SOTES
Nov., 1963
IOiXIC
H+ 17" a
Lif 11 14 See ref. 8.
Kf
Naf
8 8 10 7 See ref. 9.
Mg2+
24 28
Ca2f 27
Ba2+ 22 21
TABLE I DIELECTRIC DECREMENTS Lasf Al3f FC135 5 3 3
salt6.l0)is smaller than their partial molal volume a t infinite dilution pzo. This salting property is, however, independent of the noneleetrolyte, as their lo equation shows k,
V30(V2
-
720)
(13) 2.303p1RT (pl is the compressibility of water). Although the results for benzenelo confirm the qualitative aspects of eq. 13, many other results do not, since the same electrolyte is found to be able to salt some nonelectrolytes in, and some out (e.g., hydrocyanic acid and acetone, ref. 6, Fig. 12-10-4, p. 538, also Table 12-10-IA and 12-10-2A, p. 736-.737, for other nonelectrolytes). Furthermore, eq. 13 is sensitive to the exact value of V2, which is not always known with sufficient precision, and since the80ry6 requires Vz - pzo > 0, the cases reported where this difference is negative are possibly based on incorrect V 2values. Along with the Debye theory, eq. 12 predicts the salting coefficient, to increase with the charge on the ions and to be a function of the dielectric decrement of the nonelectrolyte 83. Salting in will occur if a3 is negative ( e . g . , as for hydrocya,nic acid), provided it is sufficiently large. Equation 13 predicts also that k, will increase with the molar volume V 3 of the nonelectrolyte, and decrease with that of the electrolyte. This result does not appear explicitly in Debye's theory, but is included in that of hlcDevit ,and Long.1o Finally, the present treatment predicts k , to increase with the dielectric decrement of the electrolyte. This feature has not appeared in previous derivations of the salting effect. Calculations have been made for the salting behavior of electrolyteis in water toward benzene. Table I1 shows the results for those electrolytes where data of VZO,hzO,8 2 , and k, are available,e,g-llusing eq. 12. :=
TABLE I1 BENZENE^
S A L T I N G OUT O F
VzO f 18hz0, Electrolyte
LiCl NaCl NaBr XaI NaN03 NaC104
ml
.
107.5 125.0 114.0 107.2 100.2 114.7 KCS 117..l XBr 106. :l HC1 144.9 HC104 134.6 Benzene, V3 = 89.4 ml., Da
62
2497
k , (calcd.),
k. (obsd.),
'I4 -1
,I4- 1
14 0.22 0.14 10 .18 .20 11 .20 .I6 12 .22 .10 10 .20 .12 11 .20 .10 7 .17 .17 8 .18 .10 20 .23 .05 19 .23 -. .01 = 2.25; mater, D1 = 78.5.
The calculated values are seen to be quite near the observed ones (considerably nearer than those of McDevit and Longlo). The calculated values for hydrochloric and peirchloric acids are relatively too high, t'he theory not being able to account for the very low salting out or in properties of acids. (11) J. H. Saylrrr, A. I. Whitten, I. Claiborne, and P. &I.Gross, ibid., 74, 1778 C1952).
0
3
Br-
0
0
I7 0
OH-
N08-
ClOr-
13 1
2
Soh214 18
For another type of nonelectrolyte, mercury(I1) halides, the experimental value for the salting coefficient by sodium perchlorate was found to bel2 k , = 0.14 M-l. The calculated values from eq. 12, using V3 = 49 ml. for HgCb, 59 ml. for HgBrz, and 72 ml. €or HgL, are k,(calcd.) = 0.123, 0.142, and 0.164 M - l , respectively; i.e., very near the experimental value. (12) Y. ;\Iarcus, Acta Chem. Scarid., 11,328 (1957).
