NOTES
733
NOTES
The Effect of Salts of Organic Acids and Bases on the Thermal Transition of Ribonuclease
by Eugene E. Bchrier and Lois D. Mackey Department of Chemistry, State University of New Y O Tat~ Binghamton, Binghamton, New York (Received May 10,1067)
In previous work, it was shown that alcohols' affected the transition temperature, T,, of ribonuclease to an extent which depends on the chain length of the added alcohol. von Hippel and Wong2 have confirmed this observation and have shown the existence of a similar trend for the tetraalkylammonium bromides. An extension of these investigations, utilizing the salts of simple organic acids and bases, is described in this paper. Of particular interest is the effect of the charge of the ions on the trend of T , values with increasing alkyl chain length.
Experimental Section Ribonuclease-A was purchased from the Sigma Chemical Co. (Type XIIa) and was used without further purification. Reagent grade or other good quality salts were employed. They were analyzed by standard methods. Stock solutions of the denaturants were prepared on a rnolarity basis. The pH values of most of the carboxylate stock solutions were adjusted to between pH 6 and 7 by adding small amounts of a carboxylic acid solution of the same concentration. For the T , measurements, samples of ribonuclease (15 mg) and the buffer salts employed previously' were diluted with a 10-ml aliquot of a salt solution. The apparent pH values of these solutions of ribonuclease were measured with a Radiometer pHM4 pH meter. Transition temperatures were obtained using the ultraviolet difference spectra method described previously.'
without added salt, TO , = 61.2", is the average result of several experiments. Measurements could not be carried out in solutions of sodium formate and acetate with concentrations greater than 2 M owing to precipitation of protein around 70". The apparent pH values of the ribonuclease solutions varied from 5.5 to 8.5. Although most of the measurements were carried out for the carboxylates between pH 6 and 7, the range of pH for the amine hydrochloride solutions was between 5.5 and 6.5. This difference was unavoidable, since the concentration of added amine hydrochloride was much larger than the 0.01 M mixed phosphates employed as a buffer. Since the presence of phosphate salts affects the transition temperature of the ribon~clease,~ the use of the same phosphate concentration as in the previous investigations with other classes of denaturants,*12was desirable. The variation in pH in these experiments was not considered to be a critical factor because von Hippel and Wong2 have shown that the transition temperature of ribonuclease in dilute buffer solutions is independent of pH in the pH range 5.5-11. However, it was important to determine whether the presence of salt induced a pH dependence in the transition behavior within the pH range of the experiments. To test this possibility, separate A(0D) us. temperature curves were obtained using 3 M sodium propionate at pH values 7.1 and 8.1. The transition temperatures obtained from these curves were the same, within experimental error. Transitiontemperature values, obtained with 3 M sodium butyrate at 7.2 and 8.4, were also commensurate. These results are consistent with the pH independence of the transition temperature reported by von Hippel and Wong.2
Discussion Reference to Figure 2 shows that T , values at a given concentration of salt generally decrease with increasing alkyl-group chain length. However, there are qualitative differences in the shapes of the curves for the two
Results Figure 1 presents representative plots of the ultraviolet difference spectrum of ribonuclease at 286 mp as a function of temperature in solutions of three different composition^.^ The T , values are obtained as the temperature corresponding to the midpoint of the total differential optical density change. The T , values are plotted as a function of solution composition for the carboxylates in Figure 2a and for ammonium chloride and the amine hydrochlorides in Figure 2b. The values of T,n are considered reproducible to *0.4". The transition temperature of ribonuclease solutions
(1) E. E. Schrier and H. A. Scheraga, Bwchem. Bwphys. Acta, 64, 406 (1962); E.E. Schrier, R. T. Ingwall, and H. A. Scheraga, J. Phys. Chem., 69,298 (1965). (2) P. H. von Hippel and K-Y. Wong, J. BbZ. Chem., 240, 3909 (1965). (3) Material supplementary to this article in the form of values of A(0D) va. temperature for ribonuclease for all the solutions used has been deposited as Document No. 9861 with the AD1 Auxiliary Publication Project, Photoduplication Service, Library of Congress, Washington 25, D. C. 20540. A copy may be secured by citing the document number and remitting in advance $2.50for photoprints or $1.75 for 35-mm microfilm. Make checks or money orders payable to: Chief, Photoduplication Service, Library of Congress. (4) J. Hermans, Jr., and H. A. Scheraga, J. A m . Chem. Soc., 83, 3283 (1961).
Volume 78, Number d February 1968
NOTES
734 -0.20
-0.15
CI 0
0 -0.10
a
-0.05
0 20
40
30
50
TEMPERATURE
70
60
80
(OC)
Figure 1. The ultraviolet difference spectrum of ribonuclease at 286 mp as a function of temperature for ribonuclease in: (a) 3.00 M butylamine hydrochloride, 0; (b) dilute buffer solution, 0;and (0) 1.43 M sodium acetate, 0.
