310
Langmuir 1986,2, 310-315
General Anesthetic Agents and the Conformation of Proteins Mohammad Abu-Hamdiyyah Department of Chemistry, University of Kuwait, Kuwait Received June 4, 1985. In Final Form: December 13, 1985 General anesthetic agents in aqueous solution affect micellization equilibria as well as the optical rotation of proteins in the direction of increasing hydrophobic bonding. Linear relations are obtained between anesthetic potency and the two effects. Further considerationsindicate that the hydrophobic moiety affected during anesthesia is approximately equivalent to the effective hydrophobic moiety in sodium hexyl sulfate which looks more like a protein side chain than that of a phospholipid. Such considerations suggest that general anesthetic agents act by causing a folding of protein side chains rather than an unfolding as has been proposed.
Introduction Anesthetic agents comprise a variety of compounds ranging from inert gases to steroids, all of which produce anesthesia, a reversible suppression of consciousness. It is widely acknowledged that these agents exert their influence in nerve cell membranes interferring with nerve impulse conduction and transmi~sion.l-~The mode of action of these agents a t the anesthetic site has been the subject of many studies and various theories have been proposed to explain the mechanism by which these agents induce anesthesia."22 However, only two approaches are actively being discussed in the current literature, one implicating the lipid bilayer as the primary step in anesthetic action, the other the protein, in nerve cell membranes. The lipid solubility theory assumes that anesthetics dissolve in the lipid bilayer of nerve cell membranes and exert their action from there. This theory correlates anesthetic potency with solubility in an organic liquid such as olive oil. More recently lipid bilayers (e.g., phosphatidyl choline bilayer) and biomembranes have been used for correlation instead of organic liquids.6 An up to date assessment of lipid theories has recently been published! However, some (1) Larrabe, M. G.; Posternak J. M. J. Neurophysiol. 1952, 15, 91. (2) Woodbury, J. W.; D Arrigo, J. D.; Eyring, H. Prog. Anesthesiol. 1975,1, 253-275. (3) Mullins, L. J. Prog. Anesthesiol. 1975, I, 237-424. (4) Seeman, P. Pharmacol. Rev. 1972,24, 583. ( 5 ) Kaufman, R. D. Anesthesiology 1977, 46, 49. (6) Janoff, A. S.; Miller, K. W. In "Biological Membranes"; Chapman, Ed.; Academic Press: London, 1982, p 417. (7) Miller, J. C.; Miller, K. W. In Physiological and Pharmacological Biochemistry; Blaschko, H. F. K., Ed.; Butterworth: London, 1975, Biochemistry series one, Vol. 12, pp 33-76. (8) Ferguson, J. Proc. R. SOC.London, Ser. B 1939, 197, 387. (9) Brink, F.; Posternak, J. M. J. Cell. Physiol. 1948, 32, 211. (10) Mullins, L. J. Chem. Rev. 1954, 54, 289. (11) miller, K. W.; Paton, W. D. M.; Smith, E. B. Nature (London) 1975,206, 574. (12) Trudell, J. R. Anesthesiology 1977, 46, 5. (13) Haydon, D. A.; Hendry, B. M.; Levison, S. R.;Ftequena, J. Nature (London) 1977,268, 358. (14) Franks, N. P.; Lieb, W. R. Nature (London) 1978,247, 339. (15) Conrad, M. J.; Singer, S. J. Proc. Natl. Acad. Sci. U.S.A. 1979,
76, 5202. (16) Lieb, W. R.; Kovalycsik, M.; Mendelsohn, R. Biochim. Biophys. Acta 1982, 688, 388. (17) Richards, D.; Martin, K.; Gregory, S.; Keightley, C. A.; Hesketh, T. R.; Smith, G. A.; Warren, G. B.; Metacalf, J. C. Nature (London) 1978, 276, 775. (18) Ueda, I.; Kamaya, H.; Eyring, H. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 481. (19) Richards, D. In "Topical Reviews of Anesthesia"; Norman, J., Whitman J. G., Ed.; Bristol John Wright and Sons: 1976; Vol. I. (20) Pauling, L. Science (Washington, D.C.) 1961, 134, 15. (21) Miller, S. L. Proc. Natl. Acad. Sci. U.S.A. 1961, 47, 1515. (22) Abu Hamdiyyah, M. In "Solution Behavior of Surfactants"; Mittal, K. L., Fendler, E. J., Eds.; Plenum Press: New York, 1982; Vol. 1, p 697.
