Hydrophobic free energy, micelle formation and the association of proteins with amphiphiles

J. Mol. Biol. (1972) 67, 59-74

Hydrophobic Free Energy, Micelle Formation and the Association of Proteins with Amphiphiles CHARLES

TANFORD

Department of Biochemistry Duke University Medical Center Durham, N.C., U.S.A. (Received 13 September 1971, and in revised fomz 18 February 1972) Experimental data for a variety of processes, all involving the removal of a hydrocarbon chain from contact with water, have been used to calculate the free energy changes associated with such processes. The results show remarkable regularity, especially in the influence of the length of the hydrocarbon chain. In the binding of such molecules to proteins, however, this regularity extends to only short hydrocarbon chains, clearly indicating a limited size for the hydrophobic combining region. In serum albumin, the binding of fatty acids and other amphiphiles is, moreover, not a purely hydrophobic reaction and a sizable contribution to the free energy of association arises from specific atiity for the hydrophilic head groups. A conclusion of general applicability is that proteins possessing binding sites for hydrophobic substances compete for such substances with aggregates such as micelles, bilayers or biological membranes, and that specific interactions, probably involving lipid head groups, are s, likely prerequisite for association of a protein with a biological membrane.

1. Introduction The objective of this paper is to obtain quantitative data on the magnitude of the hydrophobic effect for relatively long chain aliphatic hydrocarbons, or for amphiphilic molecules containing relatively long hydrocarbon tails, with a view to assessing the role played by hydrophobic forces in the interactions of proteins with lipids, that lead to the formation of biological membranes. Results from solubility and distribution studies will be given in the form of differences in standard free energy, &,-&, of appropriate molecules in organic solvents (subscript “erg”) and in aqueous solution (subscript “IV”), respectively. The free energies will be given in unitary units, as recommended by Gurney (1953) and Kauzmann (1959), so that they reflect only the internal free energy of the solute molecule in a solution, plus the free energy of interaction of an isolated solute molecule with solvent, and exclude purely statistical contributions resulting from the entropy of mixing of solute and solvent. To obtain the results in the desired units, solute concentrations, wherever they occur in an equation for the chemical potential, must be expressed in mole fraction units. Amphiphilic molecules, i.e. molecules containing both hydrophilic and hydrophobic portions, can respond to the hydrophobic effect by formation of micelles, in which contact between hydrophobic portions and water is avoided, while the hydrophilic group remains in solution. The standard free energy of transfer of an individual amphiphile molecule to a micelle will be designated as && - &, and will again be 69

60

C. TANFORD

given in unitary units. It will be seen that the data given lend themselves to the prediction of the free energy of formation of micelles by biological lipids (see following paper; Smith & Tanford 1972), quite apart from their relevance to the problem of protein/lipid interactions. Results obtained for the foregoing processes will be compared with standard free energies (& - &,,) for binding of similar molecules to proteins, obtained from equilibrium binding studies. From the comparison, thermodynamic requirements for the incorporation of proteins into biological membranes will be considered. All calculations will focus solely on molecules containing reIatively long aliphatic hydrocarbon tails because of the prime importance of such molecules in the biological problem to which the paper attempts to relate.

2. Molecules with Single Hydrocarbon

Tails

The hydrophobic free energy due to the repulsion of hydrocarbon groups by water is measured in its purest sense by the free energy of transfer of linear hydrocarbon molecules from water to a hydrocarbon solvent. The desired quantity can be obtained from the solubility of liquid hydrocarbons in water. Assuming that non-ideality in the aqueous phase can be neglected, because the solubility is so low, $& - &, is given simply as R!Z’ In X,, where X, is the solubility in water in mole fraction units. The result obtained from the solubility measurements of McAuliffe (1966) is that &,, - &, decreases with essentially perfect linearity with the number of carbon atoms (nc) in the hydrocarbon chain. For saturated hydrocarbons (up to octane, i.e. n, = 8), a least-squares treatment leads to the relation 0 Pow - & = -2436 - 884 n,. (1) The presence of double bonds diminishes the magnitude of the hydrophobic effect, but has no influence at all on the increment produced by saturated portions of the chain. Thus, from the results of McAuliffe (1966) for solubility of alkenes in water 0 PLarg- p& = - 1503 - 884 n,, (2) whereas for dienes (with the two double bonds in terminal positions) 0 Pore - & = - 903 - 860 n,. (3) Literature data on the smaller hydrocarbons indicate that these results are in no way specific to the hydrocarbon solvents that were used, which in these experiments were the individual liquid hydrocarbons, and indicate that closely similar results would have been obtained from measurement of the distribution of solute between water and a variety of organic solvents. The major contribution to p&g - & arises from the repulsive interactions in the aqueous solvent. The attractive van der Waals forces in the organic phase seem to be quite non-specific. Measurements on amphiphilic molecules yield essentially similar results. From the solubilities of liquid aliphatic alcohols in water (Kinoshita, Ishikawa t Shinoda, 1958) one obtains 0 Porg - p& = 883 - 821 n, (4) the data extending from no = 4 to n, = 10. Short chain alcohols are, of course, very soluble in water, which is reflected in the positive value of the constant term of equation 4, but become less so as the size of the hydrocarbon moiety is increased, and the increment in pLgorg - & per carbon atom at long chain lengths is seen to be nearly the same as in pure hydrocarbons.

