J. Mol. Biol. (1990) 213,375-384
Heat Capacity of Proteins I. Partial Molar Heat Capacity of Individual A m i n o Acid Residues in A q u e o u s Solution: Hydration Effect G. I. Makhatadze and P. L. Privalovt Institute of Protein Research Academy of Sciences of the U.S.S.R. 142292 Pushchino, Moscow Region, U.S.S.R. (Received 10 July 1989; accepted 5 December 1989) The partial molar heat capacities of various peptides and various organic compounds that model the amino acid side-chains or their parts in aqueous solution have been determined by precise scanning microcalorimetry in the temperature range from 5 to 125°C. This provides an estimate of the partial molar heat capacity of the peptide - C H C O N H - group and the side-chains of all amino acid residues. The values obtained are compared with the values found for these substances in the gaseous phase, in order to define the hydration effect. It has been shown that the partial heat capacity of the non-polar groups is positive at low temperature (5°C) and decreases with increasing temperature, while for the polar and charged groups it is negative at low temperature, becomes zero at room temperature and increases further with increasing temperature. This leads to a hydrophobicity scale of the amino acid side-chains based upon the temperature dependences of their heat capacities. Due to the observed specificity in the temperature dependence, at room temperature, the heat capacities of amino acid side-chains correlate well with the non-polar surface areas.
residual structure in the denatured protein with continued increase in temperature. The possible existence of residual structure in denatured proteins has been discussed for a long time (see Tanford, 1968; Privalov, 1979; Privalov et al., 1989). The heat consumed by gradual melting of residual structure with increasing temperature could indeed raise the apparent heat capacity of a protein. However, residual structure could also decrease the heat capacity of a protein by screening some of its groups from water. On the other hand, attempts to estimate the possible contribution of the configurational freedom, gained by a protein upon denaturation, to its heat capacity were also not very convincing, being too indirect (Sturtevant, 1977). To clarify the situation with protein heat capacity, one .should know, first of all, t h e partial heat capacity of the completely unfolded polypeptide chain in aqueous solution. The knowledge of the thermodynamic properties of the non-interacting amino acid residues of the unfolded polypeptide chain has in fact much wider significance, as it is just this random coiled state that is used as a reference state in theoretical considerations of the process of formation of the native
1. I n t r o d u c t i o n
It is well known that protein denaturation is accompanied by a heat capacity increase, i.e. the heat capacity of the protein in the denatured state is significantly higher than in the native state. The importance of this general feature of proteins follows from the fact that the heat capacity increment determines the temperature dependence of the thermodynamic parameters of protein denaturation and, thus, the stability of its native structure (for a review, see Privalov, 1979). There are several explanations for the denaturation heat capacity effect. The heat capacity might increase because of (1) the increase of the configurational freedom of the polypeptide chain upon disruption of the rigid compact native structure of the protein molecule, (2) the hydration of the groups that are exposed to water upon unfolding of the polypeptide chain or (3) the gradual melting of
~fAuthor to whom all correspondence should be addressed. 0022-2836/90/100375-10 $03.00/0
375
© 199o Academic Press Limited
376
G. I. Makhatadze and P. L. Privalov
protein structure. This ideal unfolded state of the polypeptide chain is never realized in practice, since even in concentrated solutions of denaturants (such as urea or Gu" HC1) in which the protein is supposed to be unfolded, its groups are heavily solvatcd by denaturants, which change all t h e r m o d y n a m i c properties of the protein and particularly its apparent heat capacity (Pfeil & Privalov, 1976). However, the heat capacity of the unfolded polypeptide chain of a protein can be estimated if the partial heat capacities of all amino acid residues constituting the polypeptide chain are known. Since the partial heat capacity of solute is determined for infinitely dilute solutions, the heat capacity of a polypeptide chain, calculated by the simple summation of the partial heat capacities of its constituent components, will correspond to t h a t of the completely unfolded chain of non-interacting amino acid residues. There have been m a n y a t t e m p t s to determine the partial molar heat capacity of amino acid residues in aqueous solutions (see e.g. Suurkuusk, 1974; Spink & Wads5, 1975; Jolicoeur et ai., 1986), but the spread in the values is too large to consider them as reliable. Furthermore, all of these determinations were made only at room temperature, which is insufficient for t h e r m o d y n a m i c analysis of the partial heat capacity function and evaluation of its components. In these two consecutive papers we report the results of the experimental determination of the partial molar heat capacities of all amino acid residues and their constituent groups in aqueous solutions over a broad range of temperature (paper I). Then we present the calculated heat capacities of the unfolded polypeptide chains of some globular proteins in the same temperature range, from 5 to 125°C, and compare them with the measured values for these proteins in the denatured and native states (paper II; Privalov & Makhatadze, 1990). This permits us to estimate the heat capacity effects of hydration and of configurational freedom gain upon protein unfolding. The main problem in experimental determination of the heat capacities of amino acid residues is t h a t the heat capacities of simple amino acids and peptides in solution are influenced b y the end charged groups. This electrostatic effect becomes negligible when the end group is separated from the studied residue X, as it is in a tripeptide of the Gly-X-Gly type. F r o m the heat capacities of various tripeptides and homo-oligopeptides (such as Glyn) we determined the heat capacity of the peptide unit ( - C H C O N H - ) and of the side-chains of several amino acids (Pro, Met, His). The heat capacities of other side-chains and groups constituting sidechains have been determined with appropriate model compounds, which we measured or took from the literature. Unfortunately, there were too few d a t a on the heat capacity of the organic compounds in aqueous solutions over such a broad temperature range as needed. Therefore we had to perform most of the measurements.
2. Materials a n d M e t h o d s
As model compounds for amino acid residues we have selected the following 9 substances: methanol, acetic acid, propanoic acid, acetamide, n-propanamide, n-butanamine, ¢-butanamine nitrate, 4-methylphenol, ¢-propylguanidine nitrate, which are analogs of the side-chains of Ser, Asp, Glu, Ash, Gin, Lys, Lys +, Tyr, Arg +, respectively. Methanol, acetic acid, propanoic acid, acetamide, n-propanamide, n-butanamine, n-butanamine nitrate, 4-methylphenol and nitric acid were purified according to the methods described by Perrin et al. (1980). Nitrate of n-propylguanidine was synthesized as described by Pievano (1928). Aqueous solutions of these compounds were prepared by mixing the weighed components, i.e. the substance and distilled water, degassed by boiling. The pH values of the solutions were not controlled. Experiments were carried out on solutions with concentrations from 0"3 to 1"5% (w/v), except for 4-methylphenol, which was studied only at 0"2% concentration because of its limited solubility in water. The concentration of n-butylamine nitrate solution was determined by Elemental Analyzer 240-B (Perkin-Elmer, U.S.A.). Homopeptides Glyn with n = 3, 4, 5 and tripeptides Gly-X-Gly with X = Gly, Pro, His, Met were used in the studies. The compounds Gly3, Gly4, Gly s (Reanal, Hungary) were recrystallized twice from the water/ethanol mixture. The monomer units of. the peptides Gly-Pro-Gly, GlyMet-Gly, Gly-His-Gly were built in the conventional stepwise manner using pentafluorophenyl esters of protected amino acids as described (Kisfaludy et al., 1973). Protected amino acids were obtained from Reanal, Hungary. The C-terminal amino acid was protected as the benzyl ester (Bergmann et al., 1933) (Gly-His-Gly) or phenacyl ester (Stelakatos et al., 1966) (the rest peptides) through the synthesis. The deprotection of the amino terminus of the growing peptide chain was carried out by cleaving the Boc group with 4 M-HC1/dioxane and the benzyloxycarbonyl group by hydrogenation (Willstatter & Waldschmidt-Leitz, 1921). The reductive cleavage of the OPhac group was made with Zn dust in 50% acetic acid/water (Hendrickson & Kandall, 1970) or by means of hydrogenation (Willstatter & Waldsehmidt-Leitz, 1921). Hydrogenation was used for reductive cleavage of the benzyl ester group and 4 M-HCl/dioxane for tret-butyltype protecting groups. The conversion was monitored by means of thin-layer chromatography on commercial pre-coated silica gel plates (Silica Gel 60 F-254 from Merck, W. Germany). The ratio of the solvent mixture chloroform/methanol was 9 : 1 (v/v). The purification of protected peptides was made by means of column chromatography on Silica gel L 40/100 (Chemapol, Czechoslovakia). The purity of the peptides after deblocking was checked by paper electrophoresis in 30% CH3COOH. Solutions of peptides were prepared by weighing the components and using double-distilled degassed water, and passing through a Sephadex G-10 (Pharmacia) column (3 cm x 100 cm) equilibrated with 5% CH3COOH, followed by lyophilization and chromatography on the same column equilibrated with 0"5 M-Na acetate/acetic acid buffer at pH 4"0. The concentrations of the peptides in solutions were determined from glycine content after 24 h hydrolysis in 6M-HCI using a Durrum D500 (U.S.A.) amino acid analyzer. The concentrations used for the calorimetric measurements varied from 3 to 9 mg/ml.
Heat Capacity of Proteins
377
Table 1
Partial molar heat capacities COp,÷ (J K-1 tool-1) for some amino acid side-chain analogs in aqueous solution Temperature (°C) Substance Methanol Acetic acid Propanoic acid Acetamide n-Propanamide 4-Methylphenol n-Butanamine n-Butanamine nitrate n-Propylguanidine nitrate Nitric acid n-Butanamine (ionized) n-Propylguanidine (ionized)
5
25
50
75
100
125
155"2 155'1 250.6 155"2 250"3 392.9 411"1 297"4 286"9 -95"3 392-7 382"2
158"3 167"0 257"0 166"8 258-2 379"7 410"5 327"8 351"4 -44.2 372.0 395"6
159"1 177"9 263"7 181.5 265"1 366"8 405"7 338'6 377"5 - 18"5 357"1 396"0
160.4 190.9 270"1 191.6 269"7 360"9 403"9 340"8 381-5 - 15-1 355"9 396"6
162"9 200.4 271"1 200"2 270"5 359'8 399"3 337"8 378"4 - 15-4 353"2 393"8
165"6 209.7 271"2 207"6 272-1 357"4 397"0 328.3 372"4 - 19"0 347"3 391"4
3.2 6"0 7"4 5"9 7"3 10"6 7'3 7"4 7"1 4"7 l 1-7 11-8
a, standard deviation. The apparent molar heat capacity Cp, ~t of the solute at temperature T was determined by measuring the apparent heat capacity difference ACp.¢ between the solution and solvent at this temperature by a precise scanning microcalorimeter DASM-4 (Special Construction Bureau of Biological Instrumentation of the Academy of Sciences of the U.S.S.R.) with a fixed operational volume of calorimetric cells, using the equation (Privalov & Potekhin, 1986): C p . , ~- Cp. 1 V~] V 1 - MACp. ¢/m,
(1)
where Cp. 1 and V1 are the molar heat capacity and molar volume of water, respectively (Weast, 1970), V~ is the partial molar volume of the solute, m is the mass of the solute in the calorimetric cell, M is the molar mass. All experiments were carried with the heating rate 1 K min-l, which is optimal for the instrument used. In the range of the concentrations employed no dependence of the apparent molar heat capacity on concentration has been found. This permits one to consider the values determined for Cp,¢ as corresponding to infinite dilution, i.e. Cp,~ = C~.,. The apparent molar volume of the studied compounds in water, V~, was determined from the density, p, of the solution and the density, Po, of the solvent, according to Freadman & Scheraga (1965), by the equation: V~ =
M
1000. (p-po)
p
c'p'po
(2)
where c is the molality of the solution. The density was measured by a precise vibrational densimeter DMA-60 (Anton Paar, Austria) at a fixed temperature (+0"01 K) using water and air as a standard for calibration. The partial molar volume at infinite dilution, V$, was obtained by linear extrapolation of the apparent molar volumes, Vv, to zero concentration. Measurements have t Abbreviations used: C~.~, partial molar heat capacity of a solute; ASA, water-accessible surface area; V~, partial molar volume of a solute; Cp, 1, molar heat capacity of a solvent; m, mass of a solute; C~,¢ ( - R), partial molar heat capacity of the amino acid side-chain in aqueous solution; ACp,i, hydration heat capacity per a given type of the ASA unit; C~, molar heat capacity in the gaseous phase.
been done for the temperature range 5 to 85°C, with a 10-deg. spacing. At higher temperatures (above 85°C) extrapolated values were used. The details of the partial molar volume determinations in a broad temperature range are considered elsewhere (Makhatadze et al., 1990).
3. Results (a) Partial molar heat capacity of simple organic
compounds and peptides The partial molar heat capacity of the simple organic solutes determined from our calorimetric experiments in the temperature range 5 to 125°C are given in Table 1. The partial molar heat capacity of n-propylguanidine and n-butylamine ions were calculated from the Cp,~ values of the corresponding nitrates and t h a t of nitric acid. The values calculated in this w a y are also listed in Table 1. The partial molar heat capacities of the studied compounds for 25°C reported in the literature are listed in T a b l e 2 , together with our data. The observed good correspondence between our and published d a t a at 25 °C permits one to suppose t h a t our d a t a are reliable for all other temperatures as well. The partial molar h e a t capacity of the - C H 2group was calculated from the partial molar heat capacity of homologous substances (i.e. acetic acid and propanoic acid, acetamide and n-propanamide). The values obtained, given in Table 3, are in good agreement with those reported for the homologous series of alcohols (Makhatadze & Privalov, 1989). This means t h a t the partial heat capacity of the - C H 2- group at all temperatures in the studied region is not affected by the presence of the polar group. The partial molar heat capacity of the - C H a group at 25°C was obtained b y addition of the heat capacity of the hydrogen a t o m to the heat capacity of the - C H 2- group and b y subtraction of the hydrogen a t o m heat capacity from the k n o w n h e a t
G. I. Makhatadze and P. L. Privalov
378
2 0 0 | (a)
Table 2
Comparison of the partial molar heat capacities of some compounds at 25 °C obtained in this study with literature values Cp.~b/JK -1 mol-I Compound
This work
Methanol Acetic acid Propanoic acid Aeetamide n-Propanamide n-Butanamine 4-Methylphenol
158.3_.+3-2 167"0__+6'0 257"0-+7"4 166-8-+5-9 258.2_+7-3 410"5_+7-3 379"7-+ 10"9 175.3_+5-0 264'5 -+6-2 361 '7 _+7-4 93"2_+6-2
Triglycine
Tetraglycine Pentaglycine -CH2CONH-
I001- Ib)
Literature 158.2_+0.1= 165_+3b 253-+ 3b 162-4_+1~ 253"6_+1: 422__.4 b 384 _ 2d 189.5+0.9 r 283 -+2f 373 _+6f 98___5f, 128-+21h 106-+ 10 ~, 50_+21s
0
25
50
75
I00
t25
Temperoture (°C)
References: "Jolicoeur & Lacroix (1976), bKonicek & Wads5 {1971). =Roux et al. (1978), dNichols & Wads5 (1975), =Cabani et a/. (1977), tjolicoeur & Boileau (1978), gBello (1978), hKreshek & Benjamin (1964).
F i g u r e I. Temperature dependence of the partial molar heat capacity of: (a)-CHa, (b)-CH2-, (c)-OH, (d)-COOH, (e)-CONH 2, (f)-NHz, ( g ) - N H C N H N H ~ groups calculated from the heat capacity values of mode[ compounds. For errors see Table 3.
c a p a c i t y of m e t h a n e , 244.7 J K - z t o o l - z (Naghibi et
al., 1986). T h e heat c a p a c i t y of the H a t o m at 25°C according to G u t h r i e (1977) is 7 5 J K - l m o 1 - 1 , while, according to Lilley's calorimetric s t u d y of a m i n o acid amides and peptides (personal c o m m u n i cation), it is 78"6 J K -1 tool - l . Close values were o b t a i n e d b y Nichols et al. (1976) a n d R o u x et al. (1978), 6 7 J K -1 tool -1 and 9 0 J K - t mol - t , respectively. I n our calculations we h a v e t a k e n the m e a n value o f all these, 78 J K - 1 m o l - 1. T h e n for the h e a t c a p a c i t y of the - C H s g r o u p at 25°C we get 1 6 9 " 2 J K -~ mol -~ f r o m the h e a t c a p a c i t y of the - C H 2- g r o u p a n d 166-7 J K -1 mol -~ f r o m the h e a t c a p a c i t y o f t h e CH 4 group. T h e c o r r e s p o n d e n c e of these two values shows t h a t the h e a t c a p a c i t y 78 J K - 1 m o l - z for the h y d r o g e n a t o m is correct. F o r the calculation o f the t e m p e r a t u r e d e p e n d e n c e
o f the h e a t c a p a c i t y of the - C H 3 g r o u p one can use the fact t h a t t h e h e a t c a p a c i t y o f a n o n - p o l a r g r o u p is p r o p o r t i o n a l to its size (Naghibi et al., 1986, 1987a,b), i.e. t h a t the ratio: O
O
Cp, ~(-CH3)/Cp, ,~(--CH2- ) -- c o n s t
is valid a t a n y t e m p e r a t u r e . T h e t e m p e r a t u r e d e p e n d e n c e o f t h e partial m o l a r h e a t c a p a c i t y per - C H 3 g r o u p o b t a i n e d in this w a y is p r e s e n t e d in Table 3. F r o m the difference in the slopes o f these f u n c t i o n s (Fig. 1) we d e t e r m i n e d also the t e m p e r a t u r e d e p e n d e n c e o f the h y d r o g e n h e a t c a p a c i t y . U s i n g the partial m o l a r h e a t c a p a c i t y o f the - C H 2 - a n d - C H s groups, the h e a t c a p a c i t y of -COOH,-CONH2,-NH2, NH~" a n d - N H C N H N H ~ g r o u p s can be easily o b t a i n e d b y a g r o u p a d d i t i v i t y
Table 3
Partial molar heat capacity (C°p,~) per -CH2-, -OH, - C H 3, -COOH, - C O N H 2, - N H 2, N H J and - N H C N H N H J groups at various temperatures Temperature (°C) Group
5
25
50
75
100
125
a
95.1 95-5 97"9 96"2 178"5
91"4 90'0 92"3 91.2 169"2
83-6 85"8 82.3 83"9 155"6
78"1 79"2 75"9 77"7 144-1
70"3 70"7 68"3 69-8 129"5
64.5 61.5 63"1 63"0 116-9
9'5 9'4 8-1 9'0 12"1
-OH -COOH -CONH2
-5"3 - 23"8 --23.9
-3"6 - 2-8 --2.3
13"7 23'3
30-4 47-6
58"0 71-4
89"6 92" 1
8-1 9'4
-NH~ -NHz -NHCNHNH~
-74"4 --56"0 -70"8
--70"8 -32"3 -34"0
25.8 -50'2 1"6 0"9
48"7 21"3 26"7 30"7
71"0 14"3 60"4 65"0
91"5 41"4 91"1 94"6
9"5 8"8 7"3 8"5
-CH 2- amides -CHz- acids - C H 2- alcohols=
-CH 2- (mean) -CH3
All heat capacity values are given in J K -1 tool -1 . • Calculated from the data reported by Makhatadze & Privalov (1989).
Heat Capacity of Proteins
379
Table 4
Temperature dependence of the partial molar heat capacity (C°p,~) of some peptides in aqueous solution Temperature (°C) Peptide Gly-Gly-Gly" Gly-Gly-Gly-Gly~ Gly-Gly-Gly-Gly-Gly~ Gly-Gly-Glyb Gly-His-Gly b Gly-Pro-Gly b Gly-Met-Gly b
5
25
50
75
100
125
a
98.2 182-8 271.3 93.1 216.5 161.4 207"9
175-3 264.6 361.7 194.6 296.2 230'3 292.5
239.5 338.2 430.9 275.7 381.2 292"3 362"1
285.1 382.3 473.5 348.9 462.1 361"3 432"8
322.7 412.0 497.1 413"1 540.5 425"0 503-5
360.6 438.5 520.4 474.2 617.6 486.9 564.7
5.0 6.2 7.4 9.1 13.6 11"0 9"0
All heat capacity values are given in J K -I mol -I. a In pure water as a solvent. b In 0"5 M - N a C H 3 C 0 0 - C H 3 C O O H (pH 4"0) buffer solution; a, is standard deviation.
scheme. In the case of the -COOH group the temperature-dependent dissociation has not been taken into account. Temperature dependences of these values are presented in Table 3 and Figure 1. It is notable that the partial heat capacity of the non-polar groups - C H 2- and - C H 3 is positive and decreases with increasing temperature, while the partial heat capacity of the polar groups, -COOH, -CONH2, -NH2 and - N H C N H N H ~ , is negative at room temperature and increases with temperature. The other notable fact is that the values of the partial molar heat capacity o f - C O O H and -CONH 2 are very similar at all studied temperatures although the partial heat capacity of the - O H and - N H 2 groups differ significantly. One can conclude from this that there is a strong interaction between the - C O - group and - O H or -NH2 groups. It should be noted that the heat capacity of all the above mentioned groups at 25°C are in good agreement with the values obtained earlier (Guthrie, 1977; Nichols et al., 1976; Roux et al., 1978). The main importance of our data in this respect is that they were obtained not only at standard temperature (25°C) but in a broad temperature range. The partial molar heat capacities of the studied peptides at different temperatures are given in Table 4. Table 2 shows a comparison of the partial molar heat capacities for Gly 3, Gly 4, Gly 5 at 25°C with available literature data. As seen, the values of C~, for peptides determined in this work agree with those obtained earlier within the limits of error of determination.
(b) Heat capacity of the protein constituent groups All the studied simple organic compounds are analogs of the side-chains of various amino acids differing only by an extra hydrogen atom. Therefore, the heat capacity of the amino acid side-chain, C~.~(-R), can be calculated from the heat capacity of the analogs, C~,,(A), by extracting the heat capacity of the hydrogen atom, C~,,( - H):
C~,A-R) = C~,AA)--V~.A-H).
(3)
Using the above heat capacity function of C~. ~ ( - H) {which is in itself the heat capacity of the Gly sidechain) and the heat capacity of the analogs, we calculate the heat capacity of the following amino acid side-chains: Ser, Asp, Glu, Asn, Gin, Lys, Tyr and Arg, which are listed in Table 5. The Table also presents the heat capacities of the Thr and Phe sidechains calculated from the heat capacities of their analogs, ethanol and toluene, which were determined earlier (Makhatadze & Privalov, 1988, 1 9 8 9 ) . Comparison of the heat capacities of the Lys sidechain in ionized and non-ionized forms, shows that there is a significant decrease of the heat capacity due to ionization. At 25°C it amounts to 38"5 J K -1 mo1-1, which significantly exceeds the value 14"9 J K - 1 mol- 1 obtained earlier by Riedl & Jolicoeur (1984). With increasing temperature the heat capacity effect of ionization increases but non-linearly. Unfortunately the scanning microcalorimetric technique cannot be used for the determination of the heat capacity of the side-chains of the amino acids Ala, Val, Leu and Ile. The analogs of these side-chains, which are methane, propane, n-butane and /-butane, have too low solubility in water to measure their heat capacities with sufficient accuracy even by the most precise contemporary scanning microcalorimetric instrument. For these substances the partial molar heat capacity in aqueous solution was determined by Naghibi et al. (1986, 1987a,b) in the temperature range from 0 to 50°C using a flow microcalorimetric technique. Assuming that above 50°C the temperature dependence of the partial molar heat capacity is the same as it is below this temperature, one can calculate by equation (3) the partial molar heat capacity of the side-chain of the above amino acids. The obtained values are given in Table 5. From the obtained data on the partial molar heat capacity of tripeptides and homopeptides we can determine the partial molar heat capacity of the internal amino acid residues, the peptide unit - C H z C O N H - and the corresponding side-chains. From the linear dependence of the partial molar heat capacity of homopeptides on the number of
-
~
o~
~
~
~
~
~
~
~
~
~.
~
~
o.
I
I
I
I
I
~'.
.
Heat Capacity of Proteins
381
Table 5 (continued) Side-chain of amino acid residue Heminec
Temperature (°C) ASA"p (A 2)
ASA ~1 (A2)
--
--
C~. ~ A~Cp
5
25
50
75
621 499
600 467
575 432
553 398
100 530 367
125 512 338
All heat capacity values are given in J K-1 mol-l. C~. ~ and A~'Cp values are precise within 5 %, C~ within 0"5 %. Water-accessible surface areas were taken from Miller et al. {1987) and are precise within l0 to 20% . "Total ASA. b Side-chain in a nitrate form. c Side-chain in an ionized form. d These values refer to the modified cysteine residue Cys-CH2CONH 2. The heat capacity of this residue at 25°C was calculated by the equation: C;.,(Cys-CH2CONH2) = C;. ,(CysH) - C;. ,(Gly) + C;. ,(CH2CONH2). The values for C~.÷(CysH) and C~.~(Gty) were taken from Joticoeur et al. {1986). The temperature dependence of this modified amino acid side-chain was supposed to be the same as that for the Glu side-chain. " Partial molar heat capacity of hemine at 25°C was calculated from the difference of the partial molar heat capacities of myoglobin and apomyoglobin taken from Privalov et al. (1989}. Temperature dependence of the Up.$ function was obtained from the partial molar heat capacities of toluene (Makhatadze & Privalov, 1988) assuming that the ratio C~.,(hemine)/C~. ¢(toluene) ffi 1-3 is valid for the whole temperature range from 5 to 125°C. The temperature dependence of the hydration heat capacity increment of heroine was obtained by comparison with the A~Cp values for toluene (Makhat~ize & Privalov, 1988).
Gly units, N~ly, it follows that at any temperature studied the glycyl residue contributes additively to the heat capacity of the peptide molecule. The slope of the plot of C;.e versus NGly at a given temperature corresponds to the heat capacity contribution of a single glycyl residue. Table 2 shows the comparison of the partial molar heat capacity of -CH2CONH- obtained in this way with the literature data at 25°C. As seen, our value 93.2(_+6-2) agrees within experimental error with those obtained by Jolicoeur & Boileau (1978) 98{_+5) and Bello (1978) 106(_+10), can be compared with the data of Kresheck & Benjamin (1964) 128(_+25) and differs significantly from that obtained by Cabani et al. (1977) 50(+21). From the determined temperature dependence of the heat capacity of the -CH2CONH- group one can easily obtain the heat capacity o f - C H C O N H - :
The heat capacity contributions of the amino acid side-chains are listed in Table 5. So, almost all values listed in Table 5, except for side-chains of Trp, were determined by calorimetric studies of the model compounds and tripeptides. In all cases the standard deviation of these determinations does not exceed 5%. The partial molar heat capacity of the Trp sidechain at 25°C was calculated from the calorimetric data obtained by Jolicoeur et al. (1986). The temperature dependence of its heat capacity was assumed to be the same as that for Phe, another aromatic side-chain that we have studied (Makhatadze & Privalov, 1989). 4. Discussion
(a) Temperature dependence of the partial heat
capacity
C~,,(-CHCONH-) = C;.e(-CH2CONH-)-C~.e(-H).
(4)
The partial molar heat capacities of the - C H C O N H - group calculated by equation (4) are listed in Table 5. Additivity of the heat capacity contribution of the peptide groups permits one to calculate the sum of the heat capacity contributions of the - N H 2 and -CHCOOH groups. The values obtained for these groups from the data for the glycine homopeptides are given in Table 5 for the temperatures studied. From the partial molar heat capacity of tripeptides we calculated the heat capacity contribution of the amino acid side-chains, C;, e ( - R ) , as: C~. ~( - R) = C~. ~(Gly-CH-Gly)
As seen from Table 5, the temperature dependence of the heat capacity of various amino acid side-chains is very different: the values decrease with increasing temperature for non-polar sidechains of Pro, Met. Trp, Ala, Val, Leu, Ile, Phe and Gly. They increase for such polar side-chains as those of Ser, Asp, Asn, Gin, Glu, Lys, Arg. As for the His, Thr and Tyr side-chains, the dependence is rather complicated and actually represents a superposition of temperature dependences specific for both the non-polar and polar groups. Therefore, it looks as though one can use the temperature dependences of the partial heat capacity for classifying the amino acids according to their polarity or hydropathy. The amino acid side-chains can be regarded as:
I
E -C;, AGIy-CH-Gly)+ C;.,(-H).
I
H
Hydrophobic if (5)
oc;.,
Hydrophilic if ~
< 0 in 5 to 125°C > 0 in 5 to 125°C
(6) (7)
G. I. Makhatadze and P. L. Privalov
382
value at 25°C: a smaller value will mean a more hydrophilic residue. In order to increase the hydrophilicity, amino acid side-chains are listed in Table 6. I n this Table the proposed h y d r o p a t h y scale is c o m p a r e d with those reported in the literature, (Bull & Breese, 1974; Chothia, 1976; M a n a n v a l a n & P o n n u s w a m y , 1978; H o p p & Woods, 1981; Janin, 1979; Levitt, 1976; P a r k e r et al., 1986). According to the calculated correlation coefficients there is the best correlation with the scale of P a r k e r et al. (1986), 0.92. However, even in this case the main exceptions are non-polar Gly and a m b i v a l e n t Tyr.
Table 6
Comparison of the hydropathy scale derived in this work with those obtained earlier Side-chain
Hydropathy scale according to various authors:
of amino acid residue
a
b
c
d
e
f
g
h
Trp Phe Ile Leu Val Pro Met Ala Gly Tyr Thr His Lys Arg Glu Gin Asp Asn Ser
l 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19
5 2 3 l 6 8 7 13 17 4 9 15 11 16 12 19 14 18 10
8 3 1 4 2 11 5 6 7 14 9 13 18 19 12 17 15 16 10
l 3 2 6 7 4 8 I0 15 5 17 II 9 12 13 19 14 16 18
8 4 1 3 2 12 5 7 6 ll 13 9 19 18 17 16 15 14 10
1 2 5 4 6 11 7 8 12 3 10 9 19 18 17 14 16 13 15
1 2 4 5 6 7 8 9 12 3 11 10 19 18 17 14 16 13 15
1 2 4 3 6 9 5 8 14 7 12 10 13 11 18 15 19 17 16
(b) Correlation between water-accessible surface area
and partial heat capacity of the amino acid side -chains I t is known from the calorimetric studies (see e.g. Gill & WadsS, 1976; Gill et al., 1976; Olofsson et al., 1984; Dec & Gill, 1984, 1985a,b) t h a t the partial molar heat c a p a c i t y of h y d r o c a r b o n s in aqueous solution is proportional to their water-accessible surface area (ASA). Therefore one is interested in possible correlations between the heat c a p a c i t y of amino acid side-chains and their non-polar wateraccessible surface area. A plot of the observed C ~ . ~ ( - R ) values at 25°C versus non-polar ASA values t a k e n from Miller et al. (1987) is presented in Figure 2. One notices two distinct correlations for these compounds. The first of these groups includes the ring-containing amino acid side-chains (Pro, His, Tyr, Phe} and the sulfur-containing Met. The other group includes linear amino acid side-chains. The correlation within each of these groups is r a t h e r good and does not exceed the error in the determination of the ASA values, which is, according to Miller et al. (1987), within 10 to 2 0 % for an individual amino acid residue. I t is notable t h a t a good correlation between the non-polar ASA and C ~ , ~ ( - R ) values is observed only at.25°C, while for higher t e m p e r a t u r e s it significantly decreases for the
Correlation coefficient
1"00 0"75 0"76 0-86 0"75 0'82 0"85 0.92
'This work; bBull and Breese (1974), :Chothia (1976), dMananvalan & Ponnuswamy (1978), "Janin (1979), rHopp & Woods (1981), BLevitt (1976}. hparker et al. (1986).
(8)
A m b i v a l e n t ff 6C~. ~ = 0 in 50 to 75 °C
aT
According to this criterion, one should consider the side-chains of Ala, Val, Leu, Ile, Phe, Trp, Pro, Met, Gly as hydrophobic and of Ser, Asn, Asp, Glu, Gin, Lys, Arg as hydrophilic; while the side-chains of Thr, T y r and His, which show the m i n i m u m heat capacity at a b o u t 60°C, a p p e a r to be ambivalent. Inside each group defined b y equations (6) to (8) the relative h y d r o p a t h y of the side-chains of amino acid residues can be defined using the C~,,~,(--R,)
500 125°C
L~/
400
L, y
I1~,~/
/
/~Phe
L6 E T 300
•
eo/
Arg[]
/2
le
Phe
~200
IOO
I
I 50
I
I I O0
t
I 150
t
I
200
I
I
50
t
I
100
I
I
150
i
I
200
Non-polor ASA (~2)
Figul~ 2. Correlation between the heat capacity of the amino acid side-chains C~, ¢ ( - R) and non-polar ASA at various temperatures. The lines are drawn by least-squares calculations using the C~, ¢ ( - R) and ASA "p values for Ala, Val, Leu, Ile (line 1) and Pro, Phe, Trp (line 2). The ASAnp values are taken from Miller et al. (1987).
Heat Capacity of Proteins
383
Table 7
Hydration heat capacity, ACp,~, per unit of surface-accessible area for various constituent groups of protein Group or side-chain of amino acid residue
Aliphatic Aromatic -CHCONH-CH2CONHPolar parts of: Met His Ser Asn Asp Gin Glu Lys Tyr Art Thr Trp
Temperature (*C) 5
25
50
75
100
125
2"23 1.32 - 1-16 0"32
2.13 1"20 - 0"97 0"37
2'00 - 0"80 0"38
1"88 1'02 - 0"79 0.44
1.77 0'95 - 0'78 0.25
1-64 0"88 - 0"86 0"14
--4-03 -0"96 - 1"60 - 1"29 - 1"74 -- 0"39 - 0'73 - 1'32 -0'06 -0'39 - l'13 3"87
--3-66 -- 1-36 - 1"43 -- 1"02 - 1"42 - 0"24 - 0"56 - 1"53 0'0 -0'20 - 1"32 3'94
-3"I9 -- 1.42 - 1"20 -0"67 - 1-07 - 0"06 - 0"35 -- 1"54 -0'02 -0.11 -- 1"19 3"72
-2-75 -- 1-36 - 0-95 --0"41 -0"71 0"08 - 0"16 - 1.30 0"09 -0"03 --0"85 3"53
--2"33 -- 1-23 - 0'71 --0.15 --0"39 0"18 - 0"03 -- 1"10 0'27 0"03 -0"25 3"27
--1"86 -- 1"06 - 0-47 0"09 --0"11 0"30 0'09 --0'90 0'43 0'09 0"59 3"05
1-11
All heat capacity values are given in J K - t mol -I A -2 and are precise within 20%.
side-chains that are not completely non-polar (Fig. 2). This is u n d e r s t a n d a b l e since w i t h increasing temperature the heat capacity contribut i o n o f t h e p o l a r p a r t i n c r e a s e s , while t h e c o n t r i b u t i o n o f t h e n o n - p o l a r p a r t o f t h e a m i n o a c i d sidechain decreases.
(c)
=
g i -Cp,
Aw~np i"
(10)
--gvp,
=
-np A S A inp "ACp, i.
(11)
C o r r e s p o n d i n g l y , for t h e r e d u c e d h y d r a t i o n effect o f t h e p o l a r g r o u p we h a v e : AW~pOl A(~,poi __ --gvp. i -- v p.. ASA•Ol
(9)
T h e h e a t c a p a c i t y o f a s m a l l o r g a n i c m o l e c u l e in t h e gaseous phase could be easily calculated from the known heat capacities of the constituent groups (Benson, 1968), as t h e h e a t c a p a c i t i e s o f o r g a n i c c o m p o u n d s i n t h e g a s e o u s p h a s e a r e u s u a l l y close to the sum of the heat capacities of their components. The values of the hydration heat capacities of amino a c i d s i d e - c h a i n s a r e l i s t e d in T a b l e 5 t o g e t h e r w i t h t h e c o r r e s p o n d i n g v a l u e s in t h e g a s e o u s p h a s e . I t is k n o w n t h a t for a n o n - p o l a r s u b s t a n c e , t h e h y d r a t i o n h e a t c a p a c i t y effect is p r o p o r t i o n a l t o t h e water-accessible surface area of the substance ( N a g h i b i et al., 1986, 1987a,b). W e c a n o b s e r v e t h i s p r o p o r t i o n a l i t y in T a b l e 5 for t h e n o n - p o l a r a m i n o a c i d s i d e - c h a i n s . T h e r a t i o o f A~Cp. i / A S A i = A C-"P p . i, w h i c h is t h e h y d r a t i o n h e a t c a p a c i t y effect, r e d u c e d t o s u r f a c e u n i t s , is s i m i l a r for v a r i o u s t y p e s o f n o n p o l a r g r o u p s , differing s l i g h t l y for a l i p h a t i c a n d r i n g c o m p o u n d s ( M a k h a t a d z e & P r i v a l o v , 1989). T o e s t i m a t e t h e h y d r a t i o n effect o f s i d e - c h a i n s c o n t a i n i n g p o l a r p a r t s , o n e s h o u l d first o f all
o
C p , ~! - -
If the water-accessible surface area of the non-polar p a r t , A S A ~ p, is k n o w n , t h e n t h e h y d r a t i o n effect o f t h e n o n - p o l a r p a r t c a n b e d e r i v e d as: g~p,i
T h e h y d r a t i o n effect o f h e a t c a p a c i t y is t h e difference b e t w e e n t h e h e a t c a p a c i t y o f a s o l u t e m o l e c u l e in t h e g a s e o u s p h a s e , Cgp, a n d i t s p a r t i a l h e a t c a p a c i t y in a q u e o u s s o l u t i o n ( P r i v a l o v & Gill, 1989): Cp,~p--Cp. ° '
AW~pOl gvp, i
Awf?np
Hydration heat capacity effect
a c. =
s e p a r a t e t h e effect o f t h e n o n - p o l a r p a r t o f a sidec h a i n f r o m t h e effect o f i t s p o l a r p a r t . T h e l a t t e r is given by:
o
=
g
•
np.
Cp. 4,,- CD, i - A S A i ASApOt
--rip
A Cp, i (12)
W a t e r - a c c e s s i b l e s u r f a c e a r e a v a l u e s for t h e n o n polar and polar parts of the amino acid side-chains g i v e n in T a b l e 5 w e r e t a k e n f r o m M i l l e r et al. (1987). T h e ACp p v a l u e s for a r o m a t i c s i d e - c h a i n s (Trp, T y r , His) were taken from Makhatadze & Privalov (1988), a n d for a l i p h a t i c s i d e - c h a i n s t h e y w e r e c a l c u l a t e d a s t h e m e a n v a l u e o f ACp p for A l a , Val, Leu and Ile side-chains. The obtained values of the hydration heat capacities reduced to surface area a r e l i s t e d in T a b l e 7. A s c a n b e seen in o u r a c c o m p a n y i n g p a p e r ( P r i v a l o v & M a k h a t a d z e , 1990), t h e s e v a l u e s p e r m i t us t o e s t i m a t e t h e h y d r a t i o n effect o f p r o t e i n u n f o l d i n g . We are grateful to Dr T . H . Lilley for sending us unpublished results of his heat capacity studies on a m i n o acid amides and peptides, to Dr K . H . Zikherman for synthesis of n-propylguanidine nitrate, to Dr V . N . Medvedkin for the help in peptide synthesis.
384
G. I. Makhatadze and P. L. Privalov
References Bello, J. (1978). J. Phys. Chem. 82, 1607-1609. Benson, S. W. (1968). Thermochemical Kinetics, John Wiley & Sons, Inc., New York, London, Sydney. Bergmann, M., Zarvas, L. & Salzmann, L. (1933). Chem. Bet. 66, 1288-1290. Bull, H. B. & Breese, K. (1974). Arch. Biochem. Biophys. 161,665-670. Cabani, S., Conti, G. & Matteoli, E. (1977). Biopolymers, 16, 465-467. Chothia, C. (1976). J. Mol. Biol. 105, 1-14. Dec, S. F. & Gill, S. J. (1984). J. Solution Chem. 13, 27-41. Dec, S. F. & Gill, S. J. (1985a). J. Solution Chem. 14, 417-429. Dec, S. F. & Gill, S. J. (1985b). J. Solution Chem. 14, 827-836. Freadman, M. E. & Scheraga, H. S. (1965). J. Phys. Chem. 69, 3795-3800. Gill, S. J. & WadsS, I. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 2955-2958. Gill, S. J., Nichols, N. F. & WadsS, I. (1976). J. Chem. Thermodynam. 8, 445-452. Guthrie, J. P. (1977). Can. J. Chem. 55, 3700-3706. Hendrickson, J. B. & Kandall, C. (1970). Tetrahedron Letters, 5, 343-344. Hopp, T. P. & Woods, K: 1%. (1981). Proc. Nat. Acad. Sci., U.S.A. 78, 3824-3828. Janin, J. (1979). Nature (London), 277, 491-492. Jolicouer, C. & Boileau, J. {1978). Can. J. Chem. 56, 2707-2713. Jolicoeur, C. & Lacroix, G. (1976). Can. J. Chem. 54, 624-628. Jolicoeur, C., 1%iedl, B., Desrochers, D., Lemelin, L.L., Zamojska, R. & Enea, O. (1986). J. Solution Chem. 15, 109-128. Kisfaludy, L., Low, M., Nyeki, 0., Szirtes, T. & Schon, I. (1973). Lieb. Ann. Chem. 9, 1421-1429. Konicek, J. & WadsS, I. ~I. (1971). Acta Chem. Scand. 25, I541-1551. Kresheck, G. C. & Benjamin, L. (1964). J. Phys. Chem. 68, 2476-2486. Levitt, M. (1976). J. Mol. Biol. 104, 59-107. Makhatadze, G. I. & Privalov, P. L. (1988). J. Chem. Themodynam. 20, 405-412. Makhatadze, G. I. & Privalov, P. L. (1989). J. Solution Chem. 18, 927-937. Makhatadze, G. I., Medvedkin, V. N. & Privalov, P.L. (1990). Biopolymers, in the press. Mananvalan, P. & Ponnuswamy, P. K. (1978). Nature (London), 275, 673-674.
Miller, S., Janin, J., Lesk, A. M. & Chothia, C. (1987). J. Mol. Biol. 195, 641-656. Naghibi, H., Dec, S. F. & Gill, S. J. (1986). J. Phys. Chem. 90, 4621-4623. Maghibi, H., Dec, S. F. & Gill, S.J. (1987a). J. Phys. Chem. 91,245-248. Naghibi, H., Dec, S. F. & Gill, S. J. (1987b). J. Chem. Ing. Data, 32, 422-425. Nichols, N. & WadsS, I. (1975). J. Chem. Thermodynam. 7, 329-336. Nichols, N., ScSld, R., Spink, C., Suurkuusk, J. & WadsS, I. J. (1976). J. Chem. Thermodynam. 8, 1081-1093. Olofsson, G., Oshodj, A. A., Qvarnstr5m, E. & Wads5, I. (1984). J. Chem. Thermodynam. 16, 1041-1052. Parker, J. M. 1%., Guo, D. & Hodges, R.S. (1986). Biochemistry, 25, 5425-5432. Perrin, D. D., Armarego, W. L. F. & Perrin, D. R. (1980). Purification of Laboratory Compounds, Pergamon Press, Oxford, New York, Toronto, Sydney, Paris, Frankfurt. Pfeil, W. & Privalov, P. L. (1976). Biophys. Chem. 4, 33-40. Pievano, M. (1928). Gazz. Chim. Italiana, 58, 247-249. Privalov, P. L. (1979). Advan. Protein Chem. 33,167-241. Privaiov, P. L. & Gill, S. J. (1989). Advan. Protein Chem. 39, 191-234. Privalov, P. L. & Makhatadze, G. I. (1990). d. Mol. Biol. 213, 385-391. Privalov, P. L. & Potekhin, S. A. (1986). Methods Enzymol. 131, 4-51. Privalov, P. L., Tiktopulo, E. I., Venyaminov, S. Yu., Griko, Yu V., Makhatadze, G.I. & Khechinashvili, N. N. (1989). J. Mol. Biol. 205, 737-750. Riedl, B. & Jolicoeur, C. (1984). J. Phys. Chem. 88, 3348-3356. Roux, G., Perron, G. & Desnoyers, J. E. {1978). Can. J. Chem. 56, 2808-2814. Spink, C. H. & WadsS, I. (1975). J. Chem. Thermodynam. 7, 561-572. Stelakatos, G. S., Panganou, A. & Zarvas, L. (1966). J. Chem. Soc. 21, 1191-1199. Sturtevant, J. M. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 2236-2240. Suurkuusk, J. (1974). Acta Chem. Scand. B, 28, 409-417. Tanford, C. (1968). Advan. Protein Chem. 23, 121-275. Weast, 1%. W. (1970). Handbook of Chemistry & Physics, The Chemical 1%ubber Co., Cleveland, U.S.A. Willstatter, 1%. & Waldschmidt-Leitz, E. (1921). Chem. Bet. 54, 113-138.
Edited by R. Huber
Heat Capacity of Proteins I. Partial Molar Heat Capacity of Individual A m i n o Acid Residues in A q u e o u s Solution: Hydration Effect G. I. Makhatadze and P. L. Privalovt Institute of Protein Research Academy of Sciences of the U.S.S.R. 142292 Pushchino, Moscow Region, U.S.S.R. (Received 10 July 1989; accepted 5 December 1989) The partial molar heat capacities of various peptides and various organic compounds that model the amino acid side-chains or their parts in aqueous solution have been determined by precise scanning microcalorimetry in the temperature range from 5 to 125°C. This provides an estimate of the partial molar heat capacity of the peptide - C H C O N H - group and the side-chains of all amino acid residues. The values obtained are compared with the values found for these substances in the gaseous phase, in order to define the hydration effect. It has been shown that the partial heat capacity of the non-polar groups is positive at low temperature (5°C) and decreases with increasing temperature, while for the polar and charged groups it is negative at low temperature, becomes zero at room temperature and increases further with increasing temperature. This leads to a hydrophobicity scale of the amino acid side-chains based upon the temperature dependences of their heat capacities. Due to the observed specificity in the temperature dependence, at room temperature, the heat capacities of amino acid side-chains correlate well with the non-polar surface areas.
residual structure in the denatured protein with continued increase in temperature. The possible existence of residual structure in denatured proteins has been discussed for a long time (see Tanford, 1968; Privalov, 1979; Privalov et al., 1989). The heat consumed by gradual melting of residual structure with increasing temperature could indeed raise the apparent heat capacity of a protein. However, residual structure could also decrease the heat capacity of a protein by screening some of its groups from water. On the other hand, attempts to estimate the possible contribution of the configurational freedom, gained by a protein upon denaturation, to its heat capacity were also not very convincing, being too indirect (Sturtevant, 1977). To clarify the situation with protein heat capacity, one .should know, first of all, t h e partial heat capacity of the completely unfolded polypeptide chain in aqueous solution. The knowledge of the thermodynamic properties of the non-interacting amino acid residues of the unfolded polypeptide chain has in fact much wider significance, as it is just this random coiled state that is used as a reference state in theoretical considerations of the process of formation of the native
1. I n t r o d u c t i o n
It is well known that protein denaturation is accompanied by a heat capacity increase, i.e. the heat capacity of the protein in the denatured state is significantly higher than in the native state. The importance of this general feature of proteins follows from the fact that the heat capacity increment determines the temperature dependence of the thermodynamic parameters of protein denaturation and, thus, the stability of its native structure (for a review, see Privalov, 1979). There are several explanations for the denaturation heat capacity effect. The heat capacity might increase because of (1) the increase of the configurational freedom of the polypeptide chain upon disruption of the rigid compact native structure of the protein molecule, (2) the hydration of the groups that are exposed to water upon unfolding of the polypeptide chain or (3) the gradual melting of
~fAuthor to whom all correspondence should be addressed. 0022-2836/90/100375-10 $03.00/0
375
© 199o Academic Press Limited
376
G. I. Makhatadze and P. L. Privalov
protein structure. This ideal unfolded state of the polypeptide chain is never realized in practice, since even in concentrated solutions of denaturants (such as urea or Gu" HC1) in which the protein is supposed to be unfolded, its groups are heavily solvatcd by denaturants, which change all t h e r m o d y n a m i c properties of the protein and particularly its apparent heat capacity (Pfeil & Privalov, 1976). However, the heat capacity of the unfolded polypeptide chain of a protein can be estimated if the partial heat capacities of all amino acid residues constituting the polypeptide chain are known. Since the partial heat capacity of solute is determined for infinitely dilute solutions, the heat capacity of a polypeptide chain, calculated by the simple summation of the partial heat capacities of its constituent components, will correspond to t h a t of the completely unfolded chain of non-interacting amino acid residues. There have been m a n y a t t e m p t s to determine the partial molar heat capacity of amino acid residues in aqueous solutions (see e.g. Suurkuusk, 1974; Spink & Wads5, 1975; Jolicoeur et ai., 1986), but the spread in the values is too large to consider them as reliable. Furthermore, all of these determinations were made only at room temperature, which is insufficient for t h e r m o d y n a m i c analysis of the partial heat capacity function and evaluation of its components. In these two consecutive papers we report the results of the experimental determination of the partial molar heat capacities of all amino acid residues and their constituent groups in aqueous solutions over a broad range of temperature (paper I). Then we present the calculated heat capacities of the unfolded polypeptide chains of some globular proteins in the same temperature range, from 5 to 125°C, and compare them with the measured values for these proteins in the denatured and native states (paper II; Privalov & Makhatadze, 1990). This permits us to estimate the heat capacity effects of hydration and of configurational freedom gain upon protein unfolding. The main problem in experimental determination of the heat capacities of amino acid residues is t h a t the heat capacities of simple amino acids and peptides in solution are influenced b y the end charged groups. This electrostatic effect becomes negligible when the end group is separated from the studied residue X, as it is in a tripeptide of the Gly-X-Gly type. F r o m the heat capacities of various tripeptides and homo-oligopeptides (such as Glyn) we determined the heat capacity of the peptide unit ( - C H C O N H - ) and of the side-chains of several amino acids (Pro, Met, His). The heat capacities of other side-chains and groups constituting sidechains have been determined with appropriate model compounds, which we measured or took from the literature. Unfortunately, there were too few d a t a on the heat capacity of the organic compounds in aqueous solutions over such a broad temperature range as needed. Therefore we had to perform most of the measurements.
2. Materials a n d M e t h o d s
As model compounds for amino acid residues we have selected the following 9 substances: methanol, acetic acid, propanoic acid, acetamide, n-propanamide, n-butanamine, ¢-butanamine nitrate, 4-methylphenol, ¢-propylguanidine nitrate, which are analogs of the side-chains of Ser, Asp, Glu, Ash, Gin, Lys, Lys +, Tyr, Arg +, respectively. Methanol, acetic acid, propanoic acid, acetamide, n-propanamide, n-butanamine, n-butanamine nitrate, 4-methylphenol and nitric acid were purified according to the methods described by Perrin et al. (1980). Nitrate of n-propylguanidine was synthesized as described by Pievano (1928). Aqueous solutions of these compounds were prepared by mixing the weighed components, i.e. the substance and distilled water, degassed by boiling. The pH values of the solutions were not controlled. Experiments were carried out on solutions with concentrations from 0"3 to 1"5% (w/v), except for 4-methylphenol, which was studied only at 0"2% concentration because of its limited solubility in water. The concentration of n-butylamine nitrate solution was determined by Elemental Analyzer 240-B (Perkin-Elmer, U.S.A.). Homopeptides Glyn with n = 3, 4, 5 and tripeptides Gly-X-Gly with X = Gly, Pro, His, Met were used in the studies. The compounds Gly3, Gly4, Gly s (Reanal, Hungary) were recrystallized twice from the water/ethanol mixture. The monomer units of. the peptides Gly-Pro-Gly, GlyMet-Gly, Gly-His-Gly were built in the conventional stepwise manner using pentafluorophenyl esters of protected amino acids as described (Kisfaludy et al., 1973). Protected amino acids were obtained from Reanal, Hungary. The C-terminal amino acid was protected as the benzyl ester (Bergmann et al., 1933) (Gly-His-Gly) or phenacyl ester (Stelakatos et al., 1966) (the rest peptides) through the synthesis. The deprotection of the amino terminus of the growing peptide chain was carried out by cleaving the Boc group with 4 M-HC1/dioxane and the benzyloxycarbonyl group by hydrogenation (Willstatter & Waldschmidt-Leitz, 1921). The reductive cleavage of the OPhac group was made with Zn dust in 50% acetic acid/water (Hendrickson & Kandall, 1970) or by means of hydrogenation (Willstatter & Waldsehmidt-Leitz, 1921). Hydrogenation was used for reductive cleavage of the benzyl ester group and 4 M-HCl/dioxane for tret-butyltype protecting groups. The conversion was monitored by means of thin-layer chromatography on commercial pre-coated silica gel plates (Silica Gel 60 F-254 from Merck, W. Germany). The ratio of the solvent mixture chloroform/methanol was 9 : 1 (v/v). The purification of protected peptides was made by means of column chromatography on Silica gel L 40/100 (Chemapol, Czechoslovakia). The purity of the peptides after deblocking was checked by paper electrophoresis in 30% CH3COOH. Solutions of peptides were prepared by weighing the components and using double-distilled degassed water, and passing through a Sephadex G-10 (Pharmacia) column (3 cm x 100 cm) equilibrated with 5% CH3COOH, followed by lyophilization and chromatography on the same column equilibrated with 0"5 M-Na acetate/acetic acid buffer at pH 4"0. The concentrations of the peptides in solutions were determined from glycine content after 24 h hydrolysis in 6M-HCI using a Durrum D500 (U.S.A.) amino acid analyzer. The concentrations used for the calorimetric measurements varied from 3 to 9 mg/ml.
Heat Capacity of Proteins
377
Table 1
Partial molar heat capacities COp,÷ (J K-1 tool-1) for some amino acid side-chain analogs in aqueous solution Temperature (°C) Substance Methanol Acetic acid Propanoic acid Acetamide n-Propanamide 4-Methylphenol n-Butanamine n-Butanamine nitrate n-Propylguanidine nitrate Nitric acid n-Butanamine (ionized) n-Propylguanidine (ionized)
5
25
50
75
100
125
155"2 155'1 250.6 155"2 250"3 392.9 411"1 297"4 286"9 -95"3 392-7 382"2
158"3 167"0 257"0 166"8 258-2 379"7 410"5 327"8 351"4 -44.2 372.0 395"6
159"1 177"9 263"7 181.5 265"1 366"8 405"7 338'6 377"5 - 18"5 357"1 396"0
160.4 190.9 270"1 191.6 269"7 360"9 403"9 340"8 381-5 - 15-1 355"9 396"6
162"9 200.4 271"1 200"2 270"5 359'8 399"3 337"8 378"4 - 15-4 353"2 393"8
165"6 209.7 271"2 207"6 272-1 357"4 397"0 328.3 372"4 - 19"0 347"3 391"4
3.2 6"0 7"4 5"9 7"3 10"6 7'3 7"4 7"1 4"7 l 1-7 11-8
a, standard deviation. The apparent molar heat capacity Cp, ~t of the solute at temperature T was determined by measuring the apparent heat capacity difference ACp.¢ between the solution and solvent at this temperature by a precise scanning microcalorimeter DASM-4 (Special Construction Bureau of Biological Instrumentation of the Academy of Sciences of the U.S.S.R.) with a fixed operational volume of calorimetric cells, using the equation (Privalov & Potekhin, 1986): C p . , ~- Cp. 1 V~] V 1 - MACp. ¢/m,
(1)
where Cp. 1 and V1 are the molar heat capacity and molar volume of water, respectively (Weast, 1970), V~ is the partial molar volume of the solute, m is the mass of the solute in the calorimetric cell, M is the molar mass. All experiments were carried with the heating rate 1 K min-l, which is optimal for the instrument used. In the range of the concentrations employed no dependence of the apparent molar heat capacity on concentration has been found. This permits one to consider the values determined for Cp,¢ as corresponding to infinite dilution, i.e. Cp,~ = C~.,. The apparent molar volume of the studied compounds in water, V~, was determined from the density, p, of the solution and the density, Po, of the solvent, according to Freadman & Scheraga (1965), by the equation: V~ =
M
1000. (p-po)
p
c'p'po
(2)
where c is the molality of the solution. The density was measured by a precise vibrational densimeter DMA-60 (Anton Paar, Austria) at a fixed temperature (+0"01 K) using water and air as a standard for calibration. The partial molar volume at infinite dilution, V$, was obtained by linear extrapolation of the apparent molar volumes, Vv, to zero concentration. Measurements have t Abbreviations used: C~.~, partial molar heat capacity of a solute; ASA, water-accessible surface area; V~, partial molar volume of a solute; Cp, 1, molar heat capacity of a solvent; m, mass of a solute; C~,¢ ( - R), partial molar heat capacity of the amino acid side-chain in aqueous solution; ACp,i, hydration heat capacity per a given type of the ASA unit; C~, molar heat capacity in the gaseous phase.
been done for the temperature range 5 to 85°C, with a 10-deg. spacing. At higher temperatures (above 85°C) extrapolated values were used. The details of the partial molar volume determinations in a broad temperature range are considered elsewhere (Makhatadze et al., 1990).
3. Results (a) Partial molar heat capacity of simple organic
compounds and peptides The partial molar heat capacity of the simple organic solutes determined from our calorimetric experiments in the temperature range 5 to 125°C are given in Table 1. The partial molar heat capacity of n-propylguanidine and n-butylamine ions were calculated from the Cp,~ values of the corresponding nitrates and t h a t of nitric acid. The values calculated in this w a y are also listed in Table 1. The partial molar heat capacities of the studied compounds for 25°C reported in the literature are listed in T a b l e 2 , together with our data. The observed good correspondence between our and published d a t a at 25 °C permits one to suppose t h a t our d a t a are reliable for all other temperatures as well. The partial molar h e a t capacity of the - C H 2group was calculated from the partial molar heat capacity of homologous substances (i.e. acetic acid and propanoic acid, acetamide and n-propanamide). The values obtained, given in Table 3, are in good agreement with those reported for the homologous series of alcohols (Makhatadze & Privalov, 1989). This means t h a t the partial heat capacity of the - C H 2- group at all temperatures in the studied region is not affected by the presence of the polar group. The partial molar heat capacity of the - C H a group at 25°C was obtained b y addition of the heat capacity of the hydrogen a t o m to the heat capacity of the - C H 2- group and b y subtraction of the hydrogen a t o m heat capacity from the k n o w n h e a t
G. I. Makhatadze and P. L. Privalov
378
2 0 0 | (a)
Table 2
Comparison of the partial molar heat capacities of some compounds at 25 °C obtained in this study with literature values Cp.~b/JK -1 mol-I Compound
This work
Methanol Acetic acid Propanoic acid Aeetamide n-Propanamide n-Butanamine 4-Methylphenol
158.3_.+3-2 167"0__+6'0 257"0-+7"4 166-8-+5-9 258.2_+7-3 410"5_+7-3 379"7-+ 10"9 175.3_+5-0 264'5 -+6-2 361 '7 _+7-4 93"2_+6-2
Triglycine
Tetraglycine Pentaglycine -CH2CONH-
I001- Ib)
Literature 158.2_+0.1= 165_+3b 253-+ 3b 162-4_+1~ 253"6_+1: 422__.4 b 384 _ 2d 189.5+0.9 r 283 -+2f 373 _+6f 98___5f, 128-+21h 106-+ 10 ~, 50_+21s
0
25
50
75
I00
t25
Temperoture (°C)
References: "Jolicoeur & Lacroix (1976), bKonicek & Wads5 {1971). =Roux et al. (1978), dNichols & Wads5 (1975), =Cabani et a/. (1977), tjolicoeur & Boileau (1978), gBello (1978), hKreshek & Benjamin (1964).
F i g u r e I. Temperature dependence of the partial molar heat capacity of: (a)-CHa, (b)-CH2-, (c)-OH, (d)-COOH, (e)-CONH 2, (f)-NHz, ( g ) - N H C N H N H ~ groups calculated from the heat capacity values of mode[ compounds. For errors see Table 3.
c a p a c i t y of m e t h a n e , 244.7 J K - z t o o l - z (Naghibi et
al., 1986). T h e heat c a p a c i t y of the H a t o m at 25°C according to G u t h r i e (1977) is 7 5 J K - l m o 1 - 1 , while, according to Lilley's calorimetric s t u d y of a m i n o acid amides and peptides (personal c o m m u n i cation), it is 78"6 J K -1 tool - l . Close values were o b t a i n e d b y Nichols et al. (1976) a n d R o u x et al. (1978), 6 7 J K -1 tool -1 and 9 0 J K - t mol - t , respectively. I n our calculations we h a v e t a k e n the m e a n value o f all these, 78 J K - 1 m o l - 1. T h e n for the h e a t c a p a c i t y of the - C H s g r o u p at 25°C we get 1 6 9 " 2 J K -~ mol -~ f r o m the h e a t c a p a c i t y of the - C H 2- g r o u p a n d 166-7 J K -1 mol -~ f r o m the h e a t c a p a c i t y o f t h e CH 4 group. T h e c o r r e s p o n d e n c e of these two values shows t h a t the h e a t c a p a c i t y 78 J K - 1 m o l - z for the h y d r o g e n a t o m is correct. F o r the calculation o f the t e m p e r a t u r e d e p e n d e n c e
o f the h e a t c a p a c i t y of the - C H 3 g r o u p one can use the fact t h a t t h e h e a t c a p a c i t y o f a n o n - p o l a r g r o u p is p r o p o r t i o n a l to its size (Naghibi et al., 1986, 1987a,b), i.e. t h a t the ratio: O
O
Cp, ~(-CH3)/Cp, ,~(--CH2- ) -- c o n s t
is valid a t a n y t e m p e r a t u r e . T h e t e m p e r a t u r e d e p e n d e n c e o f t h e partial m o l a r h e a t c a p a c i t y per - C H 3 g r o u p o b t a i n e d in this w a y is p r e s e n t e d in Table 3. F r o m the difference in the slopes o f these f u n c t i o n s (Fig. 1) we d e t e r m i n e d also the t e m p e r a t u r e d e p e n d e n c e o f the h y d r o g e n h e a t c a p a c i t y . U s i n g the partial m o l a r h e a t c a p a c i t y o f the - C H 2 - a n d - C H s groups, the h e a t c a p a c i t y of -COOH,-CONH2,-NH2, NH~" a n d - N H C N H N H ~ g r o u p s can be easily o b t a i n e d b y a g r o u p a d d i t i v i t y
Table 3
Partial molar heat capacity (C°p,~) per -CH2-, -OH, - C H 3, -COOH, - C O N H 2, - N H 2, N H J and - N H C N H N H J groups at various temperatures Temperature (°C) Group
5
25
50
75
100
125
a
95.1 95-5 97"9 96"2 178"5
91"4 90'0 92"3 91.2 169"2
83-6 85"8 82.3 83"9 155"6
78"1 79"2 75"9 77"7 144-1
70"3 70"7 68"3 69-8 129"5
64.5 61.5 63"1 63"0 116-9
9'5 9'4 8-1 9'0 12"1
-OH -COOH -CONH2
-5"3 - 23"8 --23.9
-3"6 - 2-8 --2.3
13"7 23'3
30-4 47-6
58"0 71-4
89"6 92" 1
8-1 9'4
-NH~ -NHz -NHCNHNH~
-74"4 --56"0 -70"8
--70"8 -32"3 -34"0
25.8 -50'2 1"6 0"9
48"7 21"3 26"7 30"7
71"0 14"3 60"4 65"0
91"5 41"4 91"1 94"6
9"5 8"8 7"3 8"5
-CH 2- amides -CHz- acids - C H 2- alcohols=
-CH 2- (mean) -CH3
All heat capacity values are given in J K -1 tool -1 . • Calculated from the data reported by Makhatadze & Privalov (1989).
Heat Capacity of Proteins
379
Table 4
Temperature dependence of the partial molar heat capacity (C°p,~) of some peptides in aqueous solution Temperature (°C) Peptide Gly-Gly-Gly" Gly-Gly-Gly-Gly~ Gly-Gly-Gly-Gly-Gly~ Gly-Gly-Glyb Gly-His-Gly b Gly-Pro-Gly b Gly-Met-Gly b
5
25
50
75
100
125
a
98.2 182-8 271.3 93.1 216.5 161.4 207"9
175-3 264.6 361.7 194.6 296.2 230'3 292.5
239.5 338.2 430.9 275.7 381.2 292"3 362"1
285.1 382.3 473.5 348.9 462.1 361"3 432"8
322.7 412.0 497.1 413"1 540.5 425"0 503-5
360.6 438.5 520.4 474.2 617.6 486.9 564.7
5.0 6.2 7.4 9.1 13.6 11"0 9"0
All heat capacity values are given in J K -I mol -I. a In pure water as a solvent. b In 0"5 M - N a C H 3 C 0 0 - C H 3 C O O H (pH 4"0) buffer solution; a, is standard deviation.
scheme. In the case of the -COOH group the temperature-dependent dissociation has not been taken into account. Temperature dependences of these values are presented in Table 3 and Figure 1. It is notable that the partial heat capacity of the non-polar groups - C H 2- and - C H 3 is positive and decreases with increasing temperature, while the partial heat capacity of the polar groups, -COOH, -CONH2, -NH2 and - N H C N H N H ~ , is negative at room temperature and increases with temperature. The other notable fact is that the values of the partial molar heat capacity o f - C O O H and -CONH 2 are very similar at all studied temperatures although the partial heat capacity of the - O H and - N H 2 groups differ significantly. One can conclude from this that there is a strong interaction between the - C O - group and - O H or -NH2 groups. It should be noted that the heat capacity of all the above mentioned groups at 25°C are in good agreement with the values obtained earlier (Guthrie, 1977; Nichols et al., 1976; Roux et al., 1978). The main importance of our data in this respect is that they were obtained not only at standard temperature (25°C) but in a broad temperature range. The partial molar heat capacities of the studied peptides at different temperatures are given in Table 4. Table 2 shows a comparison of the partial molar heat capacities for Gly 3, Gly 4, Gly 5 at 25°C with available literature data. As seen, the values of C~, for peptides determined in this work agree with those obtained earlier within the limits of error of determination.
(b) Heat capacity of the protein constituent groups All the studied simple organic compounds are analogs of the side-chains of various amino acids differing only by an extra hydrogen atom. Therefore, the heat capacity of the amino acid side-chain, C~.~(-R), can be calculated from the heat capacity of the analogs, C~,,(A), by extracting the heat capacity of the hydrogen atom, C~,,( - H):
C~,A-R) = C~,AA)--V~.A-H).
(3)
Using the above heat capacity function of C~. ~ ( - H) {which is in itself the heat capacity of the Gly sidechain) and the heat capacity of the analogs, we calculate the heat capacity of the following amino acid side-chains: Ser, Asp, Glu, Asn, Gin, Lys, Tyr and Arg, which are listed in Table 5. The Table also presents the heat capacities of the Thr and Phe sidechains calculated from the heat capacities of their analogs, ethanol and toluene, which were determined earlier (Makhatadze & Privalov, 1988, 1 9 8 9 ) . Comparison of the heat capacities of the Lys sidechain in ionized and non-ionized forms, shows that there is a significant decrease of the heat capacity due to ionization. At 25°C it amounts to 38"5 J K -1 mo1-1, which significantly exceeds the value 14"9 J K - 1 mol- 1 obtained earlier by Riedl & Jolicoeur (1984). With increasing temperature the heat capacity effect of ionization increases but non-linearly. Unfortunately the scanning microcalorimetric technique cannot be used for the determination of the heat capacity of the side-chains of the amino acids Ala, Val, Leu and Ile. The analogs of these side-chains, which are methane, propane, n-butane and /-butane, have too low solubility in water to measure their heat capacities with sufficient accuracy even by the most precise contemporary scanning microcalorimetric instrument. For these substances the partial molar heat capacity in aqueous solution was determined by Naghibi et al. (1986, 1987a,b) in the temperature range from 0 to 50°C using a flow microcalorimetric technique. Assuming that above 50°C the temperature dependence of the partial molar heat capacity is the same as it is below this temperature, one can calculate by equation (3) the partial molar heat capacity of the side-chain of the above amino acids. The obtained values are given in Table 5. From the obtained data on the partial molar heat capacity of tripeptides and homopeptides we can determine the partial molar heat capacity of the internal amino acid residues, the peptide unit - C H z C O N H - and the corresponding side-chains. From the linear dependence of the partial molar heat capacity of homopeptides on the number of
-
~
o~
~
~
~
~
~
~
~
~
~.
~
~
o.
I
I
I
I
I
~'.
.
Heat Capacity of Proteins
381
Table 5 (continued) Side-chain of amino acid residue Heminec
Temperature (°C) ASA"p (A 2)
ASA ~1 (A2)
--
--
C~. ~ A~Cp
5
25
50
75
621 499
600 467
575 432
553 398
100 530 367
125 512 338
All heat capacity values are given in J K-1 mol-l. C~. ~ and A~'Cp values are precise within 5 %, C~ within 0"5 %. Water-accessible surface areas were taken from Miller et al. {1987) and are precise within l0 to 20% . "Total ASA. b Side-chain in a nitrate form. c Side-chain in an ionized form. d These values refer to the modified cysteine residue Cys-CH2CONH 2. The heat capacity of this residue at 25°C was calculated by the equation: C;.,(Cys-CH2CONH2) = C;. ,(CysH) - C;. ,(Gly) + C;. ,(CH2CONH2). The values for C~.÷(CysH) and C~.~(Gty) were taken from Joticoeur et al. {1986). The temperature dependence of this modified amino acid side-chain was supposed to be the same as that for the Glu side-chain. " Partial molar heat capacity of hemine at 25°C was calculated from the difference of the partial molar heat capacities of myoglobin and apomyoglobin taken from Privalov et al. (1989}. Temperature dependence of the Up.$ function was obtained from the partial molar heat capacities of toluene (Makhatadze & Privalov, 1988) assuming that the ratio C~.,(hemine)/C~. ¢(toluene) ffi 1-3 is valid for the whole temperature range from 5 to 125°C. The temperature dependence of the hydration heat capacity increment of heroine was obtained by comparison with the A~Cp values for toluene (Makhat~ize & Privalov, 1988).
Gly units, N~ly, it follows that at any temperature studied the glycyl residue contributes additively to the heat capacity of the peptide molecule. The slope of the plot of C;.e versus NGly at a given temperature corresponds to the heat capacity contribution of a single glycyl residue. Table 2 shows the comparison of the partial molar heat capacity of -CH2CONH- obtained in this way with the literature data at 25°C. As seen, our value 93.2(_+6-2) agrees within experimental error with those obtained by Jolicoeur & Boileau (1978) 98{_+5) and Bello (1978) 106(_+10), can be compared with the data of Kresheck & Benjamin (1964) 128(_+25) and differs significantly from that obtained by Cabani et al. (1977) 50(+21). From the determined temperature dependence of the heat capacity of the -CH2CONH- group one can easily obtain the heat capacity o f - C H C O N H - :
The heat capacity contributions of the amino acid side-chains are listed in Table 5. So, almost all values listed in Table 5, except for side-chains of Trp, were determined by calorimetric studies of the model compounds and tripeptides. In all cases the standard deviation of these determinations does not exceed 5%. The partial molar heat capacity of the Trp sidechain at 25°C was calculated from the calorimetric data obtained by Jolicoeur et al. (1986). The temperature dependence of its heat capacity was assumed to be the same as that for Phe, another aromatic side-chain that we have studied (Makhatadze & Privalov, 1989). 4. Discussion
(a) Temperature dependence of the partial heat
capacity
C~,,(-CHCONH-) = C;.e(-CH2CONH-)-C~.e(-H).
(4)
The partial molar heat capacities of the - C H C O N H - group calculated by equation (4) are listed in Table 5. Additivity of the heat capacity contribution of the peptide groups permits one to calculate the sum of the heat capacity contributions of the - N H 2 and -CHCOOH groups. The values obtained for these groups from the data for the glycine homopeptides are given in Table 5 for the temperatures studied. From the partial molar heat capacity of tripeptides we calculated the heat capacity contribution of the amino acid side-chains, C;, e ( - R ) , as: C~. ~( - R) = C~. ~(Gly-CH-Gly)
As seen from Table 5, the temperature dependence of the heat capacity of various amino acid side-chains is very different: the values decrease with increasing temperature for non-polar sidechains of Pro, Met. Trp, Ala, Val, Leu, Ile, Phe and Gly. They increase for such polar side-chains as those of Ser, Asp, Asn, Gin, Glu, Lys, Arg. As for the His, Thr and Tyr side-chains, the dependence is rather complicated and actually represents a superposition of temperature dependences specific for both the non-polar and polar groups. Therefore, it looks as though one can use the temperature dependences of the partial heat capacity for classifying the amino acids according to their polarity or hydropathy. The amino acid side-chains can be regarded as:
I
E -C;, AGIy-CH-Gly)+ C;.,(-H).
I
H
Hydrophobic if (5)
oc;.,
Hydrophilic if ~
< 0 in 5 to 125°C > 0 in 5 to 125°C
(6) (7)
G. I. Makhatadze and P. L. Privalov
382
value at 25°C: a smaller value will mean a more hydrophilic residue. In order to increase the hydrophilicity, amino acid side-chains are listed in Table 6. I n this Table the proposed h y d r o p a t h y scale is c o m p a r e d with those reported in the literature, (Bull & Breese, 1974; Chothia, 1976; M a n a n v a l a n & P o n n u s w a m y , 1978; H o p p & Woods, 1981; Janin, 1979; Levitt, 1976; P a r k e r et al., 1986). According to the calculated correlation coefficients there is the best correlation with the scale of P a r k e r et al. (1986), 0.92. However, even in this case the main exceptions are non-polar Gly and a m b i v a l e n t Tyr.
Table 6
Comparison of the hydropathy scale derived in this work with those obtained earlier Side-chain
Hydropathy scale according to various authors:
of amino acid residue
a
b
c
d
e
f
g
h
Trp Phe Ile Leu Val Pro Met Ala Gly Tyr Thr His Lys Arg Glu Gin Asp Asn Ser
l 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19
5 2 3 l 6 8 7 13 17 4 9 15 11 16 12 19 14 18 10
8 3 1 4 2 11 5 6 7 14 9 13 18 19 12 17 15 16 10
l 3 2 6 7 4 8 I0 15 5 17 II 9 12 13 19 14 16 18
8 4 1 3 2 12 5 7 6 ll 13 9 19 18 17 16 15 14 10
1 2 5 4 6 11 7 8 12 3 10 9 19 18 17 14 16 13 15
1 2 4 5 6 7 8 9 12 3 11 10 19 18 17 14 16 13 15
1 2 4 3 6 9 5 8 14 7 12 10 13 11 18 15 19 17 16
(b) Correlation between water-accessible surface area
and partial heat capacity of the amino acid side -chains I t is known from the calorimetric studies (see e.g. Gill & WadsS, 1976; Gill et al., 1976; Olofsson et al., 1984; Dec & Gill, 1984, 1985a,b) t h a t the partial molar heat c a p a c i t y of h y d r o c a r b o n s in aqueous solution is proportional to their water-accessible surface area (ASA). Therefore one is interested in possible correlations between the heat c a p a c i t y of amino acid side-chains and their non-polar wateraccessible surface area. A plot of the observed C ~ . ~ ( - R ) values at 25°C versus non-polar ASA values t a k e n from Miller et al. (1987) is presented in Figure 2. One notices two distinct correlations for these compounds. The first of these groups includes the ring-containing amino acid side-chains (Pro, His, Tyr, Phe} and the sulfur-containing Met. The other group includes linear amino acid side-chains. The correlation within each of these groups is r a t h e r good and does not exceed the error in the determination of the ASA values, which is, according to Miller et al. (1987), within 10 to 2 0 % for an individual amino acid residue. I t is notable t h a t a good correlation between the non-polar ASA and C ~ , ~ ( - R ) values is observed only at.25°C, while for higher t e m p e r a t u r e s it significantly decreases for the
Correlation coefficient
1"00 0"75 0"76 0-86 0"75 0'82 0"85 0.92
'This work; bBull and Breese (1974), :Chothia (1976), dMananvalan & Ponnuswamy (1978), "Janin (1979), rHopp & Woods (1981), BLevitt (1976}. hparker et al. (1986).
(8)
A m b i v a l e n t ff 6C~. ~ = 0 in 50 to 75 °C
aT
According to this criterion, one should consider the side-chains of Ala, Val, Leu, Ile, Phe, Trp, Pro, Met, Gly as hydrophobic and of Ser, Asn, Asp, Glu, Gin, Lys, Arg as hydrophilic; while the side-chains of Thr, T y r and His, which show the m i n i m u m heat capacity at a b o u t 60°C, a p p e a r to be ambivalent. Inside each group defined b y equations (6) to (8) the relative h y d r o p a t h y of the side-chains of amino acid residues can be defined using the C~,,~,(--R,)
500 125°C
L~/
400
L, y
I1~,~/
/
/~Phe
L6 E T 300
•
eo/
Arg[]
/2
le
Phe
~200
IOO
I
I 50
I
I I O0
t
I 150
t
I
200
I
I
50
t
I
100
I
I
150
i
I
200
Non-polor ASA (~2)
Figul~ 2. Correlation between the heat capacity of the amino acid side-chains C~, ¢ ( - R) and non-polar ASA at various temperatures. The lines are drawn by least-squares calculations using the C~, ¢ ( - R) and ASA "p values for Ala, Val, Leu, Ile (line 1) and Pro, Phe, Trp (line 2). The ASAnp values are taken from Miller et al. (1987).
Heat Capacity of Proteins
383
Table 7
Hydration heat capacity, ACp,~, per unit of surface-accessible area for various constituent groups of protein Group or side-chain of amino acid residue
Aliphatic Aromatic -CHCONH-CH2CONHPolar parts of: Met His Ser Asn Asp Gin Glu Lys Tyr Art Thr Trp
Temperature (*C) 5
25
50
75
100
125
2"23 1.32 - 1-16 0"32
2.13 1"20 - 0"97 0"37
2'00 - 0"80 0"38
1"88 1'02 - 0"79 0.44
1.77 0'95 - 0'78 0.25
1-64 0"88 - 0"86 0"14
--4-03 -0"96 - 1"60 - 1"29 - 1"74 -- 0"39 - 0'73 - 1'32 -0'06 -0'39 - l'13 3"87
--3-66 -- 1-36 - 1"43 -- 1"02 - 1"42 - 0"24 - 0"56 - 1"53 0'0 -0'20 - 1"32 3'94
-3"I9 -- 1.42 - 1"20 -0"67 - 1-07 - 0"06 - 0"35 -- 1"54 -0'02 -0.11 -- 1"19 3"72
-2-75 -- 1-36 - 0-95 --0"41 -0"71 0"08 - 0"16 - 1.30 0"09 -0"03 --0"85 3"53
--2"33 -- 1-23 - 0'71 --0.15 --0"39 0"18 - 0"03 -- 1"10 0'27 0"03 -0"25 3"27
--1"86 -- 1"06 - 0-47 0"09 --0"11 0"30 0'09 --0'90 0'43 0'09 0"59 3"05
1-11
All heat capacity values are given in J K - t mol -I A -2 and are precise within 20%.
side-chains that are not completely non-polar (Fig. 2). This is u n d e r s t a n d a b l e since w i t h increasing temperature the heat capacity contribut i o n o f t h e p o l a r p a r t i n c r e a s e s , while t h e c o n t r i b u t i o n o f t h e n o n - p o l a r p a r t o f t h e a m i n o a c i d sidechain decreases.
(c)
=
g i -Cp,
Aw~np i"
(10)
--gvp,
=
-np A S A inp "ACp, i.
(11)
C o r r e s p o n d i n g l y , for t h e r e d u c e d h y d r a t i o n effect o f t h e p o l a r g r o u p we h a v e : AW~pOl A(~,poi __ --gvp. i -- v p.. ASA•Ol
(9)
T h e h e a t c a p a c i t y o f a s m a l l o r g a n i c m o l e c u l e in t h e gaseous phase could be easily calculated from the known heat capacities of the constituent groups (Benson, 1968), as t h e h e a t c a p a c i t i e s o f o r g a n i c c o m p o u n d s i n t h e g a s e o u s p h a s e a r e u s u a l l y close to the sum of the heat capacities of their components. The values of the hydration heat capacities of amino a c i d s i d e - c h a i n s a r e l i s t e d in T a b l e 5 t o g e t h e r w i t h t h e c o r r e s p o n d i n g v a l u e s in t h e g a s e o u s p h a s e . I t is k n o w n t h a t for a n o n - p o l a r s u b s t a n c e , t h e h y d r a t i o n h e a t c a p a c i t y effect is p r o p o r t i o n a l t o t h e water-accessible surface area of the substance ( N a g h i b i et al., 1986, 1987a,b). W e c a n o b s e r v e t h i s p r o p o r t i o n a l i t y in T a b l e 5 for t h e n o n - p o l a r a m i n o a c i d s i d e - c h a i n s . T h e r a t i o o f A~Cp. i / A S A i = A C-"P p . i, w h i c h is t h e h y d r a t i o n h e a t c a p a c i t y effect, r e d u c e d t o s u r f a c e u n i t s , is s i m i l a r for v a r i o u s t y p e s o f n o n p o l a r g r o u p s , differing s l i g h t l y for a l i p h a t i c a n d r i n g c o m p o u n d s ( M a k h a t a d z e & P r i v a l o v , 1989). T o e s t i m a t e t h e h y d r a t i o n effect o f s i d e - c h a i n s c o n t a i n i n g p o l a r p a r t s , o n e s h o u l d first o f all
o
C p , ~! - -
If the water-accessible surface area of the non-polar p a r t , A S A ~ p, is k n o w n , t h e n t h e h y d r a t i o n effect o f t h e n o n - p o l a r p a r t c a n b e d e r i v e d as: g~p,i
T h e h y d r a t i o n effect o f h e a t c a p a c i t y is t h e difference b e t w e e n t h e h e a t c a p a c i t y o f a s o l u t e m o l e c u l e in t h e g a s e o u s p h a s e , Cgp, a n d i t s p a r t i a l h e a t c a p a c i t y in a q u e o u s s o l u t i o n ( P r i v a l o v & Gill, 1989): Cp,~p--Cp. ° '
AW~pOl gvp, i
Awf?np
Hydration heat capacity effect
a c. =
s e p a r a t e t h e effect o f t h e n o n - p o l a r p a r t o f a sidec h a i n f r o m t h e effect o f i t s p o l a r p a r t . T h e l a t t e r is given by:
o
=
g
•
np.
Cp. 4,,- CD, i - A S A i ASApOt
--rip
A Cp, i (12)
W a t e r - a c c e s s i b l e s u r f a c e a r e a v a l u e s for t h e n o n polar and polar parts of the amino acid side-chains g i v e n in T a b l e 5 w e r e t a k e n f r o m M i l l e r et al. (1987). T h e ACp p v a l u e s for a r o m a t i c s i d e - c h a i n s (Trp, T y r , His) were taken from Makhatadze & Privalov (1988), a n d for a l i p h a t i c s i d e - c h a i n s t h e y w e r e c a l c u l a t e d a s t h e m e a n v a l u e o f ACp p for A l a , Val, Leu and Ile side-chains. The obtained values of the hydration heat capacities reduced to surface area a r e l i s t e d in T a b l e 7. A s c a n b e seen in o u r a c c o m p a n y i n g p a p e r ( P r i v a l o v & M a k h a t a d z e , 1990), t h e s e v a l u e s p e r m i t us t o e s t i m a t e t h e h y d r a t i o n effect o f p r o t e i n u n f o l d i n g . We are grateful to Dr T . H . Lilley for sending us unpublished results of his heat capacity studies on a m i n o acid amides and peptides, to Dr K . H . Zikherman for synthesis of n-propylguanidine nitrate, to Dr V . N . Medvedkin for the help in peptide synthesis.
384
G. I. Makhatadze and P. L. Privalov
References Bello, J. (1978). J. Phys. Chem. 82, 1607-1609. Benson, S. W. (1968). Thermochemical Kinetics, John Wiley & Sons, Inc., New York, London, Sydney. Bergmann, M., Zarvas, L. & Salzmann, L. (1933). Chem. Bet. 66, 1288-1290. Bull, H. B. & Breese, K. (1974). Arch. Biochem. Biophys. 161,665-670. Cabani, S., Conti, G. & Matteoli, E. (1977). Biopolymers, 16, 465-467. Chothia, C. (1976). J. Mol. Biol. 105, 1-14. Dec, S. F. & Gill, S. J. (1984). J. Solution Chem. 13, 27-41. Dec, S. F. & Gill, S. J. (1985a). J. Solution Chem. 14, 417-429. Dec, S. F. & Gill, S. J. (1985b). J. Solution Chem. 14, 827-836. Freadman, M. E. & Scheraga, H. S. (1965). J. Phys. Chem. 69, 3795-3800. Gill, S. J. & WadsS, I. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 2955-2958. Gill, S. J., Nichols, N. F. & WadsS, I. (1976). J. Chem. Thermodynam. 8, 445-452. Guthrie, J. P. (1977). Can. J. