Enthalpies of
Transfer of Ribonuclease A
The Journal of Physical Chemistry, Vol. 82,
No. 15, 1978 1703
Thermodynamic Quantities of Interaction and Unfolding in the Transfer of Ribonuclease A from Dilute Buffer to Aqueous Cosolute Solutions Ram1 Almog, Martha Y. Schrier, and Eugene E. Schrier" Department of Chemistry, State University of New York at Binghamton, Binghamton, New York 1390 1 (Received November 23, 1977; Revised Manuscript Received March 20, 1978) Publication costs assisted by the U S . Public Health Service
The enthalpy of solution of ribonuclease A was measured at 25 "C for solutions containing guanidinium chloride (GuCl),urea, calcium chloride, lithium chloride, and sodium chloride over a wide range of cosolute molalities. Enthalpies of transfer of ribonuclease A from dilute buffer to cosolute solutions were calculated from these data. Use of previously published free energies of transfer allowed the evaluation of the entropies of transfer for these systems. For all cosolutes except sodium chloride,plots of both the enthalpy and entropy as a function of cosolute molality showed striking changes in the cosolute molality ranges where protein unfolding occurs. Values of AH,,,,, and AS,, can be calculated from these changes. For the unfolding of ribonuclease A in GuCl solution, AH,, and AS,,f differ from the same quantities obtained previously at 25 "C by extrapolation of the results for the thermal unfolding of the protein. These differences are used together with data for the transfer of model compounds from water to GuCl solutions to estimate the number of peptide backbone units and nonpolar side chains newly exposed in the unfolding process. These estimates are consistent with previously published data and lead to a value of the free energy of unfolding of the protein in dilute buffer which is in agreement with literature results. Unfolding to a final state different from that in GuCl is suggested by the parameters obtained for the other three unfolding cosolutes. Thermodynamic quantities for the transfer of ribonuclease A to 2 m solutions of the unfolding cosolutes are shown to obey a straight line on an enthalpy-entropy compensation plot.
Introduction The thermodynamic stability of the collection of native conformations of a protein in solution can be reduced sufficiently by the addition of perturbing cosolutes to generate a preference for the collection of unfolded conformations. The underlying basis for the phenomenon is the change in the nature of the solvation of the constituent groups of the protein as the concentration of perturbing cosolute is increased. Recently, we measuredlJ the free energy changes attending the transfer of ribonuclease A from dilute buffer t o aqueous solutions of various cosolutes including guanidinium chloride (GuCl), urea, and calcium, lithium, and sodium chlorides. The free energies of transfer were negative for those cosolutes which unfolded the protein but the plots of AGt, vs. molality for these cosolutes showed no features characteristic of the unfolding transition which takes place. This was thought to be due in part to enthalpy-entropy compen~ation.~ The present research was undertaken in order to visualize the unfolding transition using calorimetric measurements and to continue our attempt to distinguish between the properties of perturbing and nonperturbing cosolutes on thermodynamic grounds. Experimental Section Materials. GuCl was prepared from the carbonate (Eastman) and purified following the procedure of Nozaki and T a n f ~ r d .Urea ~ was recrystallized from methanol. These substances and LiCl were dried under vacuum before use. The salts CaCl2.2H20and NaCl were reagent grade materials and were used as received. Ribonuclease A was obtained and treated in a manner described in the previous paperas All salt solutions into which solid ribonuclease A was dissolved in the calorimetric experiments also contained Tris buffer (0.025 m) a t p H 7.0. Methods. The general experimental procedure is described in the accompanying paper.5 The heat of solution 0022-3654/78/20821703$01 .OO/O
of solid ribonuclease A with a known water content was measured in cosolute solutions generally over the entire range of solubility of the cosolute.
