Interactions of a nonionic detergent. III. Further observations on hydrophobic interactions

Interactions of a N o n i o n i c D e t e r g e n t lU. Further Observations on Hydrophobic Interactions 1 F L O Y D A. G R E E N Department of Medicine, State University of New York at Buffalo and Veterans Administration Hospital, Buffalo, New York 14215

Received July 27, 1971; accepted November 5, 1971 Nonionic detergents of the Triton series are widely used as dispersing agents, but little is known about their capacity to interact with proteins and other molecules of biological interest. The criticM micellar concentration of this detergent can be accurately measured taking advantage of an ultraviolet spectral shift during micelle formation, and this procedure was adapted to measure the amount of detergent bound to human and bovine serum albumin. In addition, the effects of the lower straight-chain alcohols on detergent mieelle formation were investigated. Triton X-100 has an 8-carbon tertiary alkyl chain and 9-10 ethylene oxide units as the hydrophile. Triton X-102 has the same alkyl group, but 12-13 ethylene oxide units, and Triton N-101 has a 9-carbon alkyl chain and 9-10 ethylene oxide units. It was found that Triton N-101 bound almost as well to the albumins as the other two, but at a free concentration of approximately one-third of the others. The difference between the binding of Triton X-100 and Triton X-102 was only slight. This strongly suggests that hydrophobic interactions were most significant in the binding. Furthermore, low concentrations of n-butanol, but not secondary nor tertiary butanol, appeared to hydrophobically favor micelle formation of the Triton X-100 molecules. In comparable concentrations, methanol, ethanol and propanol had no such effect, tending rather to raise the criticM micell~r concentrations. This may have some relevance to the unique sohbilizing role of n-butanol with retention of biological activities of proteins. Studies aimed at furthering unders Landing of the role of hydrophobic interactions in biological systems are complicated by the diffcult:y in isolating the types of forces involved here from electrostatic interactions, hydrogen bonding and other types of interaction. Micelle building is a relatively clearcut case of hydrophobic interactions (1), although modified in some cases by both polar and nonpolar environments (2). I n the case of nonionic deteIgents, the very low capacity for electrostatic interactions allows for easier identification of the forces responsible for both micellar aggregation and other binding phenomena. 1 Supported in part by a research grant (lid 02370) from the National Institutes of Health, U.S.P.H.S.

A method was described by Gratzer and Beaven (3) by means of which the critical micelle concentration (CMC) of the nonionic detergen~ Triton X-100 could be measured in a simple way using ultraviolet spectroscopy and without the addition of a third substance as a chromophore. This method was adapted for previous studies (4), and interactions with bile salts and certain soluble proteins were measured by means of displacement of the sharp transition point corresponding to the concentration at which micelles formed (Fig. 1). Certain features of the absorption-concentration plot, from which the CMC was derived, were further analyzed and changes in the first portion of the curve, referred to as the premicellar slope, seemed to be as-

Journal of Colloid and Interface Science, Vol. 41, No. 1, October 1972

124

Copyright © 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.

H Y D R O P H O B I C I N T E R A C T I O N S OF D E T E R G E N T

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sociated with a more hydrophobic environment of the detergent ehromophore even before mieelles were formed. Such slope changes were not seen with the group of proteins which manifested no transition point shift, i.e., no binding. This suggested that hydrophobie interactions were involved in the binding of this nonionie detergent with human and bovine serum albumin and betalactoglobulin (5). The present studies extend the previous data using Triton X-100 to two other Triton

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closely related detergents, which differ in the one case by an addiional methyl group on the hydrophobie end (N-101) and, in the other ease, by an average of two more ethylene oxide groups on the hydrophilic end (X-102). By comparing the amount of binding with these three related detergents and correlating this with their individual critical mieelle concentrations (free concentrations), it was hoped to show more precisely the nature of the binding forces. A further aim of this present investigation was to study the effect on Triton X-100 mieellization of low concentrations of organic solvents from methanol to butanol. This is of interest because of the empirical methods which have been developed using organic solvents, such as butanol, to solubilize portions of mammalian cell membranes (15, 16). Why butanol should be less "denaturing" to membrane protein, including enzymes and antigens, than the lower alcohols has remained unclear. It was found in these present investigations that methanol, ethanol and propanol, as might be anticipated, make it more difficult for mieelles to form, raising the critical mieelle concentration. On the other hand, with n-butanol, the longer hyrophobie end appeared to favor aggregation of the h?drophobie ends of the Triton detergent molecules and in a partielar range of concentration lowered the critical mieelle concentration significantly. METHODS

