i l i t y p r o f i l e . The e f f e c t of CaCl2 on the s o l u b i l i t y of soy p r o t e i n s f a l l s i n t o t h i s category (4). However, i t i s not expected that N a or NH4"" w i l l be a p p r e c i a b l y bound by soy p r o t e i n s . Thus, t h i s e f f e c t should not be of great importance here. +

1

In the context of t h i s expanded theory, i t i s p o s s i b l e t o compare the exposed hydrophobic area of the native soy p r o t e i n s w i t h t h a t of the denatured p r o t e i n s . I t i s evident from the f a i r l y l a r g e s a l t i n g out e f f e c t of NPI at low s a l t c o n c e n t r a t i o n s t h a t n a t i v e soy p r o t e i n s have appreciable amounts of exposed hydrophobic surface a r e a . If i t i s assumed that the e l e c t r o s t a t i c term i s n e g l i g i b l e at low s a l t c o n c e n t r a t i o n s , the i n i t i a l slopes of the curves i n Figure 11 are p r o p o r t i o n a l to the exposed hydrophobic surface a r e a . Using the slopes i t i s estimated that the exposed hydrophobic surface area of n a t i v e soy p r o t e i n s i s 50 percent of that of the denatured p r o t e i n s . This r e s u l t i s i n agreement with the c o n c l u s i o n of Takagi et a l . , ( 5 h who used the binding of a f l u o r e s c e n t hydrophobic dye to probe the hydrophobic surface of n a t i v e and heat denatured soy p r o t e i n s . T h i s i s a r a t h e r g r a t i f y i n g agreement c o n s i d e r i n g the u n c e r t a i n t i e s involved i n the interpretations. Viscosity As was the case f o r s o l u b i l i t y , apparent v i s c o s i t y of concentrated soy p r o t e i n s l u r r i e s i s a n o n - e q u i l i b r i u m property that i s h i g h l y s e n s i t i v e t o past h i s t o r y of the s l u r r y and to the techniques of i t s measurement.


SHEN

Solubility

and

103

Viscosity


Elect r o i t a t i c S a l t i n g - in

\ N

v

^Hydrophobic S a l t i n g - out

1 (NH )j 4

S 0

4

Figure 10. Illustration of the fit of Melander and Horvath's theory to the experimental data for the solubility of carboxyhemoglobin (1)

2 (m )

Archives of Biochemistry and Biophysics

Protein Functionality in Foods - American Chemical Society

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