Results The enthalpy of transfer of ribonuclease A from dilute buffer to cosolute solution was calculated with
m,,= msop - AH,,^^
(1)
where AHsol~B is the enthalpy of solution of solid protein in the cosolute solution containing buffer while mHaolnB is the enthalpy of solution of the solid protein in buffer alone. Care was taken to ensure that the water content of the solid samples was exactly the same in the two measurements since the enthalpy of solution of the solid protein depends to a significant degree on the water content of the samples. That the enthalpy of transfer itself does not depend on the water content of the samples was demonstrated by the constancy of a set of measurements of the enthalpy of transfer of ribonuclease A from buffer to 4.56 m CaClz solution. For those measurements, the water content of the solid ribonuclease A was varied from 16 to 89 mol of water per mol of ribonuclease A. Since the molality of ribonuclease A in the final solutions ranged from 0.6 to 1.1X m, all enthalpies of transfer derived here are considered to be infinite dilution values. Figures 1and 3a-6a depict the negative of the enthalpies for the transfer of ribonuclease A from dilute buffer to solutions of GuC1, urea, CaCl,, LiC1, and NaC1, respectively, as a function of cosolute molality at 25.0 oC.6 These data were employed together with AG,, values previously obtained for the same systems1$2 to calculate aS,according to AGt, = AH,, - TASt, (2) Plots of -ASt, vs. cosolute molality are shown in Figures 2 and 3b-6b for transfers to the same cosolute systems. The points on the -AS, curves correspond to the molalities 0 1978 American Chemical Society
1704
The Journal of Physical Chemistry, Vol. 82, No. 15, 1978
R. Almog,
50
M. Y. Schrier, and E. E. Schrier
30r
/
- 20 2
4 6 MOLALITY
8 IO OF GuCl
12
14
6
I
\
Flgure 1. The negative of the enthalpy of transfer of ribonuclease A from dilute buffer to GuCl solutions as a function of GuCl molality. The significance of the dotted lines and the vertical arrow is explained In the text. -25 ,201
8
4
I
M O L A L I T Y OF
12 UREA
20
16
Flgure 3. The negative of the enthalpy of transfer (A) and the negatlve of the entropy of transfer (B) of ribonuclease A from dilute buffer to urea solutions as a function of urea molality. 200-
150-
'7 '
g I
1
2
4 6 8 IO M O L A L I T Y OF GuCl
12
14
II L
Flgure 2. The negative of the entropy of transfer of ribonuclease A from dilute buffer to GuCl solutions as a function of GuCl molality. The significance of the dotted lines and the vertical arrow is explained in the text.
a t which the enthalpies of transfer were obtained. The precision of the present calorimetric results and the derived entropies is not as high as for past results on urea7 and other model compound^.^^^ The precision in this case is limited primarily by the small absolute heat output and by the difficulty of filling the ampoules and weighing the samples. We estimate the uncertainty in the values of AHb as &3 kcal mol-l and the uncertainties in AS,, as 425 cal mol-l K-l. The size of the points on the curves corresponds approximately to these magnitudes.
Discussion The shapes of the curves in Figures 1-5 give evidence for the existence of at least three distinct processes occurring as the molality of perturbing cosolutes is increased in these systems. Consider Figure 1. There is an initial region of increasingly exothermic enthalpies of transfer with increasing GuCl molality. Energetically favorable solvation of the folded protein is taking place here. In the second region, the direction of change of the enthalpy of transfer becomes positive and, at its end, the values of the enthalpy of transfer are actually positive. We interpret the behavior of the curve in this region as indicating an unfolding transition. The midpoint molalitylO for this transition corresponds closely to that obtained previously by optical methods.ll While a similar concordance of midpoints is obtained for LiC12 and CaClz,Zin the case of unfolding by urea, the transition midpoint molality is 7.5
-1001
I 1
2 3 4 5 M O L A L I T Y OF CaC12
6
Flgure 4. The negative of the enthalpy of transfer (A) and the negative of the entropy of transfer (B) of ribonuclease A from dilute buffer to CaCI, solutions as a function of CaCI, molality.
m for the calorimetric measurements while it is nearly 11 m by optical r0tation.l' The reason for this difference is not readily apparent. In the third region the enthalpy of transfer resumes its exothermic direction with increasing cosolute molality, This region is, in a sense, a continuation of the first region except that it is now the unfolded protein which is being solvated. The flattening out of this region at higher GuCl molalities is suggestive of diminishing tendency toward energetically favorable solvation and has been observed with model compounds as well.9 While a similar phenomenon occurs with urea as the cosolute
Enthalpies of Transfer of Ribonuclease A
The Journal of Physical Chemistry, Vol. 82, No. 15, 1978
1705
TABLE I: Enthalpies and Entropies for the Unfolding of Ribonuclease A in Various Media at 25 "C medium dilute buffer" 3.8 m GuCl 7.6 m urea 6.2 m LiCl 2.5 m CaCl, a
AHunf,
ASunf,
kcal mol-'
cal mol-' K-' 190
71 61 50 52 46
203 169 174 150
Data from ref 15.
