W. B. GRATZER AND G. H. BEAVEN
2270
Effect of Protein Denaturation on Micelle Stability by W. B. Gratzer and G. H. Beaven Medical Research Council Biophysics Unit, King's College, Drury Lane, London, W.C.d, England, and National Institute for Medical Reaeareh (Hampstead Laboratories), Holly Hill, London, N . W.S., England (Received November 7, 1968)
A nonionic detergent, Triton X-100, has been used as a model for the investigation of the efficacy of various protein denaturants as hydrophobic bond breakers. A change in ultraviolet absorption spectrum of the aromatic chromophore has been observed when micelles are formed, and a spectrophotometric procedure is described for the determination of the critical micelle concentration. It is shown that urea has a large disrupting effect, increasing the critical micelle concentration by a factor of 5 at a concentration of 6 M . Ethylene glycol has a rather smaller effect,and sucrose has almost none. Guanidine hydrochloride has a smaller effect than urea at concentrations below about 3 M , but at higher concentrations its effect becomes rapidly larger than that of urea. This parallels the behavior observed in the solubilizationof paraffin hydrocarbons by these solvents. The anions exert an important modulating influence on the efficacy of the guanidinium ion as a hydrophobic bond breaker: guanidinium thiocyanate has an extremely strong disruptive effect, the critical micelle concentration increasing by a factor of 5 at a molarity of only 1.5. By contrast, guanidinium sulfate actually stabilizes micelles. These results are in remarkable qualitative agreement with the effects of the same substances on the stability of the native conformation of a globular protein, ribonuclease, reported in the literature. Approximate values for the standard free energy of transfer of the detergent micelles from water to the various perturbant solutions are derived and are consistent with available data on transfer of paraffin hydrocarbons.
It is widely held that protein denaturants, such as urea or guanidinium salts, owe their function to an effect on the structure of water, such that exposure of nonpolar side chains to the medium is no longer accompanied by an unfavorable free-energy change. 1--3 On the other hand, it has also been demonstrated that important contributions can arise from the solubilization of the peptide group by the d e n a t ~ r a n t ,and ~ a change in the electrostatic free energy of the protein may also play a significant part.*Ss As the data of Tanford on the solubilities of amino acids in urea solutions show, the free energy of transfer of the nonpolar side chains from water to urea solutions is entirely sufficient to account in principle for the denaturation of a protein of typical composition. Guanidine hydrochloride has been founder7to be a much more powerful denaturant than urea, a t least a t high concentrations, but the work of von Hippel and Wongs on the effect of various denaturants on the thermal transition of ribonuclease has shown that the effect of guanidinium salts is drastically modulated by the anion. It is of interest to determine to what extent these striking effects, as well as the relative efficacies of different denaturants in general, may be correlated with their effect on hydrophobic interactions. A direct and convenient measure of a reagent to destroy hydrophobic interactions, and one which bears a formal parallelism to protein denaturation experiments, without effects involving the peptide groups, is to assess its effect on the critical micelle Concentration of a detergent. This approach has been used by Bruning and H o l t ~ e rwho ,~ obtained the anomalous result that acetone, which is a relatively poor denaturant for proteins, elevates the The Journal of Physkal Chemistry
cmc to a greater extent, mole for mole, than urea. The detergent in this case, however, was a cationic species, and the interpretation of such results is therefore complicated by the importance of the bulk dielectric constant of the medium in determining the cmc, as well as by some less critical considerations, such as the influence of the solvent on the activity coefficient of the free detergent species. With a nonionic detergent such complications should be largely obviated, and one may therefore hope that the effect of substances on the cmc may offer a good model for their denaturing action on globular proteins. We describe here some studies along these lines, using the nonionic detergent Triton X-100. One report of the effect of urea and guanidine hydrochloride on the cmc of a nonionic detergent has come to our notice,'O in which both were observed to have a marked effect.
Experimental Section The detergent, Triton X-100 (Rohm and Haas Co.), as obtained, was essentially anhydrous and free from ultraviolet-absorbing impurities. It contains a pheno(1) W. Kausmann, Advan. Protein Chem., 14, l (1959). (2) Y. Nosaki and C . Tanford, J . Biol. Chem., 238, 4074 (1963). (3) C . Tanford, J . Amer. Chem. Soc., 84, 4240 (1962). (4) D.R. Robinson and W. P. Jencks, ibid., 87, 2462 (1965). (5) C. Tanford, ibid., 86, 2050 (1964). (6) C . Tanford, K. Kawahara, and S. Lapanje, ibid., 89, 729 (1967). (7) S. Lapanje and C. Tanford, ibid., 89, 5030 (1967). (8) P. H. von Hippel and K. Y. Wong, J . Biol. Chem., 240, 3909 (1965). (9) W. Bruning and A. Holtzer, J. Amer. Chem. SOC.,83, 4865 (1961). (10) M.J. Schick and A. H. Gilbert, J . Colloid Sci., 20, 464 (1965).
