22 Equilibrium Dialysis and Viscometric Studies on the Interaction of Surfactants with Proteins W. U. MALIK and V. P. SAXENA Department of Chemistry, University of Roorkee, Roorkee, India
Introduction Ever since the discovery o f Kuhn and coworkers (1) i n 1940 that c e r t a i n c a t i o n i c soaps exert powerful d i s i n f e c t i n g a c t i o n on b a c t e r i a , presumably owing t o t h e i r combining c a p a c i t y with the cell p r o t e i n and that t h e i r a c t i o n i s p r o p o r t i o n a l to t h e i r s u r face activity, chemists have evinced keen i n t e r e s t i n studying the r o l e of synthetic detergents i n b i o l o g i c a l systems and p r o cesses. Recent studies have shown that the nonionic polysorbate 80 causes a d i s r u p t i o n o f the membrane s t r u c t u r e (2) and the non i o n i c T r i t o n X-100 exerts a s i m i l a r e f f e c t on lysosomes, m i t o c h o n d r i a , and erythrocytes ( 3 , 4 ) . The s o l u b i l i z i n g a c t i o n of detergents on c h o l e s t e r o l has been reported t o check the develop ment of atheroma (5). The formation o f bile salt-monoglyceride mixed m i c e l l e s has been shown t o be a s i g n i f i c a n t f a c t o r i n lipid absorption (6). In view of the important r o l e played by the syn t h e t i c detergents i n v a r i o u s metabolic processes and t h e i r ever -growing use i n the food and pharmaceutical i n d u s t r i e s , the need to develop a knowledge of t h e i r b i o l o g i c a l e f f e c t s acquires great s i g n i f i c a n c e p r o v i d i n g enough justification f o r c a r r y i n g out s y s tematic studies on the chemical i n t e r a c t i o n of detergents with b i o l o g i c a l e f f e c t s acquires great s i g n i f i c a n c e p r o v i d i n g enough justification f o r c a r r y i n g out systematic studies on the chemical i n t e r a c t i o n o f detergents with b i o l o g i c a l substances. In the present communication we have discussed the r e s u l t s of our in vestigations on some protein-detergent systems employing e q u i l ibrium dialysis and v i s c o m e t r i c techniques. Substituted pro t e i n s , viz., p-toluene sulphonylated g e l a t i n (hereafter c a l l e d TSG) and i o d i n a t e d casein (hereafter c a l l e d IC) were used f o r the e q u i l i b r i u m dialysis s t u d i e s which provided evidence f o r t h e i r statistical b i n d i n g with the a n i o n i c detergents: sodium dodecyl sulphate (hereafter c a l l e d SDS) and sodium o c t y l sulphate (hereafter c a l l e d (S0S). The v i s c o m e t r i c s t u d i e s were made with
299
300
COLLOIDAL DISPERSIONS AND MICELLAR
BEHAVIOR
ovalbumin (hereafter called OV), a globular protein, and trans fusion gelatin (hereafter called TG), a f i b r i l l a r protein with the anionic detergent sodium lauryl sulphate (hereafter called (SLS) and the cat ionic detergent cetylpyridinium bromide (here after called CPB) with a view to gain an insight into the nature of particle-particle interaction. Experimental Materials (a) Proteins, p-toluene sulphonylated gelatin was prepared by adding p-toluene sulphonyl chloride (lOg) in small lots to a solution of gelatin (30g) made alkaline (pH 11) with potassium hydroxide and maintaining the pH throughout at this value by the occasional addition of the a l k a l i . The re action mixture was stirred for 3 hours and then centrifuged. The centrifugate was acidified with acetic acid u n t i l the pH was 3.5 when a white coagulum formed. This was separated and cut into small pieces which were subjected to prolonged washing with cold methyl alchol ( l l i t r e ) in a continous extractor for a week when a chloride free product was obtained. It was dried in vacuo and ground to a powder. Its sulphur content was found to be 1.985$. Iodinated casein was prepared by adding chloramine-T (6g) in small lots to a solution of cow s whole milk case in (20g) in water (800ml) containing dissolved sodium bicarbonate (6g) and potassium iodide (2.5g). The reaction mixture was stirred for an hour and the protein precipitated at pH 3.8 by the addition of hydrochloric acid. The curd was subjected several times to dissolution in dilute sodium hydroxide and precipitated with hydrochloric acid u n t i l the inorganic iodide was completely removed. The product was dried at 80°C. It was found to contain 1.13% of iodine. Ovalbumin was prepared from egg white according to the procedure of S^rensen and H^yrup (j) by extraction with a saturated ammonium sulphate solution and c r y s t a l l i z i n g at a pH of about h.6 brought down by adding 0.2N sulphuric acid. Trans fusion gelatin was obtained through the courtesy of the Director, National Chemical Laboratory, Poona, India in the form of 6% so lution. (b) Detergents. A l l the four detergents (SDS, SOS, SLS, and CPB) were B r i t i s h Drug House products. These were further p u r i fied by recrystallization from acetone. 1
Preparation of solutions. Solutions of TSG and IC were pre pared by soaking the protein in doubly d i s t i l l e d water for sev eral hours and then s t i r r i n g the mixture mechanically after add ing a known amount of 0.1 Μ KOH to bring the pH to 6 . 5 . Concen trations of these solutions were determined by drying a known aliquot to constant weight in an air oven at 105°C and applying the correction for potassium hydroxide to get the absolute weight of the protein. Ovalbumin solution was prepared by shaking i t with water at room temperature. The solutions of ovalbumin and
22.
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Interaction of Surfactants with Proteins
301
transfusion gelatin were purified "by dialysis and the concentra tions of the dialysed solutions determined "by drying a known v o l ume at 105-U0°C to constant weight in an air oven. Procedure for equilibrium d i a l y s i s . 20 ml aliquot s of the protein-detergent mixture in which the concentrations of SDS and SOS varied from 0.2X10"3M to 20.0x10"% at a fixed cone. (6x10 ~5g) of the protein were made "by mixing requisite volumes of 6% protein, 0.05M detergent, and d i s t i l l e d water, and "brought up to the desired pH. These were stored for two days at 2 5 ° C 5 ml por tion from each aliquot was then pipetted into dialysis bags made from 15cm long strips of Visking Nojax sausage casing (l.8cm in diameter), freed from sulphur by repeated boiling with d i s t i l l e d water, by tying a square knot on one end. Most of the a i r inside the bags was forced out by pressing which were then closed with two overhead knots, one above another. Each bag was carefully suspended in a test tube containing 5 ml of double d i s t i l l e d water brought up to the same pH, which was then stoppered. The tubes were placed in a stand supported inside a water thermostat and gently shaken for k8 hours by means of an e l e c t r i c a l shaker. The dialysis bags were then withdrawn fromthe test tubes and the amount of the detergent present in the dialysate was estimated spectrophotometrically using the cationic dye pararosaniline hydrochloride. To 1 ml solution of pararosaniline hydrochloride (1x10 M) was added an appropriate volume of the test solution (not exceeding k ml). The t o t a l volume was made up to 5 ml with d i s t i l l e d water. 5 ml of a mixed solvent (equal volumes of chloroform and ethyl acetate) were added and the solution shaken manually about 50 times. It was then centrifuged for about a minute at 5000 r . p . m . , for complete separation of the organic and aqueous phases, the former containing the coloured complex at the bottom. Its spectral absorption was measured on Klett Summersion Photo electric Colorimeter using green f i l t e r against a reference tube f i l l e d with the solvent. Procedure for viscometric studies. Three sets of proteindetergent aliquots of 20 ml each were prepared for viscosity measurements as given below: (i) In the f i r s t set, the protein concentration was fixed (0.5$) while the concentration of the detergent varied from 2-50 millimoles per l i t r e . The viscosities of the aliquots of this set were measured at pH 2 . 