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Similar data for carboxylic acids can be obtained from the measurements of Goodman (1958a) of the equilibrium distribution ratios between heptane and aqueous phosphate buffer of ionic strength 0.16. The presence of salt in an aqueous solution will in general have an effect on the free energy of transfer (the so-called “salting-out” effect), butt the effect is negligible at the low salt concentration used in these experiments. The free energy of transfer is determined from the relation p&g - & = RT ln (X,/X,,,), where X, and X,,, are the mole fractions in equilibrium with each other. Experimental. distribution ratios are extrapolated to zero total-acid concentration to avoid nonideality corrections. Goodman’s measurements extend to longer hydrocarbon chains than other data, cited so far, and lead to the interesting observation that the regular decrease in 0 Pow - &, extends only to n, = 1.5 (i.e. palmitic acid, the terminal carbon atom not being part of the hydrocarbon chain). From nc = 7 to nc = 15, the results obey the relation 0 (5:) Pu,w - & = 3380 - 820 no, the large positive term in which reflects the great affinity of the COOH group for water. For stearic acid (n, = 17), the value of p&g - p& is virtually the same as for palmitic acid, i.e. no further increase in the hydrophobic effect has occurred. The simplest interpretation is that very long hydrocarbon chains tend to fold back upon themselves, so that the area of contact with water no longer increases significantly with chain length above a certain limit, as has been suggested by Mukerjee (1967). Since these data represent the only known experimental observation of this limitation, the conclusion should at present be regarded as tentative. Goodman’s paper includes data for two unsaturated fatty acids, and they lead qualitatively to the same conclusion as was reached from the studies of hydrocarbon solubility cited earlier, i.e. that the presence of double bonds diminishes the hydrophobic effect.

3. Micelle Formation The interior of a micelle is probably very similar to a small droplet of organic solvent. Evidence for this comes from a variety of sources, the most pertinent from a thermodynamic point of view being the measurements of Wishnia (1963) of the solubility of hydrocarbons (ethane to pentane) in the interior of dodecyl sulfate micelles. The free energies of transfer determined from these data are only about lS”/b smaller in magnitude than those calculated on the basis of equation (l), which is remarkably close considering the small volume of the interior of a micelle, and the fact that a considerable portion of this volume must be a surface layer that is highly constrained, by virtue of the attachment of the dodecyl chains to the sulfate head groups. Several different approaches can be used to calculate the free energy of transfer of an amphiphile molecule from monomeric solution in an aqueous solvent to a micelle and all lead to the same result if properly carried out (Hall & Pethics, 1967), though this is not always evident in the literature on the thermodynamics of micelle formation. The simplest approach is to consider the process as a phase transition, in which case, ignoring non-ideality of the monomeric species in solution. 0 Pmic

& = RT In c.m.c. where the symbol c.m.c. is used to represent the critical micelle concentration -

(in

62

C. TANFORD

mole fraction units), which is the monomer concentration in equilibrium with the micellar phase. Equation (6) is incorrect because it ignores the entropy of mixing of the micellar “phase” with the solvent, but correction for this is easily made, leading to the relation &,,I, - & = RT In c.m.c. - (RT/%) In X,,, (7) where fi is the average number of amphiphile molecules per micelle and X,,, is the over-all mole fraction of micelles in the solution in equilibrium with monomer at a concentration equal to the c.m.c. It should be noted that micelles are heterogeneous with respect to size, and that t&C - & must vary with micelle size, reaching a minimum at the size of highest abundance. Equation (7) yields an average value over all micelle sizes present in the equilibrium mixture. It should also be noted that the mole fraction of monomeric amphiphile in equilibrium with the micellar “phase” must increase as X micincreases, and a corollary is that, in contrast to true phase separation, there cannot be a unique “critical” concentration. The actual value of Xmic corresponding to a particular c.m.c. measurement can usually be estimated quite readily from the experimental details of the measurement. Representative c.m.c. data for both non-ionic and ionic micelles are shown in Figure 1, and again, a striking regularity with respect to the length of the hydrocarbon chain is observed. Corresponding data for micelle size are not available for most of these systems. Accurate Gi values have been obtained for the N-alkyl betaines, represented by curve E of Figure (1) (Swarbrick & Daruwala, 1969;1970) and p&C - & can therefore be obtained by equation (7). Least-squares treatment of the results show that the data obey the relation 0 Pmic - p& = 2951 - 733 n, (8) where n, is the number of carbon atoms in the alkyl chain. If the incorrect equation (6) is used for analysis of the results one obtains 0 - pk = 2514 - 709 no Pmic (9)

Number

of carbon

atoms

in hydrocarbon

chain

Fro. 1. Plots of In c.m.c. V~WUShydrocarbon chainlengthat25”C. CurveA,alkyl hexa-oxyethylene glycol monoethers from Becher (1967); Curve B, alkyl sulfinyl alcohols (Corkill, Goodman, Robson & Tate, 1966); Curve C, alkyl glucosides from Becher (1967); Curve D, alkyl trimethylammonium bromides in 0.5 M-N~B~ (Emerson & Holtzer, 1967; Geer, Eylar & Anacker, 1971); Curve E, N-alkyl betaines (Swarbrick L%Daruwala, 1969); Curve F, alkyl sulfates in the a,bsenco of added salt, at 40°C (Evans, 1966).

