G. C. KRESHECK A N D L. BESJAWIN
2476
Calorimetric Studies of the Hydrophobic Nature of Several Protein Constituents and Ovalbumin in Water and in Aqueous Urea
by G . C. Kresheck and L. Benjamin M i a m i Valley Laboratories, T h e Procter and Gamble Company, Cincinnati, Ohio (Received March 8, 1964)
46%99
Partial molal heat capacities and enthalpies of solution for L-leucine, glycylglycine, glycylnL-valine (no enthalpy data), glycyl-L-leucine, L-phenylalanine, and isobutyric acid in water and 6 144urea solution and for acetic acid and glycine in 6 M urea solution have been measured. Enthalpy results have been combined with free energy data to yield entropy values for the transfer from water to 6 M urea. The partial molal thermodynamic quantities of transfer a t infinite dilution from water to 6 M urea solution appear to be additive for the various parts of the amino acid and peptide molecules. Results have been interpreted as evidence for increased ordering of water in the solutions due to the presence of the nonpolar parts of the solute molecules. Part of this ordering is removed by urea, which acts as a structure breaker. There is evidence for the existence of specific interactions between urea and polar parts of the solute molecules, including the peptide bond. The conclusions are viewed in terms of the contribution of hydrophobic bonding to protein conformation in the presence of urea. Finally, the partial molal heat capacity of ovalbumin in water and 6 M urea and the specific heat of the solid protein were determined. The results are related to the solvation of the molecule.
Introduction The importance to the understanding of protein structure and behavior of determining the thermodynamic properties of the amino acids was recognized some 30 years ago by Schmidt and co-workers.l-s Since that time limited use has been made of this information by protein chemists. The potential value of measuring the solubility of amino acids and their derivatives in various solvents has been recognized, and the results obtained by various investigators have been compiled by Cohn and EdsallS4 The recent review article by Kauzmann5 and the contributions of Klotz6-8 stimulated interest in the interaction of the nonpolar protein constituents with the water in aqueous solution. The basic concepts employed stemmed from the contributions of Frank and Evans9 and Frank and Wen.1o A quantitative statistical niechanical study of the thermodynamic properties of hydrophobic bonds in proteins was presented by Nemethy and Scheraga.” This work was extended to include a consideration of the contribution of hydrophobic interactions to The Sou m a l of Physical Chemistrv
the conformational stability of globular proteins.12 Also, Tanford13 has used solubility data of various amino acids for the same purpose. The relative partial molal entropy of water in aqueous (1) J. B. Dalton and C. (1933).
L. A. Schmidt, J Biol. Chem.,
103, 549
(2) J. B. Dalton and C. L.A. Schmidt, ibid., 109, 241 (1935). (3) C. A. Zittle and C. L. A. Schmidt, ibid., 108, 161 (1935). (4) E. J. Cohn and J. T. Edsall, “Proteins, Amino Acids and Peptides,” Reinhold Publishing Corp., New York, N. Y., 1943, Chapters 8 and 9. (5) W. Kauzmann, Advan. Protein Chem., 14, 1 (1959). (6) I. M. Klotz, Science, 128, 815 (1958). (7) I . M. Klota and S. W. Luborsky, J . Am. Chem. SOC.,81, 5119 (1959).
(8) I . M .Klota, Brookhaven S y m p . B i d , 13, 25 (1960). (9) H. S. Frank and M .W. Evans, J . Chem. Phys., 13, 507 (1945). (10) H. S. Frank and W. Y. Wen, Discussions Faraday SOC., 24, 133 (1957). (11) G. NBmethy and H. A. Scheraga, J . Phus. Chem., 66, 1773 (1962). (12) H. A. Scheraga, G. NBmethy, and I. Z . Steinberg, J . Riol Chem., 237, 2506 (1962). (13) C. Tanford, J . Am. Chem. SOC.,84, 4240 (1962).
HYDROPHOBIC SATUEE OF PROTEIN CONSTITUENTS IN' WATERAND UREA
2477
-
-.
solutions of seven amino acids was determined by Robinson. l 4 The results were consistent with the view that glycine behaves as a structure breaker and higher aliphatic homologs behave as structure makers. The compounds which behaved as the best structuremakers vvould also be expected to have the highest heat capacities in accordance with the discussion of Frank and Evans. Nozaki and Tanfordls measured the solubility of several amino acids in aqueous urea solutions and conipared them with corresponding data in water. These results indicated that the free energy was favorable for the transfer of the nonpolar portions of the amino acids from water to the urea solutions. However, it was not possible to state i f this was an entropy- or enthalpy-governed process. Predictions in accordance with recent hydrophobic bond theory16 would favor the importance of the entropy change for this transfer. The purpose of this study was to obtain quantitative calorimetric experimental information concerning the structural changes associated with the solvent which occur upon transferring the nonpolar portions of various protein constituents from water to 6 M urea. Determination of enthalpies of solution in the two solvents permits a comparison to be made with corresponding free energy data in order to obtain entropies of transfer from water to 6 M urea. The latter presuniably reflect differences in the amount of ordered solvent associated with the solute. Also, similar information was obtained from a knowledge of the partial molal heat capacities a t infinite dilution iin both solvents where only solute-solvent interactions are involved. Attempts have been made to interpret the results obtained with the amino acids and dipeptides studied in terms of the effect of urea in accordance with hydrophobic bond theory. The experimental results should also be useful when considering the importance of hydrophobic bonding to protein structure. Finall.y, the first known partial molal heat capacity of a protein, namely, ovalbumin, was determined in aqueous and 6 d l urea solution.