THE APPAREhTT PARTIAL SPECIFIC VOLUMES OF PROTEINS IN SOLUTIONS OF GUASID LNE HYDROCHLORIDE BY F. J. REITHELASD J. D. SAKURA Department of Chemzstru, rna&eiszty of Oregon, Eugene, Oreoon Reeeated M a y 80, 196s
During the past fern years there has been a wider usage of sedimentation equilibrium in the ultracentrifuge due to the work of van Holde and Baldwin' and that of Yphantise2 The methods of calculation require reliable values for the apparent partial specific volume, 4, of the sedimenting species. In this Laboratory experinientation has been going forward on the cletermination of protein molecular weights in 6 LZ guanidine hydrochloride. This solvent was chosen because of its wide applicability in dissociating proteins. Few data have been published pertaining to 4 for proteins in three-component systems and it became necossary to examine possible variations of this quantity with respect to a number of factors. Experimental The density gradient technique of LinderstrZm-Lang4 was used in this investigation. Gradients in the solvent mixture dodecane-o-dichlorobenzene were produced as described by Miller and Gasek.j In general, the techniques employed were those detailed by Schachman6 and Miller and Gasek. One set of apparatus was assembled in an aquarium bath maintained a t 25' with a regulation of zt0.005". Another set was assembled in a water bath in the cold room and the temperature maintained at 4" with a regulation of 1.0.005". Ribonuclease A was from Sigma Type I11 ribonuclease, Lot R-22B-70. The column purification demonstrated only a very small impurity in the commercial sample. The P-lactoglobulin was a sample obtained from Dr. S. Timasheff, Eastern Regional Research Laboratory. The glutamic acid dehydrogenase used was obtained as a 2% solution in 50% glycerol as prepared by Boehringer (Calbiochem) . All solvents used were purified. The guanidine hydrochloride was purified as follows. It was dissolved in a minimum volume of absolute methanol using a 45' water bath and magnetic stirring. This solution was filtered through a jacketed funnel containing a (1) K. E. van Holde and R. L. Baldwin, J . Phys. Chem., 62, 734 (1958). (2) D. 4 . Yphantis, Ann. AT. Y . Acad. Sci., 88, 586 (1960). (3) F. J. Reithel, Abstracts, Fifth International Congress of Biochemistry, Moscow, USSR, August, 1961, p. 47. (4) K. Linderstrpim-Lang and H. Lana, Jr., Compt. rend. trau. Lob. Carlsberg, Ser. chim., 21, 315 (1936). ( 5 ) G. L. Miller and J. McG. Gasek, Anal. Biochem., 1, 78 (1960). (6) H. K. Schachman in "Methods in Enzymology," Vol. 1V. S. P. Colowick and Tu'. 0. Kaplan, Ed., Academic Press, New York, N.Y., 1987, p. 32.
(7) C. H. W. Hirs, ref. 6 Vol. I, S. D. Colowick and N. 0. Kaplan, Ed., Academic Press, New York, K. Y., 1965, p. 113. (8) G. Taborsky, J . R i d . Chem., 234, 2652 (1959).
NOTES
2498
pad of powdered cellulose. Crystallization was allowed to proceed overnight a t -5". The mother liquor was poured off, the crystals were drained, and finally pressed dry between sheets of filter paper. This procem was repeated until the methanol solution showed no yellow color. Three crystallizations were usually found to be necessary. The final product was freed of methanol by warming for a few hours in a vacuum oven a t 40' and was finally dried in a vacuum desiccator. The moisture content of dry protein samples was determined by heating a t 110" in a vacuum oven until constant weight was attained. I n the case of ribonuclease and p-lactoglobulin weight fractions of protein in the solutions could be calculated directly. In the case of glutamic acid dehydrogenase this was not possible. Spectrophotometric readings were used to estimate protein content: in buffer A279mp = 0.971 (1 mg./ml.); in 6 M guanidine hydrochloride A279mp = 0.820. Calculations of apparent specific volume employed the equation
4
= 1 PO
- 1 (L n
b)
PO
where n = weight fraction of protein, po = density of solvent or dialyeate, and p = density of the protein solution.
Results and Discussion Comparison of 6, in Buffers and in 6 M Guanidine Hydrochloride.-At a concentration of 2% ribonuclease in 6 M guanidine hydrochloride the value of 4 at 25' was 0.709 f 0.002 ml./g.; a t 4' it was 0.700. Thus A6, per degree is 4.3 X 10-4. A recent careful determinationg of 4 for ribonuclease in phosphate buffer a t 25' yielded the value 0.709 3t 0.002. The temperature coe6cient above is similar to that found by others. l 1 p-Lactoglobulin a t a concentration of 2% in 6 M guanidine hydrochloride had a 9 value of 0.756 at 25' and 0.738 a t 4'. A$ per degree was 8.6 X The value of 4, in buffer, frequent,ly quoted,12 0.7514 a t 20°, was redetermined. At a concentration of 2y0 in 0.05 144' phosphate buffer a t 25' the value of cp was 0.752. The partial specific volume of glutamic acid dehydrogenase in buffer (temperature not given) has been estimated pycn~metrically~~ as 0.75. We were not able to obtain preparations of the enzyme which could be dissolved in any buffer at a concentration of 2% and which would remain perfectly clear during overnight dialysis. We were successful in obtaining satisfactory 2% solutions in 50% glycerol. At 25' cp was 0.745. To these glycerol solutions was added solid guanidine hydrochloride to obtain a 6 M solution. Such a solution was then dialyzed for 5 days a t 4' against 6 M guanidine hydrochloride. It was necessary to add the solid reagent because the behavior of this protein at lower concentrations of the reagent is unpredictable. The resultant concentrations of protein a t 25' was 0.752 were about 1%. The value of and a t 4' it was 0.739. A+ per degree was 6.2 X low4. The Relation of Changes in 4 to Dissociation and Unfolding.-Ribonuclease was used in this investigation because it can be obtained in a state of reasonable purity in reasonable quantity and because it cannot dissociate in guanidine hydrochIoride.