The methodology (4) was modified somewhat from the original description (3). The use of a spectral shift corresponding to mieelle formation in the detergent chromophore itself had the potential difficulty that the region where this shift is measured (275.5 mu) is also an area where there is significant protein absorption. All determinations were done at least in triplicate. In previous investigations (5), appropriate controls were studied which indicated that there was no change in ultraviolet absorption of the proteins which was significant enough to interfere with the use of this method for determining the critical mieelle concentration of the detergent in the presence of the proteins studied. One problem

Journal of Colloid and Inte~'face Science, Vol. 41, No. 1, October 1972

126

GREEN

which was noted in the previous studies was that there was an effect of protein concentration on the amount of detergent bound per mole protein. This should not be the case since the free concentration of the detergent is fixed at the CMC and the amount bound per mole of protein should not differ with different protein concentrations. To obviate this complication, the studies presently reported employed identical protein concentrations for the three different detergents so that comparability would be achieved with this approach. Tritons X-100, X-102, and N-101 were all obtained from Rohm and Haas, Philadelphia, and were not purified further. For reasons previously given, the known polydispersity but not heterodispersity (6) of these detergents about the mean length of the hydrophilie chain would not interfere with the results obtained. In essence, the method consists of adding small aliquots of detergent solution (5 ill) to 3 ml of protein-containing buffer and with the appropriate protein also in the blank compartment. The optical density of 275.5 was measured with a Hitachi speetrophotometer using a constant slit width. After correcting for dilution, the results were plotted as shown in Fig. 1 in which the absorbanee is given against the concentration. The two lines obtained were always linear, and the intersection of these was the transition point which corresponded to the critical mieellar concentration. Analysis of the s]opes obtained was carried out with the hope of shedding further light on the mechanism of altered CMC. The first portion of the curve is referred to as the premieellar slope and the second portion, the postmieellar slope. In addition, it was found for certain studies that displacement of the postmieellar slope of intercept seemed to correlate best with transition point shifts. This was measured by extending the postmieellar slope down to the X axis and noting the displacement of this intersection from the same intersection where detergent was used without protein. For the studies with organic reagents, the methanol, ethanol, and propanol and nbutanol were all freshl:y distilled and these were compared on the basis of their molar fractions rather than molarities, although

the difference in this concentration region is small. Only the lower organic solvent concentrations were studied with this methodology as higher concentrations led to turbidity in the ease of butanol, and insolubility of the butanol in aqueous buffer solution. All determinations were made at 22 4- 2° . The buffer system used throughout was 0.02 M pH 7.4 Tris-HC1. It was assumed, as usually done in experiments involving micelle formation, that the free concentration of detergent does not change once micelles have formed and further that the actual free concentration in the ease of protein where mieelles have formed is the same whether protein is present or not. This neglects the possibility that the protein molecules bind to micelles. This possibility appears unlikely but cannot completely be excluded. However, for the present studies where the experiments are aimed at showing differences among the detergents with the same protein solutions, this problem has probably less significance. Previous studies (4) indicated that neither defatting (7) nor ultraeentrifugation of crystalline human (Mann Research Laboratories) or bovine serum albumin (Armour Pharmaceutical Company) seemed to have an3 measurable effects on the binding of the Triton detergents. These procedures, therefore, were not carried out for the present studies. RESULTS A. Interaction of Human and Bovine Serum Albumins with Triton Detergents The transition point of the respective alkylphenol detergents was measured in the presence of proteins at two concentrations, and in the absence of protein. The amount of detergent bound in each situation was measured and plotted in relation to the free concentration of each detergent which was taken as the CMC with no protein present. Since the amount of binding has been previously shown to be a function of protein concentrations (5), the results were not normalized for each protein concentration, but rather the data plotted as obtained. The striking finding from this data (Fig. 2) was that Triton N-101 bound to the protein