0743-7463/S6/2402-0310$01.50/0
recent experimental results as well as the results of this study throw doubt on the implication of the lipid bilayer as the site of anesthetic action.l4-l7 A more recent theory is the protein conformational change or protein unfolding theory.18 It is advocated in this theory that anesthetics interact with the hydrophobic interior of the proteins and unfold them so that the hydrophobic side chains which were buried in the interior of the protein are exposed to water and thus undergo hydrophobic hydration by the formation of clathrates around the hydrophobic sites. It will be shown in this study that anesthetic agents at clinical concentrations are expected to have the opposite effect, i.e., increased folding of proteins in aqueous solution. Richards et al.,"J9 on the other hand, proposed that anesthetics modify the function of the membrane proteins directly by reversible noncovalent binding to "specific sities" on membrane proteins which are imbedded in a lipid matrix. It will be seen from the results of this study that anesthetic action is nonspecific. It is worth mentioning at this point the aqueous-phase theories of anesthetic action proposed by Paulingm and by S. L. Millerz1 independently. Pauling suggested that nonpolar anesthetic agents operate by "increasing the impedance of the encephalonic network of conductors and this increase in impedance results from the formation in the network, presumably mainly in synaptic regions, of hydrate micro-crystals...". Miller, on the other hand, suggested the formation of 'ice-covers" or "icebergs" around the agents which lower the conductance of the membrane. Both Pauling and Miller proposed that potency is correlated with dissociation pressure of gas hydrates (clathrates). Although strengthening of water structure occurs around hydrophobic moieties in liquid water, it is suggested in this study that central to the mechanism of anesthetic action is the effect of hydrophobic groups or moieties have on the hydrophobic moieties already dissolved (projecting out) in the aqueous phase. Finally in a recent study22the author suggested that the primary step in anesthetic action appears to be akin to micelle formation. Therefore it was proposed to use the micellization process as a model for the study of the effect of anesthetic agents at the site in nerve cell membranes. The objective of this study is to estimate the size of the hydrophobic group affected at the site of anesthetic action using the micellization model. This will be shown to be more like a protein side chain than that of a phospholipid. Experimental results on &lactoglobulin as well as other arguments will be presented to bolster the above conclusion. We shall, however, first define hydrophobic bonding, 0 1986 American Chemical Society
Langmuir, Vol. 2, No. 3, 1986 311
General Anesthetic Agents and Protein Conformations strengthening of hydrophobic bonding, and its occurrence in surfactant and protein aqueous solutions as well as the relationship of strenghtening of hydrophobic bonding to the total effect as measured by the partition coefficient. Strengthening of Hydrqphobic Bonding in Surfactant and Protein Solutions and Method of Representation. Basic Concepts. Because of the strong interactions between the water molecules in liquid water, nonpolar molecules (moieties) tend to be expelled from water23-25to form a separate phase, as in the limiting solubility of inert gases or hydrocarbons, or a pseudophase, as in the aggregation of monomer surfactant molecules (ions) to form micelles, or to form an intramolecular aggregate, as occurs in the association of nonpolar side chains of amino acids in proteins. The tendency of nonpolar moieties (groups) to associate together to minimize contact with water is called hydrophobic bonding. In analyzing such equilibria it is often convenient to take the pure solute as the reference state and to assign shifts in equilibria to changes in solvent power. Our discussion will be concentrating, however, on species that remain in solution (monomers) or in contact with it (nonpolar side chains of proteins not aggregated). Under these conditions, it becomes preferable to express such shifts in equilibria in terms of strengthening or weakening of hydrophobic bonding among solutes. For example, in micellar solutions intermolecular aggregation occurs at the critical micelle concentration (cmc). The increase or decrease in the cmc of a surfactant then signifies weakening or strengthening of hydrophobic bonding in the solution, respectively. In protein solutions it is not customary to consider a critical concentration of hydrophobic side chains above which all other hydrophobic side chains in the protein molecular are intramolecularly aggregated, because all the nonpolar side chains whether aggregated or exposed to water are permanently bonded to the polypeptide backbone of the protein molecule. Instead, the coricept of conformation of the protein molecule is used such as folded or unfolded conformation, indicative of the existence of intramolecular aggregation between nonpolar side chains or its absence, respectively.26 That is, in a completely unfolded conformation all the nonpolar side chains of the protein are projecting out (i-e., dissolved) in the aqueous phase. In a solution in which the protein molecule is not completely unfolded, there is equilibrium between the nonpolar side chains which are dissolved in (exposed to) the aqueous phase and those which are aggregated, i.e., folded, which corresponds to an equilibrium between the unfolded and folded conformational states. Shifts in the structural conformations of a protein molecule in solution reflecting shifts in the extent of the nonpolar side chains projecting out, or dissolved in the aqueous phase, may be followed by observing changes in a suitable parameter which is sensitive to changes in the conformation of the protein molecules in solution. If the equilibrium is shifted toward the folded conformation then strenthening of hydrophobic bonding (Le., a decrease in the number of nonpolar side chains projecting in water) is indicated and vice versa. Strengthening of Hydrophobic Bonding. When amphiphilic substances a t low concentrations are added to protein or micellar solutions, strengthening of hydrophobic bonding occurs. For protein solutions this is well (23) Abu-Hamdiyyah, M. J.Phys. Chem. 1966,69, 2720. (24) Tanford, C. “The Hydrophobic Effect”; Wiley: New York, 1980; p 40. (25) Kauzmann, W. Adv. Protein Chem. 1959,14, 1. (26) Brandta, J. F.; Hunt, L. J. Am. Chem. SOC.1967,89, 4826.