1
I
(a)
-
I
CH3COONa
0 70-
Y
?! c 0
50
6d
I
o C3H7COONa 0
CqHgCOONo
, 1.0
2.0
C4HgNH3CI
401
3.0
0
1.0
2.0
3.0
Molarity of Added Salt
Figure 2. (a) The transition temperature of ribonuclease as a function of sodium carboxylate concentration and (b) the transition temperature of ribonuclease as a function of ammonium chloride and alkyl amine hydrochloride concentration. The points denoted 0 for NH&1 and CHaNHaCl are superimposed.
classes of denaturants. Ammonium chloride and methyl amine hydrochloride give slightly curved but nearly horizontal plots of T , us. salt concentration. Increasing the chain length of the amine hydrochloride leads to progressively greater T, decreases with salt concentration and linear concentration dependence. The Journal of Physieal Chemistry
On the other hand, the sodium carboxylates (excepting those of longest chain length) raise the transition temperature. The curvature of the plots shows an increase with chain length and is particularly evident with sodium propionate and sodium butyrate as denaturants. Consideration of these trends is aided by the following model5 for the transition behavior of a protein. In the native state, nonpolar side chains and amine groups are assumed to be buried, ie., not exposed to the solvent, while charged groups are primarily on the surface of the molecule. Thermal unfolding of the protein exposes the buried groups to the solvent. A variation in solvent composition, e.g., the addition of salts to the aqueous medium, shifts the transition temperature from its value in dilute buffer solution to a new position which is characteristic of the particular solvent mixture. It has been suggented6that this shift in transition temperature, AT, = T , T,O, arises from the transfer of the buried groups from the interior of the protein to two different environments, the dilute buffer in one case (T,O) and the salt solution in the other (T,). Since the initial state is the same in both cases, the change, AT,, may be related to a free energy of transfer, AFT, of amide groups and nonpolar side chains from water to salt solutions! Charged groups which remain on the surface in both states are assumed to be uninfluenced by this variation in solvent composition in the pH region in which the experiments were carried out. The fact2 that the transition temperature of ribonuclease is independent of pH and, therefore, does not vary with changes in its own charge from pH 5.5 to pH 11 lends support to this assumption. The value of AFT for the transfer of the buried groups from water to solutions of salts containing alkyl groups should be the summation of several effects. We separate these as the interactions of: (1) the ionic head group and its associated cbunterion with the nonpolar side chains and amide groups, and (2) the alkyl chain of the ion with the nonpolar groups of the protein. Other effects are considered to be of lesser significance. The possible importance of the free energy of transfer of amide and nonpolar groups from water to salt solution in determining the sign and magnitude of the transition temperature changes is suggested by an examination of the salting-out data for the model peptide, acetyltetraglycine ethyl ester, obtained by Robinson and Jencks.7 The values of AFT for the transfer of this peptide from water to a 1 M solution of ammonium chloride and sodium acetate may be calculated from their salting-out parameters as +48 cal/mole for NH4C1 and +314 cal/mole for CH8COONa. The negligible effect of NH&1 on the transition temperature
-
(5) E.E.Schrier and E. B. Schrier, J . Phys. Chem., 71, 1851 (1967), and references therein. (6) See ref 5, eq 7-12. (7) D. R. Robinson and W. P. Jencks, J . Am. Chem. SOC.,87, 2470 (1965).
NOTES of ribonuclease is consistent with the small value of AFT calculated from the effect of this salt on the model peptide. On the other hand, the larger positive value of AFT in the case of sodium acetate is in qualitative accord with the increased T, produced by this salt. It is assumed in these comparisons that the interactions of the model peptide with the salt solutions correspond to those of simila,rgroups on the protein. We consider, in terms of the model, the differences in the effectiveness of the initial members of the two classes of denaturanb in changing the transition temperature of ribonuclease. While sodium formate and sodium acetate raise the transition temperature, ammonium chloride and methylammonium chloride produce little change. Furthermore, as will be discussed in more detail below, the methyl groups of the acetate ion and the methylammonium ion do not appear to alter the effect on T, produced by the polar head group of the respective ions. The contrasting behavior of the sodium carboxylates and the amine hydrochlorides of alkylgroup chain length 0 or 1 toward ribonuclease appears, therefore, to depend completely on head group and counterion interaction with the protein. The effect of increasing the alkyl-group chain length of the ions on. the transition behavior can also be considered. At low concentrations of added salt (> ~ ~ ~ prevails, only a small effect is due to this term.6 The dipolar correlation time can be governed by either a diffusion , is the case for C U ( H ~ O ) ~ or~ by +,~ process ( r 0 = T ~ ) as the electron relaxation time (7c = TIJ, as is the case for M(H20)62+.6The bases for these assignments of T~ are the activation energies obtained from variable temperature studies (E+,d= 5 kcal, whileET,le= 1 kcal) and approximate calculations of values of T~ needed to account for experimental relaxation times. With the aid of these previous investigations, the relation between the viscosity of the solution and T~ has been explored for the purpose of developing a third method of assigning T~ to a physical process and to further understand the relaxation process. The viscosity of the solutions was varied by the addition of glycerin obtained from Matheson Coleman and Bell and labeled 99+%. The viscosity of the solvent mixtures at 40” has been previously reportedall (5s27O2)]
(1) Correspondence and request for reprints should be sent to Physical Chemistry Laboratory, Research Laboratory, Edgewood Arsenal, Md. 21010. (2) H.M. McConnell, J . Chem. Phys., 28, 430 (1958). (3) T . J. Swift and R. E. Connick, ibid., 37, 307 (1962). (4) A paper which can serve as a key to earlier literature is Z. Lua and S. Meiboom, ibid., 40, 2686 (1964). (5) L. 0.Morgan and A. W. Nolle, ibid., 31, 365 (1959). (6) N. Bloembergen and L. 0. Morgan, ibid., 34, 842 (1962). (7) R. A. Bernheim, T . H . Brown, H. 8. Gutowsky, and D. E. Woessner, ibid., 30, 950 (1959). (8) P. F.Cox and L. 0. Morgan, J . Am. Chem. SOC.,81,6409 (1969). (9) I. Solomon, Phys. Rev., 99, 559 (1957). (10) A small correction for the relaxation time in the absence of the transition metal is to be subtracted from Tap. To correct accurately for this, the relaxation time of all solvent mixtures was determined immediately prior to the addition of paramagnetic solute. (11) .“Handbook of Chemistry and Physics,” Chemical Rubber Publishing Co., Cleveland, Ohio.