EFFECT O F P R O T E I N DENATURATION ON nf ICELLE
2271
STABILITY
lic chromophore, and we have used this as the basis of a spectrophotometric method for determining the cmc. The molar absorptivity a t the absorption maximum (278 mp) was determined on vacuum-stripped samples, and was found to be 1670 (above the cmc) in water, assuming a molecular weight of 647. This is consistent with standard data for the same chromophore, e.g., anisole, 1480 in water." (One reported value12 for Triton X-100 of E (1%)1 cm) = 10, corresponding to E 6500 is clearly in error.) Some additional experiments were also performed with Triton X-165, which has a longer head group (n 16). The ultraviolet absorption spectrum of the detergent undergoes an appreciable perturbation on micelle formation (Figure 1). The change is maximal at 285.5 mp in water (slightly longer wavelengths in the presence of the solutes) and we have worked throughout a t this wavelength. The cmc was determined by adding known volumes of a concentrated detergent solution from a microburet (Micrometric Instrument Co.) to a volume of water, or a desired solution, in a spectrophotometer cell (usually 1-cm path, but shorter path lengths were used for high cmc values). After each addition the contents of the cell were stirred with a loop of platinum wire and the absorbance was read (Beckman DU instrument, or more usually a Cary 14 to enable measurements to be made with precision up to an absorbance of 2). The cell compartment was thermostated a t 23". After correction for dilution, plots of absorbance against
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t-I '
I
I
1
I
n
I
'
6
VOLUME TITRANT (PI.)
Figure 2. Spectrophotometric determination of cmc: typical results for Triton X-100 in urea solutions of the indicated molarities. These molarities refer to the solution a t the start of the titration, and the molarities a t the cmc are obtained by correcting for the volume of added titrant. The arrows mark the cmc values, which are obtained from the volume of titrant, rather than the absorbance. The changes in slope of the upper limb of the curve are caused by the small red shift of the spectrum induced by the perturbant, which displaces the maximum of the difference spectrum.
volume of detergent solution added were constructed (Figure 2), and a clear discontinuity gave the cmc with considerable precision. Reagents were all analytical grades. Guanidine hydrochloride was recrystallized from methanol-water; guanidinium thiocyanate was prepared by mixing equivalent amounts of guanidinium carbonate and barium thiocyanate in concentrated solution and discarding the precipitate.
Results
O'
2to
2:o
2Lo 2:o 2Lo 2 2 - 5 2 WAVELENGTH ( m y )
Figure 1. Ultraviolet absorption spectra of Triton X-100 in water: the full line shows spectrum above the cmc and the broken line below the cmc. The upper panel shows the difference spectrum generated on micelle formation (sample, above cmc; reference, below cmc).
Figure 1 shows the absorption spectra of Triton X100 in aqueous solution above and below the cmc. There is seen to be a considerable difference, both in the integrated intensity and in the development of the longwavelength fine-structure band. The concentration difference spectrum is also shown in Figure 1. This has a primary maximum at 285.5 mp and may be noted in passing to resemble closely the tyrosine perturbation difference spectrum of proteins. l 3 Figure 2 shows some typical plots of absorbance against detergent concentration. Beer's law is obeyed a t concentrations well below and well above the cmc, and the intersection of the two linear limbs of the plot gives the value of the cmc. The total volume at the (11) L. Doub and J. M. Vandenbelt, J. Amer. Chem. Soc., 71, 2414 (1949). (12) F. F. Sun, Biochim. Biophys. Acta, 153, 804 (1968). (13) D.B. Wetlaufer, Advan. Protein Chem., 17, 303 (1962). Volume 79, Number 7 July 1565
2272
W. B. GRATZER AND G. H. BEAVEN
cmc is taken into account in relating the value to the concentration of the denaturant. The effects of urea, three guanidinium salts, ethylene glycol, and sucrose on the cmc are shown in Figure 3. With sucrose, the workable upper limit of concentration was determined by the viscosity of the solution, which made mixing very difficult above about 2 M . With guanidinium sulfate, the cloud point of the detergent occurred below the working temperature when the perturbant concentration exceeded ca. 0.8 M .
c.rn.c.
k
( rng./ml
%1
O
, 0
1
2
, ; 3
4
1.6 18
PERTURBANT CONCENTRATION ( rnoles/l.l
Figure 3. Effects of a series of perturbants on the cmc of Triton X-100. The right-hand axis gives the approximate standard free energy of transfer from water to a perturbant solution (see text). Open circles: urea; filled circles: guanidine hydrochloride; open squares: guanidinium sulfate (molarities in terms of guanidinium ion; a t higher concentrations precipitation of the detergent occurred); filled squares: guanidinium thiocyanate; open triangles: ethylene glycol; filled triangles: sucrose.