illustrated by the experiments of Brandts and Hunt26on the effect of ethanol at low concentrations on ribonuclease which show how the stability of the native state increases with increasing ethanol concentration until a maximum is reached after which the stability decreases and eventually the protein is denatured. Similar behavior occurs with other amphiphiles and other proteins as reported by Brandts and Hunt in their paper and by others, for example, Herskovitz et al.27 These additives stabiiize the protein when added in small amounts to the aqueous protein solution and act as denaturants when added in large enough amounts.27 The stabilization of the protein native structure by these types of additives at low concentrations is also accompanied by a decrease in the optical rotation of the protein, i.e., it becomes less levorotatory,28s29 and no significant change in reduced viscosity occurs.2E The stabilized conformation obtained provides a protective effect against urea denaturation in a reversible manner confirming the action of the amphiphiles at low concentrations as strengthening of hydrophobic bonding in view of the fact that urea is a well-known agent for weakening of hydrophobic bonding in protein solutions. Moreover, the ability to stabilize the native state of proteins by all amphiphiles at low concentrations follows the pattern found for the strengthening of hydrophobic bonding in surfactant solutions as can be seen clearly from the experimental results of Herskovitz et However, at higher concentration of the amphiphile, unfolding occurs and optical rotation increases (becomes more levorotatory) and this is accompanied by an increase in reduced viscosity. A similar pattern of behavior in micellar solutions occ u r ~ .The ~ ~cmc decreases, reaches a minimum, and then starts to increase as the amphiphile concentration is increased and eventually the cmc disappears (no micelles form). When nonpolar additives, such as hydrocarbons, are added to protein or micellar solutions, strengthening of hydrophobic bonding also occurs. In protein solutions this can be concluded from the experiments of Wetlaufer and Lovrien31who investigated the effect of several nonpolar gases (methane, ethane, propane, c-propane, butane, and isobutane) on the conformations of @-lactoglobulinand bovine serum albumin in aqueous solutions. The results show that the gases shift the protein conformation toward the more compact or folded form. In micellar solutions the cmc is decreased, as was observed by Lin and M e t ~ e r ~ ~ (using ethane and propane in dodecylammonium chloride solution), K l e ~ e n (using s ~ ~ benzene and potassium alkanoates and sulfonates), and Rehfeld34 (using aromatic, unsaturated and saturated hydrocarbons and NaLS) and in this laboratory (using n-hexane, cyclohexane, and benzene in NaLS35and also in C6,CE, Cl0, c14, and C16 sodium alkyl sulfates.% The cmc of nonionic surfactants are also depressed by nonpolar additive^.^' Quantitative Representation of Strengthening of (27) Herskovitz, T. T.; Jaillet, H.; De Sena J.Biol. Chem. 1970,245, 6511. (28) Markus, G.; Karush, F. J. Am. Chem. SOC.1957, 79, 3264. (29) Tanford, C.; De, P. K.; Taggart, V. G. J. Am. Chem. SOC.1960, 82, 6028. (30) Abu-Hamdiyyah, M.; Al-Mansour, M. J. Phys. Chem. 1979,83, 2236. (31) Wetlaufer, D. B.; Lovrien, R. J. Biol. Chem. 1964,239, 564. (32) Lin, I. J.; Metzer, A. J. Phys. Chem. 1971, 75, 3000. (33) Klevens, H. B. J. Phys. Chem. 1950,54,1012. (34) Rehfeld, S. J. J. Phys. Chem. 1967, 71, 738. (35) Abu-Hamdiyyah,M.; El-Daneb, C. J.Phys. Chem. 1983,87,5443. (36) Abu-Hamdiyyah, M.; Rahman, I. A. J.Phys. Chem., submitted
for publication. (37) Niehikido, N.; Moro, Y.; Uehara, H.; Matuura, R. Bull. Chem.SOC. Jpri. 1974, 47, 2634.
312 Langmuir, Vol. 2, No. 3, 1986
Abu-Hamdiyyah
Hydrophobic Bonding, Its Relation to the Partition Coefficient, and Its Variation as a Function of Surfactant Chain Length. It is important to distinguish between the ability of an additive to strengthen hydrophobic bonding as represented by its ability to depress the critical micelle concentration (cmc) of a surfactant in aqueous solution and its partition coefficient ( K ) between the micelles and the surrounding aqueous phase. A quantitative relationship between these two quantities has recently been d e r i ~ e d . ~ ~ , ~ *
where xf and yf are the free monomer (i.e., the cmc) and additive mole fraction concentrations, respectively, and 8 is a constant independent of the additive as a first app r o x i m a t i ~ nand ~ ~dependent ~~~ only on the surfactant chain length for amphiphilic additives. In the case of nonpolar additivies 8 is a constant independent of the additive and of the surfactant chain length.35y36K is the mole fraction partition coefficient (y,/yf) with ymbeing the mole fraction concentration of the additive in the micelle. Taking
where x p is the cmc (in mole fraction) in the absence of the additive, we have for amphiphilic additives RTlnK= -d(cmc) - R T In x p - R T In 8 (2) RTln
[ -1
dCadd
Csdd-LO
corresponding to the following respective (standard) free energy terms:36 -AGcoago = -AGHp-Ado - AGmico- AGelecto (3)
That is, the standard free energy of coaggregation AG,' (-RT In K ) is equal to the change in the hydrophobic bonding free energy due to the presence of the amphiphilic ) plus the additive AGHP-Ado(-RT In [-d(cmc)/dC,dd]c standard free energy of micellization A G d > ( f l i x p ) plus the change in the electrostatic free energy due to the positioning of the amphiphilic additive between the ionic heads in the micelle AGel,,,"(RT In e). A similar relation is obtained for nonpolar additives except that the electrostatic term is constant. We have found experimentally that In K for a given amphiphilic additive increases with decreasing surfactant chain length39but the opposite trend is found for nonpolar additives.% However, for both types of additives, In [-d(cmc)/dCaddlc, +,, decreases with increasing surfactant chain length. ft is clear that In [-d(cmc)/dCaddlCadd4is the appropriate term to represent strengthening of hydrophobic bonding. Moreover this strengthening of hydrophobic bonding can be estimated and its value represents the effect of the additive on the hydrophobic bonding tendency in the aqueous solution and depends on the concentration of the free monomers (hydrophobic moieties) in (exposed to) the aqueous phase. This is true also in aqueous solutions of nonionic surfactants (see Figure 3). Thus strengthening of hydrophobic bonding in surfactant solutions is obtained by measuring the initial rate of (38)Abu-Hamidiyyah, M. J. Phys. Chem., in press. (39)Abu-Hamdiyyah, M.; Ftahman, I. A. J.Phys. Chem. 1985,89,2377 (1985).
-4
-2
log
0
2
4
[-d C( aCdMdC)NaLS 1Cadd --o
Figure 1. Anesthetic potency, the reciprocal of the aqueous concentration (M) needed to produce anesthesia, plotted against the abilityto strengthen hydrophobic bonding solutions of sodium lauryl sulfate at 25 "C.Anesthetics are referred to as follows: (1) ethanol; (2) 1-propanol;(3) 1-butanol;(4) 1-pentanol;(5) 1-hexanol; (6) 1-heptanol;(7) methoxyfluorane;(8) halothane; (9) enflurane; (10) oxygen; (11)argon; (12) methane; (13) ethane; (14) propane; (15) n-hexane; (16) benzene; (17) cyclohexane; (18) xenon; (19) n-pentane; (20) n-butane. Anesthetic potency data were taken for 1-5 from ref 40,6 and 15 from ref 9, 7 , 8, and 18 from ref 7 , 9 from ref 66, 10 from ref 61, 11-14 from ref 41, 15, 16, 17, 19, and 20 from ref 8 and 10. Potencies in pressure units were converted to concentration through the standard solubility.
the cmc decrease with additive concentration and by a suitable equivalent quantity such as the initial rate of change of the specific optical rotation with additive concentration, (da/dCadd)cd-LO, in protein solution (abecoming less levorotatory) or the corresponding change in the "difference spectra", for example, (d(At/dC,dd)Cadd+,.
Results and Discussions Anesthetic Potency and Strengthening of Hydrophobic Bonding in NaLS Solutions. Figure 1shows the logarithum of anesthetic potenc~~9J0~40~41 (the reciprocal of the aqueous concentration in moles per liter needed to produce anesthesia) plotted against the ability to strengthen hydrophobic bonding in aqueous solutions of NaLS at 25 O C , log (-d(cmc)/dCadd)Cd+,. The anesthetic potency data come from several sources and are not for the same subjects and therefore some error is introduced. However, this factor is not very significant compared to the 5 orders of magnitude range covered. The values for the ability to depress the cmc for ethanol and propanol were taken from Manabe and Koda,42for butanol, pentanol, hexanol, and heptanol from Hayase and Hayano4, and for methoxyfluorane, halothane, and enfluorane from Kaneshina et al.44and for n-hexane, benzene, and cyclohexane were obtained in this l a b ~ r a t o r y ~These ~. substances, which represent aqueous (nonionic), volatile, and nonpolar anesthetics, all fall reasonably on the same line with a slope, intercept, and a correlation coefficient of 1.0 f 0.1, 3.71 f 0.16, and 0.94, respectively. This correlation illustrates the nonspecificity of both action at the an(40)Pringle, M.J.; Brown, K. B.; Miller, K. W. Mol. Pharm. 1981,19, 49. (41)Hansch, C.;Vittoria, A.; Sclipa, C.; Jow, P. Y . C . J.Med. Chem. 1975,18,546. (42) Manabe, M.; Koda, M. Bull. Chem. SOC.Jpn. 1978,51, 1599. (43)Hayse, K.; Hayano, S.Bull. Chem. SOC.Jpn. 1977,50,83. (44)Kaneahina, S.; Kamaya, H.; Ueda, I. J. Colloid Interface Sci. 1981,83,589.