Journal of Colloid and D~terface Science, Vol. 41, No. 1, October 1972

HYDROPHOBIC INTERACTIONS OF DETERGENT

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The effect of low concentrations of these organic solvents on the detergent critical micelle concentration was investigated. Figure 3 shows that the effect of methanol, ethanol and n-propanol are all in the same direction and quantitatively of approximately the same magnitude. The differences among them were only marginally significant in the repeated experiments. They all tended to increase the critical micelle concentration as a function of the mole fraction of organic solvent. A completely different situation was found with n-butanol. After an initial increase in critical micelle concentration the further increase in mole fraction of butanol

127

from 0.005 to 0.010 led to a progressive fall in CMC significantly below that found with no organic solvent present. The trend was again upward when a mole fraction of 0.015 was reached. Although slightly higher concentrations of n-butanol are ordinarily soluble in the buffer system used, turbidity resulted after the addition of the Triton X100 so that highest n-butanol concentrations miscible with water could not be examined. This unique situation with n-butanol was explored with the hypothesis in mind that this difference, compared with the lower alcohols, resulted from the greater capacity of the n-butanol to participate in hydrophobic interaction with the hydrocarbon portion of ~he detergent molecule. Analysis of the pre- and postmicellar slopes, as well as the displacement of the postmicellar intercept, was made for n-butanol. The only parameter which seemed to correlate well with the fall in critical micelle concentration was the displacement of the postmicellar intercept (Fig. 4). There was no change in the premicellar slope until a higher concentration of n-butanol was reached. To further document the participation of the alkyl group of the n-butanol in the micellar stabilization, the CMC of Triton X-100 was measured in the presence of similar concentrations of secondary and tertiary b u t a n o l It was found, in general, that tertiary butanol raised the critical micelle concentrations in all concentrations similar to that with n-propano], ethanol and methanol, but t h a t the secondary butanol had very little effect on the CMC in the concentrations studied. Table I shows a progressive rise in CMC going from normal to secondary to tertiary butanol, using the same mole fraction of each. n-Pentanol was also found to depress the CMC at a mole fraction of 0.002. Unfortunately, the solubility of n-pentanol in water was very limited, and turbidity resulted in the higher miscible concentrations which precluded measurement of CMC. DISCUSSION I t has been shown that the effect on CMC is greater b y addition of a high methyl group on the hydrophobie end of a detergent than

Journal of Colloid and [nterface Science,

Vol. 41, No.

1, O c t o b e r

1972

128

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an extra hydrophilic group (8). But, it is, in general, difficult to quantitate the exact contribution of the different types of forces known to be involved in binding (9). It has been shown in many systems that the hydrocarbon portion of an organic anion contributes the major share of the binding energy during binding to proteins (10). In

TABLE I CRITICAL ~V[ICELLAR CONCENTRATION OF TRITON X-100

the case of the present series of nonionic

detergents, there is a greatly lessened potentiality for electrostatic interactions. Such charge interactions are involved in binding of the ionic detergents (11) and probably in many of the so-called hydrophobic probes as well (12). A significant contribution, however, from hydrogen bonding to the total

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free energy of binding of the Triton detergents to proteins is somewhat more difficult to exclude, since the polyethylene oxide chain is known to be strongly solvated, probably by hydrogen bonding with water