Experimental MateriaEs. All chemicals used in the study were of the highest quality commercially available. All dipeptides and amino acids were obtained from California Corporation for Biochemical Research with the exception of L-phenylalanine which was obtained from Nutritional Biochemicals Corporation. Urea and acetic acid were Baker "Analyzed" reagents and the isobutyric acid was fractionally distilled. The latter was shown to be greater than 99.5% pure by gas chromatographic analysis. Ovalbuiiiin was a Worthington
2X crystallized preparation, lot KO. 561. Deionized distilled water was used. 411 solid samples, except ovalbumin, were dried in vacuo at room temperature over Pz05prior to use. Urea solutions were freshly prepared and were never heated above room temperature. Procedure. Data were obtained using the adiabatic calorimeter recently described by Benjamin.17 All measurements were carried out a t temperatures close to 25' (generally within = t O . O 2 O ) and were not corrected for small deviations from this temperature. In all cases, water or 6 M urea was the solvent. Integral heats of solution and dilution, as well as the heat capacity of the solutions, were measured as a function of concentration. Calorimetric precision has been previously as~essed'~: specific heats are known with a precision of =kO.1% while enthalpy data in the present study are less precise because of the small heat changes measured in most cases. The only correction made to experimental data was to allow for the heat change associated with opening empty sample cells. Weights were corrected to vacuun~and the results expressed in terms of the defined calorie (4.184 absolute joules). Deyived Quantities. Partial molal heat capacities of the solute, Cp,,are given by18
where M,is the solute niolecular weight, m the molality, and cp the specific heat of the solution. In the present work values a t infinite dilution, Cp,,", have been derived using (dc,lbm), = 0. The heat of solution a t zero concentration, AH,', obtained by extrapolation, is equal to Rzo - H,* where 27,' is the partial molal enthalpy of the solute in an infinitely dilute solution and H,* the (partial) molal enthalpy of the solid solute. It follows that
AHso(as urea) - AH~O(H,O) = R Z o ( a q urea) 172Om,oj = Al;T,O
(2) where A R L O is the enthalpy of transfer of the solute from water to aqueous urea a t infinite dilution. Coniparison of solubilities in the two solvents leads to the expression
Apto = pzo(aQ. urea)
-
PZ'(H,O)= RT in
as(HzOj
as(aq
(3)
urea) ~~
~-
(14) A. L. Robinson, J . Chem. Phys., 14, 588 (1946). (15) Y . Nozaki and C. Tanford, J . R i d . Chem., 238, 4074 (1963). (16) G. NBmethy and H. A. Scheraga, J . Chem. Phys., 36, 3401
(1962). (17) L. Benjamin, Can. J . Chem., 41, 2210 (1963). (18) P. White and G. C. Benson, J . Ph,ys. Chem., 64, 599 (1960).
Volume 68, Number 9
September. 1.964
G. C. KRESHECK AND L. BENJAMIN
2478
0.996
-- - ---
0,998 0,997
1
\
0.996
I
I
808 I
0808-
b
\
0,8060,806 O
081 I
MOLALITY Ob2
Ob4
Ob6 O b 8 0 ; O MOLALITY
0;2
0;4
0116
018
Figure 1. Plots of the specific heat of solutions of L-leucine in water and in 6 M urea a t 25" as a function of molality; open circles represent trials in water and closed circles represent trials in 6 M urea.
0.999
Figure 3. Plots of the specific heat of solutions of L-phenylalanine in water and in 6 M urea a t 25" as a function of molality; open circles represent trials in water and closed circles represent trials in 6 M urea.