The results show that sucrose is almost without effect on the cmc, that urea, and to a lesser extent ethylene glycol, has a considerable effect, and that the guanidinium salts differ widely from each other. Thus, guanidine hydrochloride is a poor hydrophobic bond breaker at low concentrations, but becomes more effective than urea above about 3 M . Guanidinium thiocyanate has a very large disruptive effect indeed, whereas guanidinium sulfate actually stabilizes micelles relative to pure water. It is now of interest to compare these results with the effects of these substances on protein stability.
Discussion The change in absorbance of the detergent on micelle formation does not appear to have been noted previously. Triton X-100 has the structure
and evidently the chromophore is substantially in a hydrocarbon environment when the micelle has formed, as the considerable red shift and the emergence of viThe Journal of Physical Chemistry
brational structure indicate. This change in the spectrum affords a uniquely convenient and versatile method for determining the cmc in the presence of all manner of additives, excepting only those of high ultraviolet absorption. The relative effects of the substances shown in Figure 3 bring out a number of significant points. Sucrose, for example, has no appreciable effect on micelle formation, and is known to have no significant denaturing action of proteins. It is, for this reason appropriate as a medium for density gradient sedimentation'd and perturbation difference spectrophotometry.16 Ethylene glycol, on the other hand, is kn0wn8,~6,'7to have a denaturing tendency. As noted by previous workers, urea increases the cmc of both i o n i ~ and ~ ~nonionicI0 ' ~ ~ ~detergents, ~ and guanidine hydrochloride has a greater effect than urea on the latter. lo Our results show guanidine hydrochloride to be a much more effective disrupting agent above 3 M than urea, which is consistent with its much greater denaturing capacity.61' Both these results and the extraordinary differences between the guanidinium salts parallel in remarkable manner the relative effects of these substances on the temperature of thermal unfolding of ribonuclease.8 Thus guanidinium thiocyanate has a very large destabilizing effect in both cases, whereas the sulfate actually stabilizes both the detergent micelles and the native conformation of the globular protein. Thus, qualitatively at least, it appears that the effects of these various substances on protein stability can be directly accounted for in terms of their propensity to disrupt hydrophobic interactions. Quantitatively the argument should probably not be carried too far: on the one hand, one may expect that in addition to the primary hydrophobic effect, the perturbants will have a secondary influence on the solvation of the head group of the detergent. The stability of the protein will clearly also be affected by contributions of peptide group solubilization and electrostatic interactions, as mentioned above. It is worthwhile, however, to attempt some estimate of the order of free energies involved in the micelle destabilization. The formation of micelles may conveniently be seen as a phase equilibrium, so that the standard free energy of micelle formation, say in water, is given by A F O
=
-RT In C,yw
where C, is the cmc in water on a molar basis and ywis the activity coefficient of the free species. Thus the (14) (15) (16) (17) (18) (19)
R. G. Martin and B. N. Ames, J . Biol. Chem., 236, 1372 (1961). M. Laskowski, Jr., Fed. Proc., 25, 20 (1966). H. J. Sage and 8. J. Singer, Biochemistry, 1, 305 (1962). S. Y. Gerlsma, J . Biol. Chen., 243, 957 (1968). P. Mukerjee and A. Ray, J . Phys. Chem., 67, 190 (1963). M. F. Emerson and A. Holtzer, ibid., 71, 3320 (1967).