Langmuir, Vol. 2, No. 3, 1986 313
General Anesthetic Agents and Protein Conformations 3 r
esthetic site in the CNS and of the strengthening of hydrophobic bonding in NaLS solutions. It also suggests that as a first approximation a single site is sufficient to account for the action of all the various types of agents and that the anesthetic action is similar to the process of strengthening of hydrophobic bonding produced by the agents in NaLS solution. Furthermore, in the equation, log-
1
c*n
= log
-d(cmc NaLS) dcadd
+ 3.71 f 0.16
(4)
)cadd+
the term log (-d(cmc)/d&d) is related to the hydrophobic bonding component of the standard free energy change, due to the presence of the additives, which is a unitary free energy based on the mole fraction scale (see eq 1and 2). Therefore if the concentration C, (which is in moles per liter) is divided by 55.4 then both sides would be consistent and the intercept becomes 1.97 f 0.16. The value of this constant may now be taken as the difference in the value of the strengthening of hydrophobic bonding produced by the agent in the aqueous solution of NaLS and at the anesthetic site in the aqueous solution of CNS, due to difference in hydrophobicities. We have recently measured the effect of amphiphiles30 and nonpolar substance36on the cmc of Cl0, CI2,C14, and c16 sodium alkyl sulfate and the results show that the strengthening of hydrophobic bonding produced by a given additive decreases linearly with the number of carbon atoms in the surfactant chain. From the linear relation between log (-d(cmq)/dCdd)c and log (cmcJ or C,, the number of carbon atoms in% surfactant chain (cf. Figure 3), we find that the constant 1.97 f 0.16 is equal to the (logarithm of the) ratio of the strengthening of hydrophobic bonding in sodium pentyl (or hexyl) to sodium lauryl sulfate solutions. Thus we arrive at the relation
suggesting that anesthetic agents strengthen hydrophobic bonding of a nonpolar moiety in the aqueous phase of CNS with an effective hydrophobic chain approximately equal to that of a C5 or c6 sodium alkyl sulfate. The effective hydrophobic core of such an ionic surfactant is expected to be small,3gwhich means that the agent in the process of hydrophobic bonding in the aqueous phase of the CNS is removed from an aqueous environment to an aggregate where it would very likely be partly in contact with water. We have included in Figure 1 oxygen, argon, methane, ethane, propane, xenon, pentane, and butane for which the ability to depress the cmc was obtain indirectly by using the data of Matheson and King,45Bolden et al.,46and W i ~ h n i aand ~ ~the relation35 -d(cmc NaLS)
(
dCadd
)
C&*4
- 2 x o.o0aK 55.4
where K is the partition coefficient between the micelles and water. These extra points fall also on the same line making no significant change in the slope (0.99 f 0.07) but shifting the intercept slightly (3.63 f 0.09) to make the conclusion that anesthetic agents strengthen hydrophobic bonding of a hydrophobic moiety in the aqueous phase of CNS with an effective hydrophobic chain equal to that of (45) Matheson, I. B. C.; King, A. D., Jr. J. Colloid Interface Sci. 1978, 66, 464. (46) Bolden, P. L.; Hoskins, J. C.; King, A. D., Jr. J. Colloid Interface Sci. 1983, 91, 454. (47) Wishnia, A. J. Phys. Chem. 1963, 67, 2097.
-
1
-2
-3
/
-2
-I
0
Aa ( d r g .
+I
+2
l o g AP,,(otm.)