0.92 -
co = 100 (i101
0.90 0.880.86-
0.997 0.996
-P
t
io
0.995
, > 09941
-
0.81208110'75'
0.810-
0B09L
011
02
0!3
Oi4 d5, MOLALITY
Oh
0!7
08
29
Figure 2. Plots of the specific heat of solutions of isobutyric acid in water and in 6 hf urea a t 25" as a function of molality; open circles represent trials in water and closed circles represent trials in 6 M urea.
where Fzo is the partial molal free energy (chemical potential) of the solute in the hypothetical standard statc corresponding to an ideal solubion of unit activity The Journal of Phrjsical Chemistry
Oi2
Oi4
0:8 110 MOLALITY
016
112
1:4
116
1.8
Figure 4. Plots of the specific heat of solutions of glycylglycine in water and in 6 M urea a t 25" as a function of molality; open circles represent trials in water and closed circles represent trials in 6 M urea.
of solute but with enthalpy properties of an infinitely dilute solution. The activities a t saturation, a,,can be expressed in terms of mole fractions and activity coefficients. Transfer free energy data published using an equation equivalent to (3) have been ob-
2479
HYDROPHOBIC XATUREOF PROTEIN CONSTITUENTS IN WATERAND UREA
-
-I___
-
0.984
t
0
0.750 I
a2
0.4.0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
MOLALITY
Figure 7. Plots of the specific heat of solutions of acetic acid and glycine in 6 M urea a t 25' as a function of molality; triangles represent trials with acetic acid and circles represent trials with glycine.
0.508 0.804
MOLALITY
Figure 5 . Plots of the specific heat of solutions of glycyl-DL-valine in water and in 6 M urea a t 25" as a function of molality; open circles represent trials in water and closed circles represent trials in 6 M urea.
0.986
-9
e-
0.982 MOLALITY X IO4
0.978
Figure 8. Plot of the specific heat of solutions of ovalbumin in water at 25" as a function of molality.
0
1 ~
O
0.974 I
I
entropies of transfer, AS,', ABt' and AFto.
are readily obtained from
Results and Discussion Heat Capacities. Measured data for L-leucine, L
J
Figure 6. Plots of the specific heat of solutions of glycyl-L-leucine in water and in 6 M urea a t 25' as a function of molality: open circles represent trials in water and closed circles represent trials in 6 M urea.
tained in this way assuming deviations froin ideality in the two solvents to be the saine.15 Partial molal
isobutyric acid, L-phenylalanine, glycylglycine, glycylDL-valine, and glycyl-L-leucine both in water and in 6 M urea solutions are given in Fig. 1 4 . Data for glycine and acetic acid in G If urea are shown in Fig. 7, Data for ovalbumin in water are given in Fig. 8. In drawing the smooth curve for leucine (Fig. 1) use has been made of the increasing trend toward curvature of c, values with concentration found with lower amino acid homologs. Similarly, the variation shown for glycyl-L-leucine (Fig. 6) is thought to be consistent Volume 68, Number 9
September, 19G4
G. C. KRESHECK AND L. BENJAMIN
2480
Table I : Summary of the Partial Molal Heat Capacities a t Infinite Dilution Associated with the Transfer of Several Amino Acids, Dipeptides, and Constituent Groups from Water to 6 M Urea a t 25"
Entry
1 2
3 4 5 6 7 8 9 10 11
Exptl. compound
-
--CP,O cal./mole/°C.--Exptl. Derived
group
L-Leucine Glycine
-
-ACpt3 u r e a - ~ 2cal./mole/OC.0, Exptl. Derived
- 10 27.5
121 7.58 Isobutyl (1-2)
113.5
Glycyl-L-leucine Glycylglycine
-37.5
-46 -9
151 38 Isobutyl (4-5)
- 37
113
nL-Valine
93" Isopropyl (7-2)
85.5
Glycyl-m-valine
-33
123 Isopropyl (9-5) Glycyl-L-leucine peptide unitb (4-6-2) Glycyhbvaline peptide unitb (9-10-2) Glycylglycine peptide unitb(5-2)
12 13 a
-
Constituent
C. A. Zittle and C. L. A. Schmidt, J. Bid. Chern., 108, 161 (1935).
with the behavior of leucine and glycyl-DL-valine. I n the absence of extreme experimental precision (approximately *0.001 cal./"C./g. in the present case), accurate independent assignment of cp variation is otherwise difficult to make in these dilute solutions. (The uncertainty involved in estimating the limiting slope gives rise to the errors in CpZoindicated in the figures. In all cases the data must extrapolate to the measured specific heat of the solvent.) A summary of partial molal heat capacities a t infinite dilution for the amino acids (with the exception of phenylalanine) and dipeptides studied is given in Table I. Values for constituent hydrocarbon residues and peptide units, calculated assuming additivity of contributions, are also shown. In water, the hydrovalues of carbon groups are seen to have high about 28 cal./OC. mole-' of -CH2 or -CH3, in general agreement with other CpZovalues reflect only solute-solvent interactions; where none are present should be approximately that of the pure solute. I n the case of compounds predominantly hydrocarbon in nature, this should be close to 0.5Mz since solid and liquid hydrocarbons have specific heats of approximately 0.5 cal./g./'C. a t 2,5O. It has been found that nonpolar molecules such as hydrocarbons have particularly high heat capacities in aqueous solution while showing normal behavior in nonpolar solvents. This has been interpreted as evidence (together with entropy, viscosity, diffusion data, etc.) for the presence of regions of increased order in the water.9~10~1*~16~18~23~24 As the temperature is raised, these regions termed 'Licebergs"or "flickering clusters"
cpzo
cpzo
cpZo
The Journal of Physical Chemistry
85 30.5
- 24 -36.5
30.5
-36.5
30.5
-36.5
-CHNHCO-.