+
(9) A. M. Clarke, D, W. Kupke, and J. W. Beams, J . Phys. Chem., 67,929 (1963). (10) D. J. Cox and V. N. Schumaker, J. A m . Chem. Soc., 83, 2433 (1961). (11) D. N. Holoomb and K. E. van Holde, J . Phys. Chem., 66, 1999 (1962). (12) K. 0. Pedersen, Biochem. J., S O , 961 (1936). (13) J. A . Olson and C. B. Anfinsen, J. Bzol. Chem., 197, 67 (1952).
Vol. 67
I n contrast, p-lactoglobulin is known to dissociate in high concentrations of hydrogen The value of 4 for a 2% solution of p-lactoglobulin in 0.01 M HC1 a t 25' is 0.764. A solution similar to this but 6 M in guanidine hydrochloride as well yielded a 4 value a t 25' of 0.764 and a t 4' of 0.748. A$ per degree Preliminary results have indicated was 7.6 X that the molecular weight of p-lactoglobulin in guanidine hydrochloridea is near 36,000 but the optical rotation has a large negative value. I n this reagent some type of unfolding but no dissociation occurs. Addition of hydrogen ion does effect a dissociation with a concomitant drop in molecular weight. The change in 9 observed seems to be related to the conformation of the charged protein rather than the dissociation process. I n the case of glutamic acid dehydrogenase, the molecular weight in guanidine hydrochloride3 appears to be of the order of 100,000 and preliminary results indicated dissociation. However, as noted earlier, such dissociation did not cause a change in cp as long as the solution was kept a t 4'. When such solutions were allowed to stand a t room temperature for several hours, or if the dialyses were carried out a t room temperature, two changes were noted. First, boundaries formed in a synthetic boundary cell in the ultracentrifuge were very asymmetric. Second, cp values were low and varied with the history of treatment. Values between 0.682 and 0.725 were observed a t 25'. Some type of refolding may be responsible since p-lactoglobulin solutions (in guanidine hydrochloride) behaved similarly. The results of this investigation indicate that the apparent specific volume of a protein, dissociable or not, in 6 M guanidine hydrochloride differs very little from that in buffer. This is in agreement with those15 who have found cp to vary but little with salt concentration, but it is perhaps a t variance with the values reported for proteins in urea.16 It is possible that the low values found were due to experimentation a t room temperature. Acknowledgment.-This research was supported by Grant G-18736 from the National Science Foundation. (14) R. Townend, L. Weinberger, and S. N. Timasheff, J . A m . Chem. Soc., 82, 3175 (1960).
(15) J. E. Ifft and J. Vinograd, J . Phys. Chern., 66, 1990 (1962). (16) P. A. Charlwood, J . A m . Chem. Soc., 1 9 , 776 (1957).
-
THE SOLVATIOX OF POLBR GROUPS. 11. B PHASE STUDY OF T H E DIMETHYL SULFOXIDE-p-CHLOROBENZOIC'ITRILE SYSTEM BY C. D. RITCHIE AKD ARDENPRATT~ Department of Chemistry, State Universzty of New York at Bufalo, Bu$alo 14, N e u Yorlo Recezved M a y 86,2965
I n the first paper of this series,2 we reported the results of a study of integrated intensities of the infrared bands of a series of nitriles in binary solvent systems. The results were interpreted in terms of 1:1 complex formation of the polar nitrile group with the polar solvent component. (1) NSF Faculty Fellow, 1962-1963. (2) C. D. Ritohie, B. A. Bierl, and R. J. Honour, 4687 (1902).
J. A m . Chem. SOC.,84,