-50L------4of the enthalpy changes are consistent with their results.
,
-1001
2
,
,
,
4 6 8 MOLALITY OF LiCI
,
,
1
1 0 1 2
Figure 5. The negative of the enthalpy of transfer (A) and the negative of the entropy of transfer (B) of ribonuclease A from dilute buffer to LiCl solutions as a function of LiCl molality.
T
i
i
0
'Ty
1
80
MOLALITY OF NaCl
Flgure 8. The negative of the enthalpy of transfer (A) and the negative of the entropy of transfer (B) of ribonuclease A from dilute buffer to NaCl solutions as a function of NaCl molality.
(Figure 3), in the system containing CaCl, (Figure 4) and to a lesser extent in the LiCl system (Figure 5) there is an enormous rise in both -AH,, and -AS,, in the post transition region as a function of salt molality. The possible origin of the changes in the CaC1, and LiCl systems with ribonuclease A have been considered previously.2 Finally, Figure 6 shows that only the first region is evident for the ribonuclease A sodium chloride system. Paz Andrade, Jones, and Skinner12 have recently published a calorimetric study of the unfolding of ribonuclease A by urea. There is a considerable difference between the shape of their curves and the present data which can be tentatively explained on the basis of their neglect of the phosphate ion present in commercial ribonuclease A. Pfeil and Privalov13 have determined enthalpies of interaction of lysozyme with GuCl as a function of p H and temperature. Although the results are not strictly comparable, the shape of our curves and the sign
+
The enthalpies and entropies for the unfolding of ribonuclease A, AHunfand ASunf, in the various cosolute solutions can be obtained from the data in Figures 1-5. Since these quantities are of most interest at cosolute molalities corresponding to the midpoint of the transition, extrapolation of the pre- and post-transition baselines to the midpoint molality was required. The extrapolation procedure is illustrated in Figures 1 and 2. The dotted lines indicated the extrapolations of the pre- and posttransition regions, respectively, toward the transition midpoint. The best straight line was drawn by eye in each case. The values of AHunfand ASunf are indicated by the vertical arrows drawn at the cosolute molality corresponding to the midpoint of the transition. A similar procedure was used for systems containing urea, LiC1, and CaC1,. These extrapolations were readily accomplished with systems containing ribonuclease A and GuCl or urea but were less clear cut with LiCl and CaCl,. The empirically determined transition midpoint molalities and the values of AH,,, and ASunf determined by this extrapolation procedure are given in Table I. The uncertainties in the numbers based on plausible variations of the straight lines used in the extrapolation procedure are h3 kcal mol-I in AHunfand f10 cal mol-l K-' in ASunf. There is a larger uncertainty in the CaCl, system which, as indicated above, exhibits significant curvature in the immediate posttransition region. The values14 in Table I can be compared to AHunfand ASd for the unfolding of ribonuclease A at 25 " C in dilute buffer obtained by Privalov and Khe~hinashvilil~ from their very elegant calorimetric studies of the thermal unfolding of ribonuclease A at various pH values. Their data are given in line 1 of Table I. More recently, Pfeil and Privalov13 have demonstrated that the thermal-unfolded and GuC1-unfolded states of lysozyme are thermodynamically indistinguishable. Since the thermodynamic properties and other structural characteristics of lysozyme are very similar to those of ribonuclease A, the probability of the same concordance of states for ribonuclease A is high. Let us analyze the thermodynamic quantities obtained for the unfolding of ribonuclease A in GuCl in more detail. There is an interesting contrast between our values and the results obtained by Privalov and Khechinashvili.ls While AHd is smaller in GuCl than in dilute buffer, ASd behaves in a converse fashion being larger in GuCl than in buffer solution. A t first sight, we would expect the changes SA",,, = AHUnf(GuC1)- AHUnf(buffer) (3) 6ASu,f = ASunf(GuCl)- ASUnf(buffer)
(4) to have the same sign. Since these changes are equal to AHb and AS,, respectively, for the transfer of those groups from buffer to 3.8 m GuCl which are newly exposed to the solution as the protein unfolds,16 enthalpy-entropy
1706
The Journal of Physical Chemistry, Val. 82, No.