become less stable and extra energy is required to "melt" them. On the basis of the data in Table I a structure-making role may definitely be assigned to the nonpolar groups in the molecules studied. An apparent anomaly exists between the data for isobutyric acid (Fig. 2) and published data for carboxylic a ~ i d s . 2 " ~The ~ data of Bury and Davies were obtained tit 1 5 O and a value of 79 cal./OC. mole-' was estimated for n-butyric acid. This leads to a value of about 75 cal./'C. mole-' a t 25' upon using the observed decrease in Cpzowith temperature for surfactants.I8 The latter is in fair agreement with the apparent molal heat capacity given for butyric acid solution of mole fraction 0.02.26 Isobutyric acid has a CPto value of 100 10 cal./°C. mole-' (Fig. 2) and one would therefore conclude that branching of the hydrocarbon chain leads to increased ordering in the water compared to that associated with straight chains. This is contrary to both theory and experimental data26
cplo
cpZo
*
(19) See ref. 4, p. 168. (20) L. Benjamin, unpublished data with nonionic surfactants. (21) C. R. Bury and D. G. Davies, J . Chem. SOC.,2413 (1932). (22) Data from ref. 21 were obtained a t 15' and CP,' values of 21, 40, 62, and 79 cal./"C. mole-' for formic, acetic, propionic, and n-butyric acids, respectively, have been derived using them. Possible revision of these figures is discussed more fully later. (23) E. D. Goddard, C. A. J. Hoeve, and G. C. Benson, J . Phgs. Chem., 61, 693 (1957). (24) G. NBmethy and H. A. Scheraga, J . Chem. Phgs., 36, 3382 (1962). (25) See ref. 4, Table 4, p. 169. (26) L. Benjamin, unpublished data. Cpzo is 6 cal./"C. mole-' lower for sodium isobutyrate than for sodium butyrate.
HYDROPHOBIC NATUREOF PROTEIS CONSTITUEKTS IS WATERAND UREA
which predict the reverse effect." I n addition, cornparing for alanine with glycine the contribution of one CHZ group is 32.5 cal./OC. m ~ l e - while ~ , ~ the comparative figure obtained using valine and leucine is 28 cal./"C. mole-' (cf. Table I), Thus c,," might be expected to be 60 cal./"C. mole-' larger for valine than for alanine due to two additional carbon atoms. The observed difference of 53 cal./OC. molep1 indicates that the branched methyl group does have a little less (20%) ordering effect than an unbranched group. The data for isobutyric and n-butyric acids are therefore not reconcilable in terms of molecular structure and require explanation. I n view of the above discussion, it is considered that the measured value of for isobutyric acid is probably correct because c, data have been obtained a t low concentrations with this compound. As can be seen from Fig. 2 , c , variations show a slight maximum. All values have been derivedz1 earlier data from which were obtained at concentrations greater than 0.5 and this maximum was not observed. However, similar cp variations a t lower concentrations are probably also present with other carboxylic acids as well a s isobutyric and may be associated with dimerization phenomena which are believed to be present in aqueous solutions of carboxylic acids.27 z8 Certainly, micellztr aggregation does not, occur until considerably higher concentrations are reached.29-32 values estimated from heat capacity data extrapolated from relatively high concentrations2225are low. It may be noted that if the cp variation in the more concentrated region of Fig. 2 is used to estimate CDzo l,he value obtained is virtually identical with that derived for n-butyric acid a t 25'. Proportionate adjustments to c P , O values of the lower carboxylic acids, as in the case of the butyric acids, bring the values more in line with those of the amino acids and the increments per CHZ group are then essentially the same for the two series. Heat capacity changes observed during the transfer of solute from water to aqueous urea, are also presented in Table I ; values atre negative for the hydrocarbon residues and show that 40-50% of the excess heak capacity present in water solutiona is absent in the presence of urea. From this result it would be concluded that urea has a structure-breaking influence on the water which is associated with the solute molecule. Using heat capacity data of aqueous urea solutions a10ne,3~urea could not be classed as a structure-breaker. Thus a t 2 5 O , for urea at infinite dilution is close to that of the solid and actually increases 10 cal./°C2. mole-' from 0 to 6 M concentration instead of decreasing due to structure breaking. A structure-breaking
e,,'
cpzo
cpzo
cpao
cp,
2431
role of urea has been deduced, however, from other e v i d e n ~ e . ~ ~ -I~nO6 M urea solutions there are only seven water molecules for each urea molecule and existing ideas of cluster size and distribution in wateyZ4 would require considerable modification under these conditions even in the absence of strong urea-water interactions. I n addition, urea itself readily forms hydrogen-bonded structures, particularly around nonpolar molecules in the well-known crystalline clathr a t e ~ . ~The ~ - ~extent ~ to which such structures exist in aqueous solutions of short chain length heteropolar solutes is questionable, but it is conceivable that a competition exists between urea and water molecules for forming ordered structures around the solul e. The heat capacity data in Table I can be rationalized on such a basis if the urea structure is less thermally labile than the competitive water structure. Further discussion of urea hydrocarbon interactions in solution is presented later. The peptide bond plays an important part in the chemistry of proteins. Data obtained with dipeptides (Table I) show that the peptide unit does not have a large effect on solvent structure in water (estimated for the solid is -20 cal./'C. mole-') but the negative Ac,;,,' value suggests a peptide-urea interaction resulting in less water structure. I n this respect the peptide unit behaves like hydrocarbon residues. In view of the experimental precision of values (see figures) the observed agreement of derived data in Table I is fortuitous; the trends discussed, however, are definite. Comparing C,,," values in water for glycine, 7 . 5 cal./'C. and acetic acid (40 cal./'C. mole-' a t 15°),z2it can be seen that the a-amino substitution has a structure-breaking influence on the solvent. Glycine itself is a weak structure breaker while in urea
cp2
(27) A. Katchalsky, H. Eisenherg, and S. Lifson, J . Am. Chem. Soc., 73, 5889 (1951). (28) E. E. Schrier, M .Pottle, and H. A. Scheraga. to he published. (29) P. White, D. Moule, and G. C. Benson, Trans. Faraday SOC., 54, 1638 (1958). (30) D. Mode and G. C . Benson, Can. J . Chem., 37, 2083 (1959). (31) E. R. Jones and C. R. Bury, Phil. Mag., 4, 841 (1927). (32) J. Grindley and C R. Bury, J . Chem. Soc., 679 (1929). (33) F. T. Gucker, Jr., and F. D. Ayres, J . Am. Chem. Soc., 59, 2152 (1937).
W. Bruning and A. Holtzer, ibid., 83, 4865 (1961). P. Mukerjee and A. Ray, J . Phys. Chem., 67, 190 (1963). P. Mukerjee and A. K. Ghosh, ibid., 67, 193 (1963). D. Waugh, Adnan. Protein Chem., 9, 339 (1954). M. Hagan, "Clathrate Inclusion Compounds," Reinhold Puhlishing Corp., New Tork, N. Y.,1962. (39) W. Schlenk, Jr., Ann., 565, 204 (1949). (40) E. Terres and S. N. Sur, Brennstof-Chem.. 38, 330 (1957). (34) (35) (36) (37) (38)
Volume 68, Number 9
September, 1864
G.
2482
c. KRESHECK AND L. BEh'JAMIN
Table 11: Integral Heat8 of Solution and Dilution for Various Compounds in Water and 6 M Urea at 25"" Compound
L-Leucine
Solvent
Water
minitial
mfinal
... ...
0.04184 0.01057 0.00601 0.01390 0 0.04162 0.03122 0.02496 0.01489 0.01026 0.00609 0.00693 0 0.1115 0,04303
0.17083 6 M urea
0.07906 Glycine
Water
Ob
6
M urea
2.2774 1,7917 Acetic acid
6 M urea
Isobutyric acid
Water
6
Phenylalanine
M urea
Water
0.90643 0.51843 0.20959 0.10755 0.03958 0.03141 0.02281 0.01406 0.24150 0.1694 0 0.37501 0,10991 0 0,1913 0.10499 0 0,1835 0.0985 0 0.07.527 0,05902 0.08610 0,01866
0.15792 0.11162 6
M urea
0.15115 Glycylglycine
~
The Journal of Physical Chemistry
Water
0,00937 0.00479 0,00974 0 0.18972 0,07676 0.06153 0,05133 0.03944 0.03033 0.02618 0.02005 0 01051 0.01569 0 0.21857 0.07214 0.05119 0.04241
AHBI cal./mole
847 795 841
...
AHdil,
crtl./mole
... ...
- 49
(828 f 20) 787 833 770 739 708 688
0 (650 f 15) 3367 3367 (3376 f 10) 2492 2492 2483 2499 2481 2459 2426 2406
..
- 39 - 36
(2365 f 25) - 737 - 756 (-747 f 10) - 330 - 361 ( -360 f 30) - 765 - 845 ( -805 f 50) 2023 2003 2000 1997 2022
...
27 0
(2010 i 15) 1224 1205 1192 1207 1195 1229 1197 1144 1185
0 (1180 f 10) 2758 2680 2767 2732
HYDROPHOBIC NATUREOF PROTEIN CONSTITUENTS IN WATERAND UREA
Table I1
2483
(Continued) Compound
Glycylglycine
Solvent
minitial
Water
1.6439 1.1479 0.11992 6 M urea
1.2460 0.94186 0.56615 0.10537 Gly cyl-L-leucine
Water 0.28973 0.23761 0.18187 0.03138 6 M urea 0.20353
mfinsl
0.02874 0.02005 0.01550 0.01270 0.01109 0.00703 0,1185 0.08481 0.00817 0 0.06849 0.03064 0.01463 0.00810 0.07794 0,06102 0.04315 0.01696 0 0.01278 0.00558 0.02027 0.01929 0.01276 0.00264 0 0.00769 0.00388 0.01720 0
AHm
AHdilj
oel./mole
cal./rnole
2737 2793 2715 2597 2634 2786
...
206 159 34
(2790 f 25) 1279 1273 1299 1324
- 124 - 75 - 48 54 (1365 f 15) - 445 - 508
- 71 - 42 -215 - 232 ( -600 f 100)
- 1106 - 1107 - 73 (-1106 f 10)
* Heat of dilution reported by C. A. Zittle 5 Quantities in brackets represent values obtained by extrapolation to zero concentration. and C. L. A. Schmidt, J. Biol. Chem., 108, 161 (1935), used to obtain value a t infinite dilution. solutions Cppzofor glycine (35 cal./'C. mole-', cj'. Fig. 7) is above that of the solid, namely, 24 cal./'C. m01e-l.~ I n contrast, the heat capacity of acetic acid decreases to 33 cal./'C. mole-' in the presence of urea (Fig. 7), the magnitude of the change being in line with reduction of ordered water around a CH3 group. With isobutyric acid, ACD,,' is larger as expected (Fig. 2). Although isobutyric acid differs from valine by an a-amino carbon group, its CPp" value in water is actually higher than that of valine, again confirming the structure-breaking action of the a-amino substitution. Phenylalanine exhibits peculiar heat capacity variation. It has the potential for forming strong hydrophobic bondsll and its increased solubility in urea solutions would support such behavior.15 However, the low C,,' value in water indicates structure breaking with this amino acid, and the heat capacity isapparently increased in urea solutions (Fig. 3). These results are
the reverse of those expected for structure inalters and are contradicted by enthalpy data discussed later. An explanation for this behavior may lie in the probable tendency of this compound to dimerize or aggregate a t very low concentrations in water due to its hydrophobic character. This would be similar to the micellization of surfactants in which hydrophobic bonding is also involved and Cp2 is d e c r e a ~ e d . ' ~ , By ~ ~ ,re~~ ducing hydrophobic bonding, urea has been reported to decrease aggregation t e n d e n c i e ~ ~ ~ - ~ 6 ; data for the phenylalanine in 6 M urea (Fig. 3) are similar to those observed for surfactants during micellisation and could be interpreted as evidence for displaceiiient of the concentration of aggregation from a low indiscernable value in water to around 0.04 m in urea solutions. If this is the case, the Zp,"value for phenylalanine in 6 M urea is correct while that obtained by extrapolation of c, data in water actually refers to aggregated species. The value for monomer may Volume 68, Number 9
September, 1964
G. C. KRESHECK AND L. BENJAMIN
2484
therefore be much larger (-170 cal./'C. mole-l estimated using data for benzene (cf. ref. 16)). The results for ovalbumin in water, which are presented in Fig. 8, represent the first known determination of the partial molal heat capacity of a protein molecule. It is interesting to note that the value of 21,000 cal./niole/"C. for CP,O is considerably less than that of about 40,000 cal./mole/"C. predicted from a summation of the values for the known side-chain and backbone constituents. The specific heat of the solid material was found from a single determination to be 0.47 + 0.02 cal./g./'C. (after correcting the experimental value for the presence of 4% water in the ovalbumin). The latter value compares favorably with that of 0.46 cal./g./'C. obtained by dividing the value for by the molecular weight (46,000 used throughout this study). These results indicate that an excess heat capacity is not associated with the protein molecule in aqueous solution and suggests that all of the hydrocarbon portions of the molecule are not accessible to the solvent. The partial molal heat capacity of ovalbumin a t infinite dilution was also determined in the denaturing solvent of 6 M urea. The value of 22,000 f 2000 cal./mole/'C. obtained was independent of the period of holding a t 25' in 6 M urea up to 48 hr. within the limits of experimental error. Again this evidence suggests that the interior of the protein molecule is not solvated under the experimental conditions. However, an alternative and more plausible explanation, which would apply to both aqueous and 6 M urea solutions, would be to consider that cancelling opposing effects are occurring-as observed with the transfer of L-leucine-so that a net heat capacity change of zero is realized upon solution in either water or 6 M urea. Whatever the reason or reasons for the near identity of the Cpzovalue in water and in 6 M urea, the result was quite unexpected in view of the reasonable changes anticipated, as discussed in a later section. The indicated behavior only emphasizes the need for additional information in order to provide a more complete understanding of the denaturation process. Enthalpy Data. A summary of heats of solution and heats of dilution data both in water and in 6 M urea solutions is given in Table 11. Estimated AH,' values, obtained by extrapolation, are also shown. The measured values for the heat of solution of glycine in water are 380 cal./niole lower than those reported by Zittle and Schmidt.3 Present values have been extrapolated to zero concentration using dilution data of these authors, and the differences observed are probably due to crystal differences in the specimens used.
e,,'
The Journal of Physical Chemistrti
For consistency, the earlier data have not been used for comparative purposes. Isobutyric acid showed calorimetric behavior not noted with any of the other compounds studied. The heat change which occurred during solution in water was instantaneous. However, in 6 M urea solution, in addition to an initial rapid exothermic change, a slow subsequent evolution of heat was observed. The latter was followed in the adiabatic calorimeter for about 45 min. and the temperature change was found to correspond to a first-order reaction process. In two solution runs (isobutyric acid concentration 0.0959 and 0.1835 m) the half-lives for the slow reaction were 21 and 28 min. and the corresponding enthalpy changes extrapolated to completion of the reaction were -388 and -345 cal./mole, respectively. The initial rapid heat changes were -457 and -420 cal./mole for the respective concentrations. The heats of solution given in Table I1 represent values obtained for the total solution process. I n view of the first-order reaction kinetics observed, it is considered that an interaction between the dissolved acid and urea is involved since the latter is present in excess. Crystalline urea clathrates have only been prepared for nbutyric and higher carboxylic acid homologs39 and branching in the hydrocarbon chain reduces the stability and ease of formation of urea clathrate^.^^^^^ The stability of the latter in various solvents depends on the relative solubility of urea or the compound studied in the solvent; where both are soluble the clathrate tends to dissociate. Such dissociation is not always complete, however, and the possible existence of clathrate compounds in solution has been discu~sed.~gThe rate process observed for isobutyric acid may therefore be due to urea interaction with the hydrocarbon chain in solution, the possible existence of which has been inferred earlier in this discussion. Acetic acid did not exhibit slow enthalpy changes during solution. However, it does not form the characteristic crystalline clathrates but instead gives rise to an addition compound of different crystal struct ~ r e .Further ~ ~ investigation in this area appears to be promising, but it is outside the main purpose of this study. With L-leucine and glycyl-L-leucine, which contain an isobutyl rather than an isopropyl radical, solution was a little slow (as also found previously3). However, no slow thermal changes were observed which would compare with those noted with isobutyric acid. This may mean that with longer hydrocarbon residues interactions in solution with urea take place more rapidly or are virtually eliminated with amino acids because of head-group effects.
Table I11 : S u m m w y of the ‘rherrtiotiyriarnic: Properties Aw)ciatetI with the Transfer of Several Compounds arid Constituent, Groups from Water t,o 0 iM Urea a t 25”
1 2 3 4 3
6 7 8 9
10 11 12 13
L-Leucine Glycine Aretic) acid Isobutyric, acid L-Phenylalanine Gly c ylglycine (:lycyl-r,-leucine
828 3376 3701 - 360 2010 2790
- 101
33= ... ... - 438” - 10
..
1365 -1106 ...
...
...
...
... ...
-34
... ...
-471
- 600 Isobutyl (1-2) Isobutyl (7-6) Peptide iinitr (6-2) Peptide unitc (7-2-9) Renxyl ( 5 - 2 ) Ethylidme (4-3)
050 2365 - 747 - 805 1180
...
- 224
...
...
- 178 -1011 -1117 - 446 - 830 - 1425 - 506 833 919 -414 -414 181 672
0.04 -3.5
... ...
-1.3 -4.8 ...
3.5
... -1.3 ...
2.2 ...
* F. It. Birhowsky and F. n. Rossini, “The Thermochemistry of the 5 Y. Nozaki and C. Tanford, J . Wid. (‘hem., 238, 4074 (1963). Chernicd Substances,” Reinhold Publishing Corp., New York, N. Y., 1936, p. 46. c -CHNI-ICO-. Enthalpies of solution a t infinite dilution are surnmarized in Table 111 for the compounds studied.*’ Trarisfcr enthalpies from water to 6 ‘1.1 urea solution are listed arid thew have been combined with the free ericrgy data of n’ozaki and Tanford for the amino acidsI5 to give the thermodynaniic quantities shown. The lattcr appear to be additive and the derived contributions of various groups are also shown in the table. The increased solubility of L-leucine in urea solutions, as reflected in t,he negative Apto value, is due to a favorable enthalpy change while the entropy change during transfer is zero. Glycine is less soluhle in ti ’$1 urea due to the adverse entropy effect and, sindarly, the favorable enthalpy of transfer for the peptide unit is nearly outweighed by the unfavorable entropy decrease. Ilifferrnces between L-leucine and glycine show that the greater solubility of the isobutyl residue in urea solution is due to the increased entropy effect which more than outweighs the unfavorable enthalpy term. The latter suggests that heat is required to “melt” or break down some of the ordered water associated with the hydrocarbon. The net decrease in free rnergy of transfer increases with chain length.15 In addition, it may be noted that the benzyl group behavrs in a riianncr which resembles the isobutyl group in that the enthalpy and the entropy of transfer are both positive. However, the entropy change makes a more significant contribution to the free energy of transfcr i n the case of the benzyl group. Thus, both the benzyl and isobutyl group appear to behave as structure nialiers on the basis of this evidence. 1)ifferences in heats of solution of carboxylic acids in water and in urea solutions have been interpreted
as cvidence for specific urea-carboxylic acid interactions in solution42and the negative A R , ” values found with glycine and acetic acid also support such conclusions. The earlier d ~ t a , however, 4~ were obtained with very concentrated acid solutions which definitely contained r n i c e l l e ~ ~ ~since - ~ ~ urea ; is known to decrease niicellizatiori t e n d e n ~ i e s , ~ ~the - ~ 6 observed differences in heats of solution found by Ketelaar and Loopstra may have been cssentially heats of niicellization of the acids in question. Certainly the order of niagnitude is reasonable for such an effect. Despite this possible alternative interpretation of the data in concentrated solutions, the present enthalpy data at infinite dilution definitely indicate a favorable urea-carboxylic acid interaction. A similar interaction has also been inferred from the effect of urea on polar protein constituents.13 I n addition, since enthalpies of transfer of glycine and acetic acid are numerically similar (Table 111), this suggests that urea interacts mainly with the carboxylic acid group and that the a-aniino group is inert. Application to Proteins. Despite the fact that urea has been used extensively during the isolation and characterization of proteins, the mrchanisin by which it exerts its influence is still being discussed.5~1 3 43144 (41) Enthalpy chmges with concentration of amino acids nnd dipeptides are varied (Txble I1 and ref. 3). I’ossihle interpret:itions of s u c h datii, while being of interest ill terms of xggregsition :ind ionization phenornen:i, are outbide the scope of the present discussion. (42) J. A. A. Ketelwir nnd B. 0. Loopstm. Rev. t m , . chim., 74, 113
(1955). (43) M. L. Meyer nnd W. K:ui~m:inn,Arch. Biorht,m. Iliophvs., 99, 348 (1962).
Volume 68. N u n h e r D
September. 1964
G. C. KRESHECK AND L. BENJAMIK
2486
The effect of surface-active agents on proteins has been examined by J i r g e n ~ o n s *and ~ ~ a~ ~plausible explanation has been given for this process. lleyer and K a ~ z m a n nstudied ~ ~ the change in optical rotation of ovalbumin induced by urea and by surfactant and proposed that if urea weakens hydrophobic bonds, it must do it in a way that is entirely different from the action of surfactant. This statement appears to be true. Thus, urea has been found to yield lower partial molal heat capacities than those obtained in water for the hydrocarbon residues examined in this study, and also increases critical micelle concentration^.^*-^^ These results are consistent M. ith the concept that urea behaves as a structure breaker and meEts some of the “iceberg” water surrcunding the hydrocarbon chains. The difference between the action of structure makers and structure breakers on proteins can be seen by including the results obtained by Gordon and Jencks14’ who found that the substitution of urea and guanidinium salts by alkyl or other relatively nonpolar groups and increases in the chain length of such substituents abolishes or progressively decreases their effect on the optical rotation of bovine serum albumin. Thus, the ability of urea and guanidine to weaken hydrophobic bonding which may be instrumental in leading to changes in optical rotation of protein solutions, and presumably conformation, is destroyed by substitution of groups which themselves manifest “iceberg”
The Journal of Physical Chemistry
structure. It is interesting that the functional groups found by Gordon and Jencks t o be active for denaturing serum albumin were groups which Unieda and Wada48found to be effective in lowering the ternperature of the maximum density of water. Presumably, these two processes are related to the effect the pertinent groups have on the water structure. The feasibility of determining partial molal heat capacities of proteins has been exemplified by the data obtained with ovalbumin. The apparent additivity of the heat capacity and thermodynamic parameters associated with the transfer of amino acids and dipeptides from water to 6 M urea enhances the prospects for being able to extend these studies to polypeptides and other proteins. By examining low molecular weight polypeptides which are of chain length less than required for forming a helix and comparing these results with helical polypeptides, one could conceivably obtain the corresponding changes associated with coiling. These studies could be carried out with different solvents and under varying conditions related to specific protein interests. (44) G. Colacicco, A’ature, 198, 583 (1963). (45) B. Jirgensons, Arch. Biochem. B i o p h y ~ . ,94, 59 (1961). (46) B. Jirgensons, ibid., 96, 321 (1962). (47) J. A. Gordon and W. P. Jencks, Biochemistry, 2, 47 (1963). (48) G. Wada and S. Gmeda, Bull. Chem. Soc. J a p a n , 35, 1797
(1962).