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which shows that the last term in equation (7) has a relatively small influence on the calculated result. It is evident therefore, that all of the data, of Figure 1 imply that 0 Pm10- t& like CL& - &, decreases regularly with increasing chain length. It should be noted that line F extends without change in slope to n, = 18 and that this throws doubt on the conclusion reached earlier regarding a possible limit on the hydrophobic effect above no = 15. Equation (8) shows that the decrease in && - & per added carbon atom is somewhat less than the corresponding factor in p&g - ,G& as given by equations (1) to (4), and a similar result would clearly be obtained for the systems represented by lines A to D of Figure 1. The reason for this is that micelle formation requires the existence of two opposing forces, the hydrophobic force favoring aggregation, and a, repulsive force that prevents growth of the aggregates to large size and, in particular, prevents separation of the amphiphile into en entirely distinct phase. The repulsive force is thought to reside in the hydrophilic head groups of the amphiphile molecules, representing an increase in free energy that results from the close proximity of these groups in the micelle surface. This effect will increase as nc increases, because the average micelle size increases as no increases, and the head groups are consequently brought closer together. This leads to a positive contribution to the n, - dependent term of &ic - &. In the case of ionic micelles, the repulsive force must result primarily from electrostatic interaction, and the same is probably true for the N.alkyl betaines, since the --N(CH&groups of the beteines must be crowded closely together at the surface of the hydrophobic core of the mioelles, whereas the terminal COO- groups can extend into the surrounding solvent with some freedom of motion, i.e. these groups will resemble a diffuse layer of counterions. Approximate calculations of the effects of ionic repulsion can be made if a spherical shape for the micelles is assumed (e.g. Emerson & Holtzer, 1967) and such calculations show that a positive contribution of about 100 cal./mole per C atom to the n,-dependent term of equation (8) is reasonable. Many studies of the critical micelle concentrations of ionic amphiphiles have been carried out in the absence of added salt and line F of Figure 1 is representative of such data: both cationic and anionic amphiphiles give similar results, depending primarily on the length of the hydrocarbon chain? with minimal effect from changes in the chemical nature of the head group @evens, 1953; Shinoda, Nakagawa, Tamamushi & Isemurs, 1963). The absence of a high concentration of counterions (in contrast to line D of Fig. 1) increases the contribution from electrostatic repulsion. Moreover, the actual concentration of available counterions in the absence of added salt is determined by the c.m.c. itself. Since the c.m.c. decreases by a factor of 2 per added carbon 7 An interesting observation made by Klevens (1963) is that the c.m.c.‘s of alkyl carboxylaten, alkyl ammonium chlorides and alkyl sulfonates, for the same length of hydrocarbon tail, are nearly the same, but that alkyl sulfates of a given chain length have c.m.c.‘s comparable to those of the other amphiphiles with one additional carbon atom. In other words, the extra oxygen atom in the sulfste head group (-O-SO, ) behaves as ifit were an extra CH, group. This does not mean that this oxygen atom is hydrophobic, but probably implies that repulsion between head groups of an ionic micelle prevents the immediately adjacent part of the attached chain from becoming part of the micellar core, so that it remains in contact with water. In this situation, anoxygenatom (which is spatially equivalent to a CH2 group) can be substituted for a CHa group adjacent to the ionic site without affecting the c.m.c. This special characteristic of alkyl sulfates is also reflected in their association with proteins, as is shown in Figure 3 below, for binding to the native protein and in the fact thltt dodecyl snlfonate behaves like decyl sulfate rather than dodecyl sulfate in the formation of micelle-like protein/8mphiphile complexes,

64

C. TANFORD

atom, the contribution of the electrostatic repulsion to &,, - &, increases likewise as nc is increased and the diminished slope of line F, as compared to the other data in Figure 1, is in fact of about the expected magnitude.

4. Molecules with Two Hydrocarbon

Chains

Amphiphilic molecules with two hydrocarbon chains attached to the same head group are of particular interest because many biological phospholipids are of this type. A search of the literature has revealed only one model system from which quantitative data can be extracted, this being a study by Evans (1956) of the c.m.c. of compounds of the type R, -

CH I

OSO;

RZ

both R, and Ra representing saturated hydrocarbon chains. Some of his results are summarized in Figure 2. They represent increments in t&c - &,, (or In c.m.c.; the approximate equation (6) has been used) resulting from the lengthening of the hydrocarbon chain and demonstrate the interesting result that a second hydrocarbon chain, added to an amphiphile molecule already possessing a longer chain, makes a smaller contribution to && - &,, than is made by the longer chain. On the other hand, the increment per carbon atom added to the longer chain is not affected by the presence of the shorter chain: the data were obtained in the absence of added salt and the slope of the upper line is the same as that of line F of Figure 1, multiplied by RT.

-4 -

Number

of carbon

atoms added

chains of 1’ - (alkyl)‘--alkylFIG. 2. Increment in p& - & on lengthening the hydrocarbon 1 -sulfates. Data of Evans (1956), ctt 40°C in the absence of added salt. Major chain data are for single chain alkyl sulfates with octyl sulfate as reference and for double chain derivatives with sulfate as reference. Minor chain data represent a. 1-butyl-decyl sulfate and 1-pentyl -hexyl variety of compounds, the parent major chain in each case being the reference compound,

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These results strongly suggest that two hydrocarbon chains on the same amphiphila molecule (in the monomeric state in aqueous solution) tend to associate with each other so that the hydrophobic effect is less than it would be if the two chains did not interact. The value of the c.m.c. observed experimentally for dipalmitoyl lecithin, as described in the following paper (Smith $ Tanford, 1972) suggests that such association may also occur in biological phospholipids.

5. Association of Proteins with Hydrocarbons

and Amphiphiles

Association between proteins and hydrophobic ligands can only occur if the free energy gamed by association with the protein exceeds the free energy gained by other processes available to the ligand molecule. Association with hydrocarbons, for example, could not be observed if the free hydrocarbon concentration required for the association exceeds the saturation concentration at which separation of liquid hydrocarbon would occur. Association with amphiphiles could not be observed if the required concentration exceeds the c.m.c. Free energy contributions by the protein molecule are thus required, and one possible source arises from the protein’s hydra.. phobic side chains (Nozaki & Tanford, 1971). A substantial fraction of these side chains are typically buried in the interior of the molecule, the free energy gained thereby being a major factor in the stability of the native conformation, in water, relative to a more flexible conformation with exposed hydrophobic groups. Nevertheless, hydrophobic patches may be retained at the molecular surface or in crevices, and would constitute suitable binding sites in that their association with hydrophobic ligands would remove the exposed hydrophobic residues as well as the ligand itself (or part thereof) from contact with water. In the case of amphiphilic ligands, specific attraction for the head group could also contribute to the free energy of binding and this will in fact be seen to be a major factor in some of the results to be discussed. It should also be added, that association between proteins and hydrophobic ligands need not be confined to the native state. The free energy of formation of denatured states with the majority of hydrophobic residues exposed is relatively small (Tanford, 1970). Such states have augmented affinity for hydrophobic ligands, so that the latter can often be expected to induce formation of the altered states with an over-all gain in free energy. As might be expected from these anticipated features of the association process, experimental studies of the binding of hydrophobic ligands to proteins lead to results quite different from those discussed previously. The lack of specificity and the remarkable regularity in the effect of hydrocarbon chain length on the tendency for a hydrophobic molecule to be expelled from water are accompanied, and often overshadowed by specificity in the ability of proteins to accommodate the molecule, and limitation on the size of the hydrocarbon chain that can be accommodated is a common feature. An excellent example of such size limitation is provided by the study of Wishnia & Pinder (1966) on the binding of butane and pentane to /?-lactoglobulin. Two molecules of either hydrocarbon can be bound, with pg - &, more negative than pErg - &, for binding of the first molecule, as well as for the second molecule of butane, but not for the second molecule of pentane. The data indicate that a single hydrophobic binding site is involved, with a volume of 200 to 230 cm3/mole. This volume can readily accommodate two molecules of butane, but cannot completely contain two molecules 6

66

C. TANFORD

of pentane. Longer hydrocarbons were not studied, but one would predict that single molecules up to about decane will be bound and that the free energy of association would become unfavorable relative to phase separation for n, 5 12. It has been found that purely hydrophobic sites of even such limited extent are not a common feature of simple water-soluble protein. Serum albumin appears to have sites of only very weak afiinity for hydrocarbons (Wishnia & Pinder, 1964; Wetlaufer & Lovrien, 1964) and in several other proteins no binding at all can be detected below the solubility limit (Wishnia, 1962). There is a large body of experimental data on the binding of amphiphiles to proteins, which has been reviewed in detail by Steinhardt & Reynolds (1969). The majority of such studies have involved the binding of anionic amphiphiles to a single protein, serum albumin; this protein functions biologically as a carrier for fatty acid anions (as well as for other substances not related to the subject of this paper). Studies with this protein have yielded complex results, indicating several levels of ai&.ity. The limited data available for other proteins indicate that binding with very high affinity may be unique to serum albumin: several other common water-soluble proteins do not show it. The lowest afllnity type of association, however, which proves to be a co-operative process in which protein can bind more than its own weight of amphiphile, appears to be a non-specific process common to all proteins. Three quite distinct types of complex have been characterized to some degree, and we shall confine the discussion to them. (a) High ajinity

sites of native serum albumin

There are about 10 or 11 discrete binding sites with high association constants and considerable information concerning them is available, most of it from the work of Reynolds, Herbert, Polet & Steinhardt (1967) ; Reynolds, Herbert & Steinhardt (1968) and Reynolds, Gallagher & Steinhardt (1970). The sites are specific for anionic amphiphiles. Hydrocarbons do not bind strongly to serum albumin, and cationic amphiphiles, if they bind at all in this manner, do so with a binding constant about three orders of magnitude less than is observed for anionic ligands with a hydrocarbon chain of the same length (Few, Ottewill & Parreira, 1955). The best evidence for the active involvement of the anionic head group in the binding process is provided by the finding of Reynolds et al. (1968) that some of the binding sites, identifiable by perturbation of the tryptophan spectrum when binding takes place, do not bind alkyl carboxylates at all (or do so with greatly diminished affinity), whereas both alkyl sulfates and sulfonates are strongly bound. As previously noted, no such head-group speciiicityisobserved in micelle formation. Calculation of & - & for these sites presents something of a problem. The sites are heterogeneous, as is evident from the fact that some of them are capable of binding fatty acid anions, whereas others are not, but analysis of the binding isotherms to yield separate association constants for individual sites cannot be performed unambiguously. For comparative purposes we have set & - & equal to RT In X, at the point of half-saturation of the sites. This would be a measure of the average value of & - & for all sites if there is no interaction between them. If there is interaction, the interaction free energy at the point of half-saturation is included. The values of & - &, obtained in this way are summarized in Figure 3. Alternative procedures for analysis of the data would yield somewhat different absolute values, but the dependence of & - & on hydrocarbon chain length would be essentially the same.

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Number of carbon atoms in hydrocarbon chain

FIQ. 3. Free energy of association between anionic amphiphiles and bovine serum albumin. For RCOO - : (0) data of Teresi & Luck (1952), l”C, ionic strength 0.2; (0) data of Reynolds et al.. (1968), 2”C, ionic strength 0.03; (x) data, of Spector, Fletcher & Ashbrook (1971), 37”C, ionic strength 0.15. For ROSO; and RSO; all data are from Reynolds et al. (1967), 2”C, ionic strength. 0.03, except that the point for hexyl sulfate is a result of poorer precision, taken from Steinhardt &. Reynolds (1969). The dashed lines show the slopes of plots of pore - & t~r8us hydrocarbon chain. length a8 given by equations (1) to (5).

The values of & - & shown in Figure 3 are much more negative than the values of && - & for the same substances. Because of the specificity for anionic amphiphiles, the greater energy gain must be ascribed to the locus of attachment of the hydrophilic head group. It is possible that the binding sites seen here are the same as this protein’s binding sites for simple anions, and that an adjacent or nearly adjacent hydrophobic surface area provides the afkity for the hydrocarbon chain. The hydrophobic areas need not be exactly adjacent nor need they represent single contiguous areas: an “inch worm” type of attachment is possible, with some CH, groups not involved in the interaction. Regardless of the exact nature of the interaction, the phenomenon of size limitation is clearly evident. Whereas the dependence of & - & on hydrocarbon chain length up to n, N 8 is of the same order of magnitude as for p&g - &, (dashed lines of Fig. 3), a pronounced decrease in the slopes of the plots is observed for nc > 8. It evidently becomes increasingly difficult to find suitable locations for the hydrocarbon chain as its length increases, and little or no gain in free energy of binding occurs.

(b) Massive co-operative binding without signi$icant conformational change When binding measurements are extended to amphiphile concentrations above those required to saturate the discrete sites of serum albumin, further association. occurs. The final step is always a massive co-operative process, in which a largenumber of rtmphiphile molecules are incorporated over a very nsrrow range of free amphiphile concentration. The free energy of association for this process csn be calculated by considering the process as analogous to micelle formation, the protein molecule acting as a nucleus for the deposition of the amphiphile molecules, regardless of the mechanism

68

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TANFORD

invo1ved.t The critical concentration at which the process occurs is considered as analogous to the critical micelle concentration. It should be noted that equation (6) rather than equation (7) is the correct equation in this case because no new entropy of mixing term arises as a result of the process, since the protein molecules are already dispersed in the solvent before the association occurs. Values of & - & calculated in this way include contributions from all changes in the protein molecule that accompany the binding, including the free energy of inducing any conformational change in the protein that is required, or the free energy required to,displacepreviouslybound ligand molecules in the likely event that they enter into the co-operative mode of binding rather than retaining their previous locations. In the association of octyl and decyl sulfates and of octyl, decyl and dodecyl sulfonates with bovine serum albumin (Reynolds et al. 1967) the co-operative process occurs without drastic conformational change, as judged by intrinsic viscosity and optical rotation, i.e. the product is a compact, nearly spherical particle and most of the internal structure of the native molecule is retained. An even more remarkable finding is that it occurs at essentially the same critical amphiphile concentration for all of the ligands studied, i.e. & - & is entirely independent of the length of the hydrocarbon chain. At 2°C pH 5.6, ionic strength 0.03 (conditions under which the most satisfactory data are available) & - &, = - 5.3 f 0.5 kcal./mole. What the precise structure of this complex may be is not known, and it is not possible even to speculate as to why an increase in the length of the hydrocarbon chain above 1~~= 8 cannot be utilized in its formation. It is not known whether cationic amphiphiles are capable of forming similar kinds of complexes. There is evidence to suggest (J. A. Reynolds, personal communication) that all proteins can form complexes of this type and, if so, they could become an important biochemical tool in that they would represent a means of solubilizing otherwise insoluble proteins without drastic disruption of the native structure. The problem clearly deserves further study. (c) Massive co-operative binding accompanied by drastic conformational change The behavior of dodecyl and myristyl sulfates is quite different from that of the shorter alkyl sulfates or of the alkyl sulfonates (to n, = 12). The co-operative binding of these ligands to serum albumin is accompanied by large changes in optical and hydrodynamic properties, indicative of a major conformational change, presumably one in which many new hydrophobic residues are exposed (Reynolds et al. 1967; Steinhardt, Krijn & Leidy, 1971). The values of ,ug - & for the process can be calculated as before. At 2”C, pH 5.6, ionic strength 0.03, the values are -8-O & 0.5 kcal./mole for myristyl sulfate (nc = 14) and -6.8 f O-5 kcal./mole for dodecyl sulfate. A minimal value for decyl sulfate can be obtained, by virtue of the fact that decyl sulfate forms the compact type of co-operative complex in preference to the type being considered here. This requires that & - & for the complex being considered here cannot be more negative than -5.3 & O-5 kcal./mole. It is evident from these results that there is no significant limitation in the length of hydrocarbon t As has been pointed out by Foster & Aoki (1958) and by Reynolds et al. (1967), it is not necessarily true that the co-operativity in this process results from mioelle-like association between the amphiphilic ligands. The large number of ligands could be bound individually to discrete binding sites, the co-operativity arising in that case from the fact that an intrinsically unfavorable conformational change in the protein must occur before the new binding sites are generated. In the limiting case of a very steep transition between nearly saturated states the two mechanisms are indistinguishable and lead to identical values for & - & as defined in this paper.