Jour~ml of Colloid and Interface Science, VoI. 41, No. 1, October 1972

IXYDROPIXOBIC INTERACTIONS OF DETERGENT

(13). However, the present data with three nonionic detergents, two of which have the same hydrophobic portion and two of which have the same hydrophilic portion, point to the conclusion that hydrogen bonding is not significant in the interaction of these detergents with the proteins studied. I~ would be expected that a significant difference would be noted between Triton X-100 and Triton X-102, if the differing ethylene oxide chains were significantly involved in the bonding. On the other hand, what was found was very little difference between Tritons X-100 and X-102, when one takes into account the difference in free concentration, but a striking difference with Triton N-101, which has the same hydrophilic end as does Triton X-100. Here the addition of one methyl group to the hydrophobic end on the one hand lowered the critical micelle concentration, but on the other hand made binding much more effective at this free concentration level. These data support the view that hydrophobic interactions are the principal forces involved. The striking difference between n-butanol and the lower alcohols was an interesting finding which again points to the contribution of the hydrophobic portion of the nbutanol molecule to interaction with the Triton X-100 detergent molecule. This could be looked at alternatively as a destabilization of the monomeric form or a favoring of the micellar form. Slope analysis failed to show any change in the premicellar slope in this concentration range, as was noted with bile salts, but the smaller molecular size might make this evidence of interaction less apparent. In addition, the region of concentration was crucial for this effect to be noted. Probably at concentrations below and above this critical area the hydroxyl group is more predominant in its solvation effect. Hydroxyl groups have a special affinity for the polyoxyethylene chains which may form an extra "outer shell" solubilizing compartment in addition to the nonpolar core (14). The differences between normal, secondary, and tertiary butanol are also of interest and point again to the importance of the hydrocarbon end of the molecule, n-Pentanol also had a similar effect to n-butanol, but the range of concentration in which it could be

129

studied was seriously limited by solubility considerations. The mutual solubility of n-butanol and water is such that intermediate mixtures cannot readil~ be studied, and only in the highest n-butanol concentration regions could further observations be made. Unfortunately, micelles cannot form in such concentrations, so one can only conjecture on the difference between effects of n-butanol and the lower alcohols in this concentration range. Since butanol has been used to solubilize membranes (15) with retention of various types of activity (16), in contradistinction to the situation with lower alcohols, it may be tha~ there is some stabilization of hydrophobic interaction compared with the lower alcohols. However, this may represent only a small tendency, since, on the whole, n-butanol must be disruptive of hydrophobic interactions at high concentrations. REFERENCES 1. POLAND, D. C., AND SCtIERAGA, H. A., or. Colloid Interface Sci. 21,273 (1966). 2. SCHICK,M. J., J. Phys. Chem. 68, 3585 (1964). 3. GR.XTZER,W. B., ANDBEAVEN, G. H., J. Phys. Chem. 73, 2207 (1969). 4. GREEN, F. A., J. Colloid Interface Sci. 35,475

(1971). 5. GREEN, F. A., J. Colloid Interface Sci. 35, 481

(1971). 6. BECHE~, P., in "Nonionic Surfactants" (M. J. Schick, ed.), p. 478. Marcel Dekker, New York (1967). 7. CnEN, R. F., J. Biol. Chem. 242, 173 (1967).

8. CORI(ILL, J. M., GOODMAN,

J. •., AND HAR-

ROLD, S. P., Trans. Faraday Soc. 60, 202

(1964). 9. ARVIDSSON, E. O., Thesis, Small Molecule-

Protein Interaction, Lund, Sweden (1965).

Studentlitteratur,

10. R~Y, A., REYNOLDS, J. A., POLET, IX., AND STEINH~RDT, J., Biochemistry 5, 2606 (1966). 11. STEINHARDT,J., AND REYNOLDS, J. A., "Multiple Equilibria in Proteins," p. 234. Academic Press, New York (1969). 12. McCLURE, W. O., AND EDELM~N, G. M., Biochemistry 6, 1908 (1966). 13. MULLEY, B. A., AND METCALF, A. D., J. Pharm. Pharmacol. 8, 774 (1956). 14. NAKAG.~W~, T., in "Nonionic Surfactants" (M. J. Schick, ed.), p. 559. Marcel Dekker, New York (]967). 15. MORTON, R. K., Nature (London) 166, 1092

(1950). 16. GREEN, F. A., J. Biol. Chem. 243, 5519 (1968).

Journal of Colloid and Interface Science, Vol.41, No. 1, October1972