MARILYN F. EMERSON AND ALFREDHOLTZER
3320
The Hydrophobic Bond in Micellar Systems. Effects of Various Additives on the Stability of Micelles of Sodium Dodecyl Sulfate and of
n-Dodecyltrimethylammonium Bromide’
by Marilyn F. Emerson and Alfred Holtzer Department of Chemistry, Washington Universitg, St. Louis,Missouri 69190 (Received March 6 , 1967)
The results of measurements of the (conductivity) critical micelle concentrations (cmc) of sodium dodecyl sulfate (SDS) and n-dodecyltrimethylammonium bromide (DTAB) a t several temperatures in a wide variety of aqueous media containing protein denaturants or closely related compounds and in DzO are presented. The observed temperature dependence of the cmc in a given medium has been shown to provide the standard enthalpy and entropy changes accompanying addition of a detergent molecule to a micelle of the most probable size in that medium: the relative contributions of these over-all enthalpic and entropic terms t o the micelle stability are discussed. It is also shown that present experimental techniques are inadequate to allow assessment of the electrical and hydrophobic contributions t o these thermodynamic parameters. Qualitatively, correlations are observed between the hydrophobic nature of the additive and its micelle-breaking power, but only by confining the comparisons within a group of similar compounds and by allowing for the possibility that some compounds may penetrate the micelle. Simple experimental criteria are developed for distinguishing such penetrating additives from nonpenetrating ones. Application of even these limited, qualitative correlations to the case of hydrophobic bonds in proteins, it is made clear, will have to be done with considerable caution.
Introduction That hydrophobic bonds play an important role in stabilizing the native structures of proteins in aqueous solutions is well established.2*s Unfortunately, attempts to describe these forces molecularly and analyze how they are influenced by additives or temperature4r6 have not proved to be of very great predictive value. To choose a specific case, there is much evidence that substances like urea disrupt protein structures,2p6 a t least in part, by breaking hydrophobic bonds; in spite of claims to the contrary,6 however, the mechanism of this action is not understood sufficiently to allow a firm prediction, even a qualitative one, to be made of how the effectiveness of urea compares with, say, tetramet,hylurea.7 It is also evident that hydrophobic bonds cannot be the only important source of stabilization of native proThe Journal of Phyaicd Chemistry
tein conformations. That most proteins denature when heated immediately implies (although many authors, but not all,* have been content to ignore the implication) that forces other than hydrophobic bonds ~
~~
~
(1) This investigation was supported by Research Grant RG-5488 from the Division of General Medical Sciences, Public Health
Service. (2) W. Kauzmann, Advan. Protein Chem., 14, 1 (1959). (3) J. A. Schellman and C. Schellman i n “The Proteins,” Vol. 11, H. Neurath, Ed., Academic Press Inc., New York, N. Y., 1964, Chapter 7, p 1. (4) G. NBmethy and H. A. Scheraga, J. Phys. Chem., 66, 1773 (1962).
(5) M. Abu-Hamdiyyah, ibid., 69, 2720 (1965). (6) D.F.Waugh, Advan. Protein Chem., 9 , 325 (1954). (7) M. F. Emerson, Ph.D. Thesis, Washington University, St. Louis, Mo., 1966. (8) H. A. Scheraga, G. NBmethy, and I. Z. Steinberg, J. BWZ. Chem., 237, 2506 (1962).
THEHYDROPHOBIC BONDIN MICELLAR SYSTEMS
dominate the enthalpy of unfolding; disruption of hydrophobic bonds, as far as we know, is either slightly exothermic or athermal, a t least near room temperat ~ r e . ~ ,Furthermore, ’~ there is experimental evidence that substances such as urea can lower the free energy of an exposed peptide group in aqueous solution relative to a group that is hydrogen bonded to another peptide group;’: thus, the action of this denaturant is not exclusively confined to hydrophobic bond breakage. I n view of these ambiguities concerning the role of additives and temperature in protein denaturation, it seemed desirable to carry out an extensive investigation of the effect of temperature, urea, and other organic additives (“denaturants”) on a “simple” model system containing hydrophobic bonds-aqueous solutions of detergent micelles. Detergent systems are attractive as models, not only for their simplicity, but because the hydrophobic bonding can be assessed relatively easily through measurement of the critical micelle concentration (cmc). The particular detergents chosen in this investigation were sodium dodecyl sulfate (SDS) and n-dodecyltrimet hylammonium bromide (DTAB), and their cmc’s were measured over a wide range of conditions of solvent :tnd temperature. As will be seen, the results allow qualitative conclusions t o be drawn about hydrophobic bonds in micelles, a system of considerable intrinsic interest, but the extension of these conclusions to include hydrophobic bonds in proteins will have to be made with considerable circumspection; indeed, the results indicate that interpretation of protein denaturation experiments may be even more equivocal than has been suspected.
Experimental Section Unless otherwise specified, all chemicals used were reagent grade and were used without further purification. Tetramethylurea (boiling range, 176.5-177.5’ ; obtained from John Deere Chemical Co., Pryor, Okla.) was used without further purification. N,N’-Dimethylurea (Matheson Coleman and Bell; Practical grade) was recrystallized twice from hot ethanol. The recrystallized product had a melting range of 107-108”, as compared to the reported value of 107’. Deuterium Oxide (99.8% D) was obtained from Stohler Isotope Chemicals, Rlontreal, Quebec, Canada. The preparative procedures for the detergents in this study have been described elsewhere.7812 These references also contain a description of the conductivity apparatus used for determination of critical micelle concentrations.
3321
* 70
40
0
5
10
15
102(C”2)
Figure 1. Conductometric titration curves for DTAB at 25‘; concentration (C) is in moles per liter: 0, HtO; 0, 2.51 M methanol; A, 2.00 M 1-propanol.
Results The conductivity critical micelle concentrations (cmc) of DTAB and SDS in a wide variety of aqueous media were measured a t several temperatures. The temperature dependence of the cmc of DTAB in DzO was also determined. Some typical plots of the conductance data from which the cmc’s were derived are presented in Figure 1. The numerical values of all the measured cmc’s, along with estimates of the uncertainties involved in determining them from the data, are catalogued elsewhere? To conserve space, we present here only enough of the data to illuminate the points in question. It was found that, in general, the change in slope of the equivalent conductivity a t the cmc becomes more and more gradual with increasing concentration and hydrophobic character of the additive, and, usually, with increasing temperature. I n extreme cases, the break in the curve is sufficiently gradual that the cmc is very difficult to assess. Thus, for example, the cmc can be determined with reasonable accuracy for a 6.00 M urea solution a t 25”, but not for a 1.00 IM tetra(9) (a) J. A. V. Butler, Trans. Faraday SOC.,33, 229 (1937); (b) D* D. ibid*9 35* 1281i1421 (lg3’). (10) J. T.Edsall and G. Scatchard in “Proteins, Amino Acids and Peptides as Ions and Dipolar Ions,’’ E. J. Cohn and J. T. Edsall, Ed., Reinhold Publishing Gorp., New York, N. Y., 1943,p 183. (11) D. P. Robinson and W.P. Jencks, J. B i d . Chem., 238, PC1558 (1963). (12) M. F. Emerson and A. Holtzer, J. Phys. Chem., 71, 1898 (1967).
Volume 71, Number 10 September 1967
MARILYN F. EMERSON AND ALFRED HOLTZER
3322
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Figure 2 . Cmc's for DTAB in aqueous organic solvents a t 25": 0, urea; 0, N-methylurea; A, N,N'-dimethylurea; H, thiourea; 0, sucrose.
0
5
10
15
102(molc fraction of additive)
Figure 4. Stability of DTAB micelles in aqueous organic solvents a t 25": 0, urea; A, N,N'dimethylurea; 0, tetramethylurea; A, acetamide; H, acetone.
2.5
-
x'
c I-
K
I
102(rnole fraction of additive)
Figure 5. Stability of DTAB micelles in aqueous organic solvents a t 25": 0, methanol; a, ethylene glycol; A, glycerol; H, 1,3-propanediol; I, 2-propanol; 8 , 1-propanol; 0, ethanol; a, dioxane. 0
10
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30
40
50
60
70
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t("C)
Figure 3. Dependence of cmc on temperatore for DTAB in: 0, H20: D, D20; A, 1.00 M dioxane; A, 2.00 M dioxane; 0, 3.00 M urea.
methylurea solution at the same temperature. This result, somewhat surprisingly, is independent of the micelle-breaking power of the solutions. Thus, a 2 M methanol solution destroys micelles (compared to wat'er), whereas 2 M 1-propanol stabilizes them; nevertheless, the observed slope change is considerably sharper in the case of methanol (Figure 1). A few typical plots of the experimental cmc's are The Journal of Physical Chemistry
presented directly in Figure 2, which shows the molar crnc for DTAB a t 25" as a function of the molarity of the organic additive, and in Figure 3, which shows the variation with temperature of the molar cmc of DTAB in several solvent media. Although the results for SDS show the same general trends as for DTAB, some differences do exist between the two detergents. I n general, the organic additives are not as effective in breaking up SDS micelles as they are in breaking up DTAB micelles. Urea at 25" presents a striking case of this-2.00 M urea raises the cmc of DTAB by 46%, and 6.00 M urea raises it by 306%; the corresponding values for SDS are 16 and 10401,. Most of the sub-
THEHYDROPHOBIC BONDIN MICELLARSYSTEMS
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20
30
2.9
3.0
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40
50
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70
80
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3.3
3.4
3.5
3.6
Figure 6. Stability of SDS micelles in aqueous organic solvents a t 25': 0, urea; 0, acet,amide; A, acetone; 0, tetramethylurea; m, N,N'-dimethylurea. 1700
-
1600
c Ri I 1500
1400
1031~( O K - ' ) Figure 8. Dependence of cmc on temperature for DTAB in: 0, H20; 0, D20; A, 1.00 M dioxane; A, 2.00 M dioxane.
0
5
10
15
102(mole fraction of additive)
Figure 7. Stability of SDS micelles in aqueous organic solvents a t 25": 0, methanol; A, et,hylene glycol; V, glycerol; m, ethanol; 0, dioxane; A, 1- and 2-propanol.
stances which increase t,he cmc's of both detergents a t 25" are not only less effective with both detergents a t low temperatures, but actually decrease the cmc of SDS at 0". The effects of various additives on the erne a t 25" are displayed in a somewhat different manner in Figures 4 and 5 (DTAR) and in Figures 6 and 7 (SDS), i.e., as R T (In Xo) os. mole fraction of additive, where Xo represents the cmc in base mole fraction units. The temperature dependence of the cmc is presented in Figures 8-10 (DTAB) and in Figure 11 (SDS) as -RT(ln X o )vs. T("I