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chain (at least to n, = 14) that can be effectively utilized in the formation of this complex, in sharp contrast to what was observed for the compact type of complex.. It has been established (for dodecyl sulfate) that this type of complex can be formed with all proteins and that the maximum amount of ligand that can be bound per gram of protein is the same for all of them (Pitt-Rivers & Impiombato, 1968; Reynolds & Tanford, 1970a). For proteins without disulfide bonds, or with disulfide bonds reduced if originally present, there are actually two forms of the complex, one containing 0.4 g dodecyl sulfate per gram of protein (one amphiphile per 7 amino-acid residues), the other 1.4 g dodecyl sulfate per gram of protein (one amphiphile per 2 amino-acid residues), the transition between them occurring at 6 to 7 x 10e4 Mdodecyl sulfate at 25°C pH 5.6 to 7.2, with little dependence on ionic strength. All proteins are dissociated to their constituent polypeptide chains, and each chain forms an individual asymmetric particle, approximately rod-shaped, with length roughly proportional to molecular weight (Reynolds & Tanford, 1970b). A model in which the dodecyl sulfate forms a cylindrical micellar shell about a somewhat flexible extended protein core would be consistent with the results and, if correct, would suggest that the two forma of the complex might represent different ways of packing the detergent about the protein core. Results for proteins with intact disulfide bonds indicate that very similar complexes are formed, subject to steric limitations imposed by the disulfide bonds. The amount of detergent bound is diminished (Pitt-Rivers $ Impiombato, 1968) and the resulting complex is not as extended (Fish, Reynolds tS Tanford, 1970). A paper by Few et al. (1955) indicates that cationic amphiphiles may be able to form a similar complex with serum albumin, but there are some difficulties in the interpretation of their results. (d) Association betweenproteins and micelles In the foregoing discussion, we have considered only the binding of amphiphile to protein molecules, at free amphiphile concentrations below the c.m.c. An entirely different type of association, between protein molecules and amphiphile micelles is possible. It has been demonstrated (Reynolds & Tanford, 1970a) that such association does not occur between proteins and dodecyl sulfate micelles when both components are present at relatively low concentrations. As the discussion below will show, this type of product is likely to become more important under other circumstances.

6. Competition

for Amphiphiles between Proteins and Micelles or Membranes

The observation has already been made that micelle formation and association with protein represent competitive phenomena. This is illustrated graphically in Figure 4, where estimates have been made of the free amphiphile concentrations required for alkyl sulfates to undergo micelle formation or co-operative association with serum albumin or to achieve half-saturation of the high energy binding sites of the native protein. All estimates are for approximately physiological conditions and are based on the previous data with the assumption that AH for all processes is zero1 and that t One of the binding sites of native serum albumin, not only for alkyl sulfates but for simple anions as well, has a far higher binding constant than the other high-energy binding sites and AH for binding to this site is - 18 kc&./mole (Lovrien & Sturtevant, 1971). The point of half-saturation of all 10 or 11 sites is not influenced by this one exceptional site, and its variation with temperature corresponds within experimental error to AH = 0 (Reynolds et al. 1967). The main conclusions drawn from Figure 4 would in any event be the same at any temperature.

C. TANFORD

-7 I

I

5

IO Number

of carbon

atoms

I

I

15

20

in hydrocarbon

chain

FIG. 4. Estimates, applicable to physiological conditions, for the free alkyl sulfate concentrations at which processes previously discussed will take place. The dotted line labeled “membrane” is the critical concentration for incorporation into a hypothetical membrane-like bilayer containing a small amount of alkyl sulfate. All data refer to alkyl sulfates with a single hydrocarbon chain, except that the dashed line is an estimate for the c.m.c. of alkyl sulfates with two such chains. BSA, bovine serum albumin.

ionic strength has no effect except on micelle formation. The Figure shows that 50% saturation of the high-energy binding sites of serum albumin can be attained at free amphiphile concentrations below the c.m.c. up to very long hydrocarbon chainlengths, despite the size limitation of the protein binding sites. If the curves for the c.m.c. and for 50% saturation of these sites are extended without change in slope, they would cross when n, N 18, and 50% saturation could not be attained for nc > 18. If, however, there is a limit to the hydrophobic effect per se, as was suggested by the distribution ratios for fatty acids, the two curves would both become nearly horizontal and increasing nc beyond the limits of the experimental data would not favor micelle formation. Whether or not the rod-like micellar complex can be formed for alkyl sulphates with very long hydrocarbon chains, depends on whether or not a size limitation would set in for this complex when nc > 14. A different result is obtained when these considerations are extended to alkyl sulfates with two hydrocarbon chains. The dashed line in Figure 4 shows the expected c.m.c. values for alkyl sulfates with two identical saturated hydrocarbon chains (the abscissa referring to n, for each chain), based on the assumption that the contribution of the second chain to p&C - & is 60% of the contribution of the fist chain, as indicated by the data of Figure 2. If the second chain were to make a similar contribution to & - &, for binding t.o the high energy sites of native serum albumin, the 50% saturation curve for these sites would be displaced downward to about the same extent as the c.m.c. line and the competitive situation would not be greatly altered. In view of the difficulty of finding favorable locations for even one hydrocarbon chain near the point of attachment of the anionic head groups to the protein binding sites, it is, however, unreasonable to expect muchcontribution to & -&from a second hydrocarbon chain, so that the 50% saturation curve for a two-chain alkyl sulfate would probably not differ greatly from the curve of the Figure for the single chain amphiphile. In that event, the competitive situation would shift in favor of micelle formation: the two curves would cross near nc = 12 and 50% saturation of the high energy sites could not occur when nc > 12.