Figure 2. Anesthetic potency, the reciprocal of the partial
pressure in atmospheres needed to produce general anesthesia, of methoxyfluorane (l),chloroform (2), halothane (31,n-butane (4), ethyl ether (5), propane (6),ethane ( 7 ) , xenon (8),nitrous oxide, (9),and methane (lo), plotted against the ability to strengthen hydrophobic bonding in P-lactoglobulin in aqueous solution at 25 "C,pH 7.75, as measured by the rate of change of the optical rotation a 5460A (becoming less levorotatory) with the partial pressure of the anesthetic. The data were taken from ref 31 and 48. sodium hexyl sulfate more favorable. The nonpolar groups in nerve cell membrances that have sizes comparable to the effective hydrophobic groups in C5 or c6 sodium alkyl sulfates are those of protein side chains. Anesthetic Potency and Strengthening of Hydrophobic Bonding in a Soluble Protein. Balasubramanian and Wetlaufer4 investigated the effect of anesthetic agents on ,&lactoglobulin and bovine serum albumin solutions at 25 OC by following the changes in the optical rotation of the protein solution in the visible spectrum. This rotation became more positive (less levorotatory), opposite to the direction observed on denaturation by urea~8~29~49 indicative of strengthening of hydrophobic bonding in view of the fact that urea weakens hydrophobic bonding in these protein solutions. The measured change in the optical rotation (becoming less levorotatory in the visible) is taken as a measure of the extent to which the nonpolar side chains exposed to the aqueous phase are aggregated-coaggregated. This conclusion is in accord with the views of Tanford et al.29 regarding the origin of the difference in the value of the optical rotation of 8-lactoglobulin in the native and denatured states and also with the general view expressed by Hermanso regarding the contribution of folding to the value of optical rotation. As a first appr~ximation~l the specific rotation in the visible region may be considered to be made up of two contributions, a levorotatory one from the amino acid residues and a dextrorotatory one arising from secondary and tertiary structures. If A ( a ) is the change in optical rotation when the partial pressure of the anesthetic changes by AP(atm) then A ( a ) / A Pmeasures the effectiveness of the anesthetic in strengthening hydrophobic bonding in the protein (48) Balasubramanian, D.; Wetlaufer, D. B. h o c . Natl. Acad. Sci.
U.S.A.1958, 55, 762.
(49) Greene, R. F.; Page,C. N. J . Biol. Chem. 1974, 249, 5388. (50) Herman, J. R. J. Methods Biochem. Anal. 1965,13, 81. (51) Perrin, J. H.; Hart, P. A. J. Pharmacol. Sci. 1970, 59, 431.
Abu-Hamdiyyah
314 Langmuir, Vol. 2, No. 3, 1986
-1
//
4 t
f
/ -I2
protruding is equal to the difference between these two volumes. For soluble proteins the fraction of nonpolar residues p~~ exposed to water (a)has been e ~ t i m a t e d ~as~0.4-0.5. Knowing the molecular weight, partical specific volume u< (mL/g-’) and the number of nonpolar side chains in the protein molecule (n)53155-57 gives the concentration of unaggregated nonpolar side chains in aqueous solution as (na)/(l.8V) where V is the molar volume in liters. For bovine ribonuclease (MW = 13863, up- = 0.728, n = 74, a = 0.45) the concentrations is 1.9 M. For 0-lactoglobulin (35000, 0.751, 128, and 0.45) the concentration is 1.2 M; for bovine serum albumin (65000,0.734,338, and 0.45) it is 1.9 M. For elastinMwhich is more hydrophobic and less soluble with molecular weight per chain between crosslinks equal to 6000, v2- = 0.769, n = 51, and a = 0.4, the concentration is 2.4 M. If a is taken equal to 0.2 in elastin, i.e., 80% of hydrophobic side chains are not exposed to the aqueous phase, the concentration of unaggregated nonpolar side chains to the solvent would be 1.2 M. Turning now to membrane proteins, the primary structure of the eels sodium channel protein was recently determined.59aib It has 1820 amino acids of which 1340 are nonpolar. The proposed transmembrane topology of this protein shows four homology units spanning the membrane “presumably surround the ionic channel”. Assuming the number of nonpolar side chains taking part in constructing the inner wall of the ionic channel n = nt(Vch/V)where nt is the total number of hydrophobic side chains in the protein, Vchthe volume of the ionic channel, and V the molar volume of the protein, the concentration of hydrophobic side chains exposed to the solvent in the ionic channel is equal to (na)/Vch= (nt(a)/V. Taking the molecular weight59to be 260000 and the specific volume 0.75 and a = 0.4 as in the case of globular proteins, the concentration of hydrophobic side chains exposed to water is 2.7 M. If a = 0.1 then the concentration would be about 0.7 M. These values are comparable to those obtained for globular soluble proteins and therefore the strengthening of hydrophobic bonding effect would also be correspondingly similar in both types of proteins. These estimated values of the concentrations of unaggregated hydrophobic side chains in the aqueous phase are comparable to the cmc of a surfactant with c3-cG hydrophobic tai1.36,46,60 It is worth noting at this point that the phopholipid cmcZ4is about 10-loM and thus the strengthening of hydrophobic bonding of phospholipid ions compared to that involving the hydrophobic moieties of protein side chains