0 , 3 . 0 , 6.0 and 8.0 in the case of anionic detergent-protein system and at pH 2 . 0 , 3 . 0 , 8.0 and 9-0 in the case of cationic detergent-protein system. ( i i ) In the second set, the concentration of the protein was varied, from 0.8% to l.k% in the case of 0Y and from 0.6% to 1.2% in the case of TG using the same concentrations of detergent solutions. Viscosity measurements were made at pH 8.0 for the anionic detergent-protein system and at pH 3.0 for the cationic detergent-pirotein system, ( i i i ) The third set consisted of s o l -
302
C O L L O I D A L DISPERSIONS A N D M I C E L L A R
BEHAVIOR
u t i o n s having d i f f e r e n t p r o t e i n - d e t e r g e n t r a t i o s . Viscositymeasurements were made at pH 8.0. The v i s c o s i t i e s were measured by Scarpa's method as modified by Prasad et a l . ( 8 ) . The v i s c o metric c o n s t a n t , k , was c a l c u l a t e d from the experimentally d e t e r mined values o f t^ and t 2 , the time o f r i s e and the time o f f a l l r e s p e c t i v e l y , f o r a given volume o f s o l u t i o n under constant p r e s sure (for double d i s t i l l e d w a t e r ) . The v i s c o s i t y , T| , o f the s o l u t i o n i s r e l a t e d to ti and t o under constant pressure as
The v i s c o s i t y o f the t e s t s o l u t i o n was determined by f i n d i n g t]_ and t2 f o r a known volume of s o l u t i o n (20ml). A l l measurements were c a r r i e d out at 30 + 0 . 1 ° C i n a thermostatic water b a t h . Calculations (i) E q u i l i b r i u m D i a l y s i s . The extent of b i n d i n g was c a l c u l a t e d by K l o t z s equation (9): 1
€
V s €
where a p p i s the molar e x t i n c t i o n molar e x t i n c t i o n t i o n o f the free of the detergent pression :
apparent molar e x t i n c t i o n c o e f f i c i e n t , *S the c o e f f i c i e n t o f the bound detergentthe c o e f f i c i e n t o f free detergent and oi i s the f r a c detergent. The v a l u e of Vm, the number of moles bound per mole o f p r o t e i n , i s given by the ex
V
m
= A
p
/ P
t
where Ap i s the number o f moles o f p r o t e i n bound detergent and Pt i s the t o t a l number of p r o t e i n molecules. ( i i ) V i s c o s i t y . The i n t r i n s i c v i s c o s i t y , [Ή] at d i f f e r e n t detergent c o n c e n t r a t i o n was obtained by p l o t t i n g 7J p/c vs c. ( 7? p i s the s p e c i f i c v i s c o s i t y and c the cone, o f the p r o t e i n i n *g/ml) and e x t r a p o l a t t i n g the curves t o zero c o n c e n t r a t i o n . The i n t e r a c t i o n index k , was c a l c u l a t e d u s i n g Kramer's equation which has been found t o be v a l i d f o r such systems over a wide range o f c o n c e n t r a t i o n s . The equation i s 9
S
S
In 7 j where
1
^rel
1
r e l
/c
= [17] - k [ T 7 ]
i s the r e l a t i v e
viscosity.
2 c
22.
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Interaction of Surfactants with Proteins
303
Results and D i s c u s s i o n E q u i l i b r i u m d i a l y s i s s t u d i e s . T y p i c a l data f o r the b i n d i n g of SDS -with TSG and of SDS with IC are given i n Table I and I I . T y p i c a l curves f o r the p l o t s o f Vm vs l o g C and p l o t s of l/\ vs 1/C are given i n F i g u r e s 1, 2 and 3. Values o f V obtained from e x t r a p o l a t i o n are given i n Table I I I . The course o f b i n d i n g o f SDS and SOS with p-toluene sulphonylated g e l a t i n and i o d i n a t e d c a s e i n as the detergent i s added i n p r o g r e s s i v e l y i n c r e a s i n g con c e n t r a t i o n to a f i x e d amount o f the p r o t e i n at d e f i n i t e values o f pH (7.7 and 9.5) has been followed by p l o t t i