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The competitive situation would also be significantly altered if the micelles into which the alkyl sulfates were incorporated were mixed micelles. The concentration of free amphiphile in equilibrium with such a micelle would be lower than the c.m.c., in analogy with the fact that the partial vapour pressure of a component of a liquid mixture is less than that of the pure liquid. (If a mixed mice110obeyed ideal solution thermodynamics, which of course cannot be expected, the equilibrium concentration of free amphiphile would be proportional to its mole fraction in the micelle.) The dotted line in Figure 4 labeled “membrane” is a hypothetical line which might represent a biological lipid bilayer, consisting primarily of phospholipids with zwitterionic head groups, and containing a small amount of dissolved alkyl sulfate. The equilibrium concentration of alkyl sulfate has been arbitrarily reduced by 8 factor of about 50 below the c.m.c., and the slope of the line has been increased in relation to the c.m.c. line, since the electrostatic repulsion factor that influences the dependence of the c.m.c. on n, would be unimportant here. Two important changes in the competitive situation are observed. (1) The rod-like micellar complexes between proteins and alkyl sulfates could not be formed at all. (2) 50% saturation of the highenergy binding sites of serum albumin could not be attained for single chain alkyl sulfates with n, > 12. For alkyl sulfates with two hydrocarbon chains there would be a similar decrease in the equilibrium concentration relative to the c.m.c., and their association with the protein binding sites would be greatly diminished, even if thta hydrocarbon chains are relatively short. The disappearance of the rod-like complex in this type of situation is in fact R common experimental experience. If a preparation of a biological membrane is suspended in an aqueous solution and enough dodecyl sulfate is added to saturate the proteins that are present, no micellar complexes are formed because the dodecyl sulfate is preferentially incorporated in the lipid bilayer. It is necessary to add a large excess of dodecyl sulfate, which destroys the bilayers and replaces them with micelles that contain a high percentage of dodecyl sulfate and relatively small amounts of phospholipid (under which conditions the equilibrium concentration of free dodecyl sulfate would approach the c.m.c.) before formation of the rod-like protein complexes can be observed. These considerations are also of interest in relation to the role of serum albumin as a carrier protein in blood, a function it must perform in the presence of cell membranes. The binding sites for amphiphiles must, therefore, be so constructed as to preclude significant binding of membrane lipids, otherwise the protein would tend to extract lipids from the membranes. This requirement is well met. The specificity of the binding sites of the native protein for anions prevents association with phospholipids with neutral or zwitterionic head groups. The fact that some of the sites combine with alkyl sulfates and sulfonates but not with alkyl carboxylates suggests that the sites may be restricted beyond the requirement for a negative charge and that the bulky head groups of anionic lipids such as phosphatidylserines may not be capable of binding. Even in the absence of this restriction, the sites would probably have poor ability to compete with the membrane. for molecules with two hydrocarbon chains. As for the function that serum albumin does carryout, i.e. transport of fattyacid sidechains, the data in Figure 4 would indicate that the ability to bind long-chain fatty acids might be relatively poor if a membrane rather than a pure fatty acid micelle is the competing location. (While the Figure itself refers to alkyl sulfates, a similar diagram for alkyl carboxylates would show similar features and would indicate that

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50% saturation of the high energy binding sites could not be attained for n, > 12.) In this regard, however, the diagram is misleading, for the 50% saturation curve refers to the average properties of the 10 or 11 strong binding sites of the protein. One or two of these sites? actually have a much higher affinity for carboxylate anions than the remainder (Goodman, 19583), and these are probably the sites that swing the competitive situation in favor of the protein.

7. Attachment

of Proteins to Membranes

The nature of the linkage between membrane-bound proteins and the lipids of the membrane is at present an unresolved problem. It is often loosely implied that the association should depend primarily on hydrophobic interactions, and the existence in membranes of some proteins with a somewhat elevated content of amino acids with hydrophobic side chains is sometimes considered to support this point of view. It is clear, however, that this idea, in its simplest form, is invalid. A protein like /3-lactoglobulin, or serum albumin, for example, but with a much larger hydrophobic binding site, would combine avidly with molecules with long hydrocarbon chains, but this process would compete with membrane formation rather than be complementary to it. Such proteins would have the effect of removing lipids from membranes and the hydrophobic binding site per se would not constitute a feature favoring association with a membrane. Another simple possibility that has sometimes been suggested is an ionic linkage between the membrane surface (which is negatively charged, owing to the presence of anionic lipids) and positively-charged areas of the protein surface. This type of linkage is not inconsistent with any of the data presented here. It was pointed out earlier that no interaction between proteins and dodecyl sulfate micelles has been observed, but under the conditions under which this observation was made (cf. Fig. 4), all proteins are converted to rod-like micellar complexes at dodecyl sulfate concentrations below the c.m.c. At sufficiently high dodecyl sulfate concentrations for the simultaneous existence of micelles, all proteins would already be in the form of complexes, with a high dodecyl sulfate content and consequent negative charge and combination with the micelle surface by simple ionic linkage would be impossible. The situation would be quite different if appropriate mixed micelles or membranes were present. The rodlike complexes would not be formed under these circumstances and micelles or membranes would coexist in the same solution with proteins in their native states. Thus, it is possible that an ionic linkage does in fact serve to attach some proteins (especially basic proteins) to membrane surfaces, though it appears unlikely that this is a common method of attachment, if for no other reason than that a protein so attached would be effectively external to the membrane proper and could have only a very limited role in functional properties. The binding sites of native serum albumin for anionic amphiphiles are the only sites of interaction between proteins and amphiphiles that have been characterized to any extent. Both the amphiphile head group and the hydrocarbon tail are involved in the association, and the interaction with the head group is not merely ionic, but has at least some chemical specificity.That portion of the binding sitewhichcombineswith the hydrocarbon chain is quite limited in size, and we have surmized that it probably could not accommodate more than one hydrocarbon chain at a time. If we use these t Seefootnote on p. 69.