compensation should presumably make the changes defined by eq 3 and 4 parallel in sign. Examination of transfer quantities for appropriate model compounds9J7 makes this point clear. For the transfer of the peptide backbone unit from water to 3.8 m GuCl a t 25 "C, AH,, = -520 cal mol-l and TAS, = -335 cal mol-l while for transfer of a leucyl side chain, i.e., that side chain which contains the number of carbons of the average nonpolar side chain in ribonuclease A, AHtr = 890 cal mol-l and TAStr = 1175 cal mol-l. If only peptide backbone units were newly exposed, the changes in eq 3 and 4 should both be negative, while if only nonpolar side chains were exposed, the converse situation should prevail. Neither of these cases corresponds to the experimental results. However, it appears possible that the exposure of both peptide backbone units and nonpolar side chains could cause unequal cancellation of effects leading to the observed signs of SA& and SASunp How might this cancellation come about? Suppose P peptide backbone units are exposed to the solution in either the thermal-unfolded state or in the GuC1-unfolded state in addition to those peptide backbone units exposed in the native state. Similarly, let N average nonpolar side chains be exposed in either final state of ribonuclease A in addition to those nonpolar side chains exposed in the native state. According to the model compound data given above, the AHtrfor the transfer of a peptide backbone unit from water18 to 3.8 m GuCl is -520 cal mol-l while AHt, for the transfer of a nonpolar side chain is 890 cal mol-l. We can then understand the difference in the enthalpy of unfolding, SA",,, defined by eq 3 as resulting from the transfer of the peptide backbone units and nonpolar side chains newly exposed in the unfolding process from the solvent medium of the thermal transition (dilute buffer) to 3.8 m GuC1.l6 An analogous argument can be made for 6TASunf.We can, therefore, write
+ 890N = -10000 cal mol-l 6TASunf= -335P + 1175N = 3880 cal mol-l SAH,,,, = -520P
R. Almog, M. Y. Schrier, and E. E. Schrler
75, 7978
(5) (6)
Simultaneous solution of these equations gives P = 48.6 and N = 17.1. The question of whether these numbers accurately depict the true situation with respect to unfolding can be addressed by further calculations employing them, For instance, we have shown that 49 out of 123 peptide backbone units and 17 out of 124 side chains are newly exposed in the unfolding process. This represents a total of 66 of the 247 groups to which separate free energies of transfer were assigned in the accessibility calculation described by Schrier and Schrier.' The percentage increase in the total number of groups exposed is (66/247) X 100 = 27% in good agreement with the value of 26% obtained by them.l A calculation of the free energy of unfolding of the protein in dilute buffer is also possible using the derived numbers and model compound data. We know that the positive free energy of unfolding of the protein in dilute buffer is reduced to zero when the protein is placed in 3.8 m GuCl since this is the midpoint molality for the unfolding transition. If unfolding is the result of the free energy change produced by the exposure of additional groups to the solution, then the free energy of transfer of 49 peptide backbone units and 17 leucyl (average) side chains from water to 3.8 m GuCl should be exactly equal in magnitude and opposite in sign to the free energy of unfolding in dilute buffer. Estimating the free energies of transfer from the data recorded by Pace,l' we obtain AGt, = -175 cal mol-l for the transfer of a peptide
r------301
GuCi
"
a/
1
/
/
I
LlLl
j
0'
1
d 20
40
-AS+,, CAL
60
80
Mori K-'
Figure 7. Enthalpy-entropy compensation plot for the transfer of ribonuclease A from dilute buffer to various 2 rn cosolute solutions.