n g the average number o f moles of the detergent bound per 10*g p r o t e i n , V , against the l o g of c o n c e n t r a t i o n o f the unbound detergent, l o g Op (Figure l ) . The general shape o f the curves obtained w i t h d i f f e r e n t detergent p r o t e i n combinations at d i f f e r e n t pH values i s the same. An ex amination of the p l o t s o f V vs l o g Cjp r e v e a l s t h a t i n each case the mode of b i n d i n g o f the surface a c t i v e molecules to the p r o t e i n v a r i e s i n three d i s t i n c t p a t t e r n s as the concentration o f the former i s g r a d u a l l y i n c r e a s e d . These p a t t e r n s o f b i n d i n g have been roughly demarcated by d i v i d i n g the p l o t s i n t o three regions A , B , and C , w i t h a view t o f a c i l i t a t i n g a b e t t e r a p p r e c i a t i o n of the mechanism of d e t e r g e n t - p r o t e i n i n t e r a c t i o n at v a r i o u s stages. In the r e g i o n A , which i s the r e g i o n o f r e l a t i v e l y high p r o p o r t i o n o f p r o t e i n to detergent, the p l o t o f V vs l o g Cp follows a l i n e a r course. T h i s i s i n d i c a t i v e of a more or l e s s s t a t i s t i c a l d i s t r i b u t i o n o f the detergent over the e n t i r e a v a i l a b l e p r o t e i n molecules i n t h i s r e g i o n . S i m i l a r observations have been r e ported by K l o t z et a l . (9) i n d y e - p r o t e i n i n t e r a c t i o n . The ap p l i c a b i l i t y o f the simple s t a t i s t i c a l theory i n the present studies may be e a s i l y t e s t e d by p l o t t i n g l / V against 1/Cp (Figure 2, 3) when a p l o t i s obtained which i s l i n e a r up t o a c e r t a i n l i m i t . The values o f the r e c i p r o c a l o f the i n t e r c e p t s on the o r d i n a t e obtained a f t e r e x t r a p o l a t i o n s o f the s t r a i g h t l i n e s w i t h v a r i o u s d e t e r g e n t - p r o t e i n combinations are given i n T a b l e l l l . These values represent the maximum number o f b i n d i n g s i t e s a v a i l a b l e , i . e . , t h e max.values o f V , i n t h i s r e g i o n . Beyond the r e g i o n A , t h e course o f the p l o t s o f V vs l o g Cp s h a r p l y deviates from the i n i t i a l l i n e a r course i n the d i r e c t i o n o f higher values o f V , r e f l e c t i n g a d i s t i n c t change i n the mode o f b i n d i n g which no l o n g e r appears to be wholly s t a t i s t i c a l . I t i s reasonable to assume that a f t e r combination with the number o f moles o f detergent as a maximum f o r s t a t i s t i c a l b i n d i n g (vide Table III) , a large number of ions get bound e s s e n t i a l l y as a u n i t . P o s s i b l y a f t e r occupying the r e a d i l y a c c e s s i b l e s i t e s (basic groups) on the surface o f the p r o t e i n molecule,the e x t r a detergent molecules penetrate the o r i g i n a l t i g h t l y f o l d e d p r o t e i n molecule causing i t to undergo a change i n p h y s i c a l o r g a n i z a t i o n i n which form i t o f f e r s h i t h e r t o f o r e i n a c c e s s i b l e s i t e s f o r combination and, t h e r e f o r e , e x h i b i t s greater b i n d i n g c a p a c i t y f o r the detergent. With the s t r u c t u r a l d i s o r d e r i n g o f the p r o t e i n molecule, the p o t e n t i a l p
m
m
m
m
m
m
m
m
20 30 1^0 60 80 100 120 200 300 i+oo 600 800 1000 1200 1600 2000
5
Cone, of SDS (C) xlO~ M
12 18 26 36 51.5 6k 75 116 150 175 26k 3k2 i+oo 1+50 520 590
F
Cone, of free SDS(C ) X10-5M 8 12 16 2k 28.5 36 1+5 8k 150 225 336 1+58 600 750 1080 11+10
Cone, of hound SDS(Cg) xlO^M
1.33 2.0 2.66 1+.0 k.75 6.0 7.5 ll+.O 25.0 37.5 56.0 78.0 100.0 125.0 180.0 235.0
¥.0792 1.2553 ΐ.3802 ΐ.5563 ΐ.7118 T. 8062 Ί+.8751 3.061+5 3.1761 3\2l*55 ΙΛ216 3.531+0 3.6021 3.6532 3.7160 3.7709
m
Moles SDS hound log Cp per lO^g TSG,V m
0.75 0.50 0.375 0.25 0.21 0.166 0.133 0.07 0.