LA4 -12
-8
-4
0
In C M C i
Figure 3. Strengthening of hydrophobic bonding produced by a given additive in an aqueous solution of ionic or nonionic surfactant 89 measured by the initial slope of the variation of the cmc with additive concentration plotted against the concentration of unaggregated monomeric surfactant molecules or ions in the aqueous solution. Data on potassium alkanoates (A)have been taken from K l e ~ e n s(the ~ ~ benzene data) and from Shinodae2 (ethanol and propanol) and data on polyoxyethylene lauryl ethers ( 0 )were taken from Nishikido et al.37 aqueous solution and therefore should correlate with its anesthetic potency. Figure 2 shows anesthetic potency (the reciprocal of the partial pressure of the anesthetic agent1°s41necessary to anesthetize 50% of subjects) of methoxyfluorane (11, chloroform (2), halothane (3), butane (4), ethyl ether (51, propane (6), ethane (7), xenon (8), nitrous oxide (91, and methane (10) plotted against A ( a ) /AP both on logarithmic scales. All the anesthetic potency data came from ref 41 except for that of butane which was taken from ref 10 the subjects being mice in all cases. A linear relation is obtained with a correlation coefficient of 0.956, slope 1.70 f 0.19, and an intercept of 2.59 f 0.23. Thus, we find that anesthetic potency is linearly related to the ability to strengthen hydrophobic bonding in the protein solution. This result is expected to be general, because the strengthening of hydrophobic bonding effect which occurs in the aqueous phase is nonspecific and ita extent depends, for a given additive, on the concentration of free nonpolar moieties in (exposed to) the aqueous phase. This dependence is illustrated in Figure 3 for nonpolar and amphiphilic additives with ionic surfactants (benzene and alkanols, respectively, in potassium alkanoates) as well as for the amphiphilic additive propanol in nonionic surfactants. The higher the concentration of unaggregated hydrophobic moieties in the aqueous phase the greater the ability of an additive to stren@hen hydrophobic bonding in the given solution. That both soluble globular proteins and transmembrane proteins have comparable concentrations of hydrophobic side chains exposed to the solvent may be shown by using published data on membrane and on soluble proteins. Considering soluble proteins first, it has been empirically found for several globular proteins that the water-accessible surface area is nearly exactly twice that if the protein was considered fi uniform sphere with the same mass and density.52 This is the surface area of a sphere having a radius 1.4 times and a volume 2.8 times larger. The excess surface area is due to the roughness of the protein surface. We shall assume for an order-of-magnitude estimate that the volume of the solvent in which the side chains are (52) Janini, J. J.Pharmacal. Sci. 1976, 105, 13.
(53) Wolfenden, R. V.; Cullis, P. M.; Southquate, C. C. C. Science (Washington,D.C.) 1979,206, 575. (54) Tanford, C. Adu. Protein Chem. 1969, 23, 121; 1970, 24, 2-95. (55) Croft, L. Handbook of Protein Sequence Analysis, 2nd ed.;Wiley: New York, 1980. (56) Putnam, F. W., Ed. The Plasma Proteins, 2nd ed.; Academic Press: New York, 1975. (57) Marshall, A. G. Biophysical Chemistry; Wiley: New York, 1979; -
p
nnn
LUL.
(58) Gosline, J. M. Adu. Exp. Med. Biol. 1977, 79. (59) (a) The author is grateful to a reviewer who pointed out Ref 59b. (b) Noda, M.; Shimizu, S.; Tanabe, T.; Takai, T.; Kayano, T.; Ikeda, T.; Tskahashi, H.; Nakayama, H.; Kanaoka, Y.; Minamino, N.; Kangawa, K.; Matauo, H.; Raftery, M. A.; Hirose, T.; Inayama, S.; Hayashida, H.; Miyata, T.; Numa, S. Nature (London) 1984, 312, 323. (60) Mukerjee, P.; Mysels, K. J. Critical Micelle Concentrations of Aqueous Surfactant Systems; National Bureau of Standards, US Department of Commerce: Washington, DC, 1971. (61) Edger, E. J. Anesthetic Uptake and Action; Williams and Nilkins: Baltimore, 1974. (62) Shinoda, K. The Formation of Micelles. Colloidal Surfactants; Hutchinson, E.; Rysselberghe, P. V. Eds.; Academic Press: New York, 1963; pp 1--88.
Langmuir 1986,2, 315-319
315
is insignificant (equal to the ratios of their cmc’s, Le., of pentyl or hexyl sulfates suggesting it to be a protein side chain. The linear relationship obtained between anesthetic the order to 1). Pressure Reversal of Anesthesia. It is well-known potency and the ability to strengthen hydrophobic bonding that hydrostatic pressure weakens hydrophobic bonding in 8-lactoglobulin aqueous solution adds support to the in surfactant solutions63and also reverses a n e ~ t h e s i a . ~ ~ ~above conclusion. Further support came from the equivalence of the effect of hydrostatic pressure on anesthetic Kaneshina et al.63measured the effect of pressure on the action and on the ability to weaken hydrophobic bonding CMC of sodium alkyl sulfates. From their results it is in sodium pentyl or hexyl sulfates. found that the pressure required to weaken hydrophobic The basic point raised in this study is that on introbonding in sodium hexyl sulfate solution just enough to counteract the strengthening of hydrophobic bonding ducing anesthetic agents in the aqueous phase containing produced at clinical concentrations is about 240 atm comfree, i.e., unaggregated, hydrophobic moieties of protein pared with the observed value of about 100-150 atm to side chains at the site of anesthetic action, aggregation of If sodium pentyl sulfate ws assumed reverse ane~thesia.”~ hydrophobic moieties occurs causing the folding of the side instead of the hexyl homologue then the pressure required chains. This action is nonspecific and depends only on the concentration of free hydrophobic moieties in the aqueous to counteract the effect produced by anesthetic agents falls phase. Thus it is unlike the “protein-unfolding” theory within the observed range (140 atm). of Ueda et al.le or the protein “specific site” theory of Summary and Conclusions Richards et al.17 According to the point of view presented in this study the lipid bilayer does not play a direct role We have used the micellization process as a model for in anesthetic action. the action of anesthetic agents at the site in nerve cell Finally, the new hypothesis may be looked upon as an membrane and arrived at the conclusion that the hydroextension of the aqueous phase theories of Paulingm and phobic moiety involved during anesthetic action is of Millerz1to which the concept of “strengthening of hyequivalent to the effective hydrophobic group in sodium drophobic bonding“ has been introduced. (63) Kaneshina, S.; Tanaka, M.; Tomido, T.; and Matuura, R. J. Colloid Interface Sci. 1974, 48,4550.
Acknowledgment. I thank Dr. Karol J. Mysels for his help and interest in this work.
Infrared Spectrum of Diborane Adsorbed on Silica B. A. Morrow*? and Richard A. McFarlane Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada KIN 9B4 Received December 19, 1985 Diborane at low pressures (10-20 torr) does not react to a significant extent with surface OH groups on silica at 20 O C . However, there is some reaction at slightly elevated temperatures (such as that due to the heat from the infrared source in a dispersive spectrometer), and all OH groups react rapidly if the silica/BzH, mixture is heated at 100 “C. Regardless of the extent of reaction, three dissociativelyadsorbed surface species, called A, B, and C, are formed. Species A, =SiOB2H5,is formed from reaction with isolated =SOH groups, or with siloxane bridges, and is stable in the presence of gas-phase diborane. Upon evacuation this converts to species B, =SiOBHz, via the equilibrium =SiOB2H5 =SiOBH2 + ‘/ZBzH6, and species A can be completely restored upon readdition of diborane. A third species, C, results from the reaction of diborane with adjacent H-bonded hydroxyls via the reaction 2SiOH + ’/&& (Si0)2BH+ 2Hz and is more abundant when the initial surface OH density is greater. Diborane also reacts with reactive siloxane sites which are created as a result of high-temperature degassing to yield A (or B after evacuation) plus SiH. A definitive spectroscopic assignment was only possible by using “B2H6, “BZD6, ‘OB2H3D3,and oxygen-18 exchanged silica.
-
Diborane was one of the first probe molecules to be used in order to determine the density of hydroxyl groups on silica surfaces.’-5 It was assummed that a determination of the ratio of Hz evolved to BzH6 consumed (called R hereafter) could be used to determine not only the number of surface OH groups but that one could also determine the relative proportions of isolated =SiOH groups and “Paired” hydroxyl groups [either as H bonded Pairs of adjacent S 3 i O H groups or geminal =Si(OH)2 groups]. The reaction with isolated groups was assumed to be SiOH + ‘/ZBZH6
-
SiOBH2 + H2& = 2
(1)
whereas with “paired” silanols the reaction would be either of Member of the Ottawa-Carleton Chemistry Institute.
0743-7463/86/2402-0315$01.50/0
=SioH ESiOH
+
’ \Si/oH
-=Si-0
9262H6
‘/282H6
\OH
=S,-O’
\E-H
\S/O\BH / \o/
+
2H,,
+
2H2, R . 4
R = 4 (2)
(3)
It further assumed that even higher values for R might arise from highly hydroxylated surfaces where three silanols might react to produce a (=Si0)3B surface species, yielding 3H2 per 1/2BzH6. (1) Weiss, H. G.; Shapiro, I. J. Am. Chem. SOC.1963,75,1221; J.Phys. them. 1953.57. . .. . -. .- , 219. (2) W e b , H. G.; Shapiro, I.; Knight, J. A. J.Am. Chem. SOC. 1969,81, 1823. 2
( 3 ) Naccache, C.; Fancoise-Rossetti,J.; Imelik, B. Bull. SOC.Chim. Fr. 1969,404. (4) Naccache, C.; Imelik, B. C. R . Hebd. Seances Acad. Sci. 1960,250, 2019; Bull. SOC.Chim. Fr. 1961, 553. (5) Fripiat, J. J.; Van Tongelen, M. J. Catal. 1966, 5, 158.
0 1986 American Chemical Society