backbone unit and AG,, = -285 cal mol-l for the transfer of a leucyl side chain from water to 3.8 m GuC1. Multiplying these estimates by the number of peptide backbone units and average nonpolar side chains exposed gives -AG,,(overall) = AG,,f(buffer) = 13.4 kcal m o P . The estimate of the free energy of unfolding in dilute buffer at 25 "C obtained in various ways by Pace17using methods developed by Tanford and co-workers is 12.2 f 3.2 kcal mol-l while Privalov and Khe~hinashvilil~ obtained 11kcal mol-I as the free energy of unfolding of ribonuclease A in dilute buffer at 25 "C from an extrapolation of thermal unfolding data. The value of AGd(buffer) estimated here is in agreement with these results. The success of these two subsidiary calculations supports the validity of the numbers of exposed groups which we have obtained. The values of AHmfand ASud obtained with the other three cosolutes are fairly consistent. The values of ASunf for them do not differ appreciably from the quantity obtained with GuCl. What difference there are may be attributable to unfolding to slightly different states than with GuC1. In the case of the salts, unfolding is likely to result in states which maximize solution contact with peptide backbone units and minimize contact with nonpolar groups. In the absence of (1)AHunfand ASunf data for the unfolding or ribonuclease A to these states in dilute buffer and (2) sufficient data for model compounds, an analysis of the type given for GuCl unfolding is not possible. Can any information be derived from the enthalpies and entropies of transfer for these systems outside the transition region? Figure 7 shows an enthalpy-entropy diagram3!' comprising these quantities for the transfer of ribonuclease A to a 2 m solution of each of the cosolutes investigated at 25 "C. It can be seen that while the quantities for the unfolding solutes fall on a straight line (slope = 420 K), the point for NaCl falls far from the line. Although there is a limited amount of data from which to draw conclusions, some empirical observations are possible. The quantity, -AHb, for NaCl solutions is the same as that for LiCl at this molality but, while this quantity continues to increase in magnitude for LiC1, it remains the same throughout the complete molality range for sodium chloride. The negative of the entropy of transfer, -AStr, is about the same for NaCl solutions as for GuCl solutions. For NaCl to unfold the protein it would have to allow somewhat more than twice as large an enthalpy of transfer than it actually does in accordance with its relation to GuCl on the compensation plot. Since it has been shown in a variety of ways that Li+ ion stands out from Na+ ion in its ability to solvate polar groups,P it is not surprising that
The Journal of Physical ChemiStty, Vol. 82, No. 15, 7978
Dipolar Association of Oxygen Bases in Solution
it falls with the unfolding ions on the plot and Na+ ion does not. It is, however, gratifying to observe the distinction between them so clearly by direct thermodynamic measurements on a protein.
1707
E. R. Stimson and E. E. Schrier, J. Chem. Eng. Data, 19, 354 (1974).
E. R. Stimson and E. E. Schrier, Biopolymers, 14, 509 (1975). The salt molality at which the transition is haif complete, Le., the midpoint molality, has been determined in the following way. Consider Figure 1. The portion of the plot in which -AH, decreases sharply forms the transition region. We have measured the distance along this straight line after extending it to each dotted extrapolation h e . The cosolute molality at the halfway point of this straight line is the midpoint molality. R. F. Green and C. N. Pace, J. Bioi. Chem., 249, 5388 (1974). M. I. Paz Andrade, M. N. Jones, and H. A. Skinner, Eur. J. Biocbem., 68, 127 (1976). W. Pfeil and P. L. Privaiov, Siophys. Chem., 4, 33 (1976). It should be noted that the vaiue of AH,,, = 61 kcal mol-’ does not agree with AH^ = 35 kcal mol-’ obtained for the GuC1-k ribonuclease A system by A. Salhuddin and C. Tanford, Biochemistry, 9, 1342 (1970). P. L. Prlvalov and N. N. Khechinashvili, J. Mol. Biol., 88, 665 (1974). This may be shown for AH, for example, as follows. We can write
Note Added in Proof. Pace and Vanderburg [Fed. Proc., 37, 1274 (1978)] have calculated that 48 additional CuCl’s are bound to ribonuclease A after the protein has unfolded. If we assume as they do that a guanidinium ion requires two peptide groups as its binding site, the groups on the protein exposed upon unfolding as calculated here would interact with 24 17 = 41 GuCl’s in good agreement with the value of Pace and Vanderburg. The similar number of GuCl’s interacting with peptide and nonpolar groups is reasonable.
+
Acknowledgment. This research was supported in part by Grant GM11762 from the Institute of General Medical Sciences, U.S. Public Health Service.
AH,,,(buffer) AH,,,(3.8
= H,(buffer)
m GuCI) = H,(3.8
- H,(buffer)
m GuCI) - HF(3.8 m GuCI)
where the subscripts D and F represent the unfokled and folded states, respectively. Substituting in eq 3
References and Notes M. Y. Schrier and E. E. Schrier, Biochemistry, 15, 2607 (1976). M. Y. Schrier, A. H.4. Ying, M. E. Ross, and E. E. Schrier, J . Phys. Chem., 81, 674 (1977). R. Lumry and S. Rajender, Biopolymers, 9, 1125 (1970). Y. Nozaki and C. Tanford, Methods Enzymoi., 11, 715 (1967). R. Almog and E. E. Schrier, J . Phys. Chem., 82, preceding paper in this issue. Tables of original data may be found in the Ph.D. Dissertation of R. Almog, State University of New York at Binghamton, 1978. The enthalpies of transfer given there refer to transfer to salt solutlons of a given molarity. M. Y. Schrier, P. J. Turner, and E. E. Schrier, J . Phys. Chem., 79, 1391 (1975).
6AH,,, = [H,(3.8 m GuCI) - HF(3.8 m GuCl)] [ H,(buffer) - HF(buffer)] = [ H,(3.8 m GuCI) - H,(buffer)] [HF(3.8 m GuCI) - HF(buffer)]
AH,,,
- AH,,F
The difference between these auantities comes about from those groups which are newly exposed in the unfoldingtransition, Le., 6AH, = AH,, of newly exposed groups from buffer to 3.8 m GuCI. (17) C. N. Pace, CRC Crit. Rev. Blochem., 3, 1 (1975). (18) In the calculation, the thermodynamic quantties of transfer of a p e p t i i backbone unit or nonpolar side chain from water to dilute buffer are assumed to be negligible.
Dipolar Association of Oxygen Bases in Solution Sherril D. Christian,” Edwin E. Tucker, and Douglas R. Brandt Depatfment of Chemistry, The University of Oklahoma, Norman, Oklahoma 730 19 (Received November 28, 1977: Revlsed Manuscript Received March 27, 1978) Publication costs assisted by the National Science Foundation
For a number of years the claim has been made that oxygen bases such as N,N-dimethylacetamide are highly self-associatedin dilute solution in hydrocarbon solvents. Vapor spectral data for solutions of dimethylacetamide in cyclohexane, in the 04.10 M region, indicate only relatively small deviations from Henry’s law for the amide solute. By considering the present results and results of several previous studies based on various techniques, we conclude that oxygen bases are only slightly associated at low concentrations in hydrocarbon or CC14 solutions.
In several recent discussions of the effects of solvents on heats of formation of molecular complexes, it has been argued that cyclohexane and similar solvents should not be chosen as media for studying adducts of oxygen ba~es.l-~ It is claimed that bases such as dialkylamides and ketones are highly self-associated in alkane solvents at very low concentrations. The belief that oxygen bases in general form high molecular weight aggregates in dilute solution in nonpolar solvents apparently derives from consideration of ebullioscopic and vapor pressure osmometry studies of only one compound, N,N-dimethylacetamide (DMA).4,5The first of these investigations, which produced activity values for solutions of DMA in CC14 at temperatures near the boiling point of carbon tetrachloride, indicated only a relatively small extent of solute self-association at DMA concentrations up to 0.4 M.4 These data do not seem out of line 0022-3654/78/2082-1707$01 .OO/O
with colligative property measurements for similar compounds in nonpolar solvents. However, the vapor pressure osmometry experiments yielded average molecular weights for DMA about three times that of the monomer amide in CC14 (0.01-0.2 M) and nearly twelve times that of the monomer in cyclohexane at comparable concentrations at room t e m p e r a t ~ r e . Such ~ results appeared to us to be incompatible with known molecular properties of N,Ndialkylamides. Moreover, the reported degrees of association for DMA are much larger than those obtained by various other techniques for DMA and several oxygen bases with similar dipole moments.6-10 We believe it is important to emphasize here how unlikely it is that degrees of aggregation as large as 12 in cyclohexane and 3 in C C 4 could occur at total amide concentrations on the order of 0.0E1.~First of all, if the aggregation number (3 or 12) were really constant over the 0 1978 American Chemical Society