EFFECTOF PROTEIN DENATURATION ON MICELLE STABILITY standard free energy of transfer of the detergent from water to a solvent, s, will be
2273
M guanidine hydrochloride, is of the order of 0.1 kcal/
-
mol per carbon atom, which compares satisfactorily with our values (Figure 3) for Triton X-100, with n 10. cs Moreover, Wetlaufer, et al., also find a steeper effect of AF," = - R T l n - R T l n 'ya cw YW guanidine hydrochloride concentration on the solubility than urea, and for butane the two curves cross at a We have no alternative but to make the assumption molarity between 2 and 3. This is very similar to the that ys = Y ~ which , one would suppose not to be far effect noted in Figure 3. in error, since the detergent molecule is uncharged. From the data of von Hippel and Wongs on the efThe values of APtoso estimated are given on the rightfects of the various denaturants on the transition temhand axis of Figure 3. perature of ribonuclease, and literature values for its Our treatment is a very simplified one, but there is entropy of unfolding, one may derive some rough estisome evidence to support the idea that the assumptions mates of the corresponding changes in the stability of are broadly reasonable. The treatment of micellizathe globular conformation in terms of a free energy of tion in terms of a phase transition has been widely distransfer to the denaturing solvents of the globular and cussed: the extent of its validity, with particular refunfolded forms. These estimates do not give any erence to some non-ionic detergent is considered for satisfactory quantitative correlation with the values of example by Corkill, et a1.,20 who give reasons why the AFto for the detergent. This is not surprising, and additional terms arising from a kinetic treatment may reflects presumably the role of factors other than weakbe neglected. For detergents, which also contain polyoxyethylene head groups, Corkill, et ~ 1show . by~ vapor ~ ~ ening of hydrophobic interactions in the denaturation of ribonuclease. It may be remarked that Tanfords has pressure measurements below the cmc that yw is innoted the apparently anomalously low degree of hydrodistinguishable from unity. There remain the assumpphobic stabilization in this particular protein (the only and that the effect of the solutes on tions that yw = one, however, for which comprehensive data are availthe solvation of the head group will indeed be secondary able). to their influence on the hydrophobic association of the We may comment finally on the effects of the differnonpolar chains. Certainly one will expect that adent anions on hydrophobic interaction. The apparent ditives will produce some change in the free energy of stabilizing effect on the sulfate ion finds application in solvent reorientation around the head groups when the the universal use of ammonium sulfate for salting-out micelle is formed. That this effect is quite small in and crystallizing proteins. The strongly disruptive the case of urea and a series of nonionic detergents of varying chain lengths has been shown by Corkill, et aLZ1 effect of thiocyanate suggests that this may be found useful as a denaturant. Guanidine hydrochloride a t They find that the free energy of micellization remains concentrations of 6 M and above is almost universally essentially constant as the length of the head group is used for the denaturation of proteins, and in particular varied, and that the increment produced by urea, even for the purpose of breaking down quaternary associaa t 6 M , is likewise independent of chain length. We tions. One case has already come to light23in which are able to support the conclusions by our own results this solvent is not effective, and at least one case has on Triton X-165, which has a much larger head group been described24 where dissociation occurs only be(n 16) than X-100: here the free energies of transtween 5.5 and 6 M guanidine hydrochloride. It is posfer estimated in the manner indicated above are essensible that other proteins exist in which a more powerful tially the same in the two detergents for urea over the denaturant is called for, and it seems that guanidinium whole concentration range, and for guanidine hydrothiocyanate should certainly fulfill this role. chloride at the highest workable concentrations, with only some deviations in the profile at low concentraAcknowledgments. We are grateful to Mr. P. RiI. tions. We therefore feel that our assumptions are Stanley of Lennig Chemicals Ltd. for generous samples justified within the limits of a n a h e model of this kind. of detergents, to Dr. Roger Woodbridge for valuable We would stress, however, that we do not pretend to discussion, and to Professor Sir John Randall for supdraw more than qualitative, or a t most semiquantitaport. tive, conclusions from these results. The most direct measure of the effect of urea and guanidine hydrochloride on hydrophobic interactions is (20) J. M. Corkill, J. F. Goodman, and S. P. Harrold, Trans. Faraday Soc., 60, 202 (1964). provided by the work of Wetlaufer, et a1.,22on the sol(21) J. M. Corkill, J. F. Goodman, S. P. Harrold, and J. R. Tate, ubility of paraffin hydrocarbons in solutions of these Trans. Faraday Soc., 63, 240 (1967). substances. The standard free energy of transfer of a (22) D. B. Wetlaufer, 6. K. Malik, L. Stoller, and R. L. Coffin, J. Amer. Chem. Sac., 86, 508 (1964). hydrocarbon from water to a urea or guanidine hydro(23) B. E. C. Banks, S. Doonan, J. Gauldie, A. J. Lawrence, and chloride solution is dominated by a favorable entropy C. A. Vernon, Eur. J . Biochem., 6,507 (1968). contribution. For the linear hydrocarbons, the value (24) V. H. Paetkau, E. S. Younathan, and H. A. Lardy, J . Mol. Biol., of this free energy, going from water to 7 M urea or 4.9 33, 721 (1968).
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Volume 73, Number 7 July 1969