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characteristics as a model for what to expect of membrane-bound proteins, it would suggest the plausibility of a protein with specificity for a particular lipid head group and a hydrophobic site for one hydrocarbon chain, which would leave the other hydrocarbon chain free to hold the resulting complex in the membrane. This model, like the preceding one, is however not satisfactory from a functional point of view because the protein is left effectively on the outside of the membrane bilayer, and its presence does little to perturb the latter. An extension of the model to a protein with several such binding sites, relatively far apart, would be more satisfactory in that it would provide a means of forcing lipid head groups apart, thereby creating a distortion of one membrane surface and a space into which parts of the protein and/or small molecules might penetrate. The limited experimental information available to us does not justify extension of this discussion to propose more complex and more speculative models. The one conclusion that seems inescapable is that a functioning membrane protein must possess a high order of specificity, in the interactions that attach it to the membrane as well as its functional site. Moreover, there is no evidence to indicate that such interactions need be exclusively or even predominantly hydrophobic in origin. The benefits of numerous discussions with Dr J.A. Reynolds and valuable advice frorn Dr J. Steinhardt are gratefully acknowledged. This work was supported by a research grant from the National Science Foundation. The author is a research Career Awardee, National Institutes of Health, United States Public Health Service. REFERENCES Becher, P. (1967). Non-ionic Surfactank, ed. by M. J. Schick. New York: Marcel Dekker, Inc. Corkill, J. M., Goodman, J. F., Robson, P. & Tate, J. R. (1966). Trans. Farad. Sot. 62, 987. Emerson, M. F. & Holtzer, A. (1967). J. Phys. Chem. 71, 1898. Evans, H. C. (1956). J. Chem. Sot. p. 579. Few, A. V., Ottewill, R. H. & Parreira, H. C. (1955). Biochim. biophys. Acta, 18, 136. Fish, W. W., Reynolds, J. A. & Tanford, C. (1970). J. Biol. Chem. 245, 5166. Foster, J. F. & Aoki, K. (1958). J. Amer. Chem. Sot. 80, 5215. Geer, R. D., Eylar, E. H. & Anacker, E. W. (1971). J. Phys. Chem. 75, 369. Goodman, D. S. (1958a). J. Amer. Chem. Sot. 80, 3887. Goodman, D. S. (1958b). J. Amer. Chem. Sot. 80, 3892. Gurney, R. W. (1953). Ionic Processes in SoEution. New York: McGraw-Hill Book Co. Hall, D. G. & Pethics, B. A. (1967). Non-ionic Surfactants, ed. by M. J. Schick. New York: Marcel Dekker, Inc. Kauzmann, W. (1959). Advances in Protein Chem. 14, 1. Kinoshita, K., Ishikawa, H. & Shinoda, K. (1958). BUZZ. Chem. Sot. Japan, 31, 1081. Klevens, H. B. (1953). J. Amer. Oil Chemists’ Sot. 30, 74. Lovrien, R. & Sturtevant, J. M. (1971). Biochemistry, 10, 3811. McAuliffe, C. (1966). J. Phys. Chem. 70, 1267. Mukerjee, P. (1967). Advances in CoZZoid and Interface Science, 1, 241. Nozaki, Y. & Tanford, C. (1971). J. BioZ. Chem. 246, 2211. Pitt-Rivers, R. & Impiombato, F. S. A. (1968). Biochem. J. 109, 825. Reynolds, J. A. & Tanford, C. (1970a). Proc. Nat. Acd Sci., Wash. 66, 1002. Reynolds, J. A. & Tanford, C. (19705). J. BioZ. Chem. 245, 5161. Reynolds, J., Herbert, S. & Steinhardt, J. (1968). Biochemistry, 7, 1357. Reynolds, J. A., Herbert, S., Polet, H. & Steinhardt, J. (1967). Biochemistry, 6, 637. Reynolds, J. A., Gallagher, J. P. & Steinhardt, J. (1970). Biochemktq, 9, 1232. Shinoda, K., Nakagawa, T., Tamamushi, B. & Isemura, T. (1963). C&oi& &‘urfactoqt~. New York: Academic Press,

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Smith, R. W. & Tanford, C. (1972). J. Mol. Bid. 67, 75. Spector, A. A. Fletcher, J. A. & Ashbrook, J. D. (1971). Biochemietry, 10, 3229. Steinhardt, J. & Reynolds, J. A. (1969). Multiple Equilibria in Proteins. New York: Academic Press. Steinhardt, J., Krijn, J. L Leidy, J. G. (1971). Biochemdry, 10, 4005. Swarbrick, J. & Daruwala, J. (1969). J. Phys. Chem. 73, 2627. Swarbrick, J. & Daruwala, J. (1970). J. Phys. Chem. 74, 1293. Tanford, C. (1970). Adwanc. Protein Chem. 24, 1. Teresi, J. D. & Luck, J. M. (1952). J. Biol. Chem. 94, 823. Wetlaufer, D. B. & Lovrien, R. (1964). J. Biol. Chem. 239, 696. Wishnia, A. (1962). Proc. Nat. Acad Sk., Wash. 43, 2200. Wishnia, A. (1963). J. Phya. Chem. 67, 2079. Wishnia, A. & Pinder, T. W., Jr. (1964). Biochemistry, 3, 1377. Wishnia, A. & Pinder, T. W., Jr. (1966). Biochemistry, 5, 1534.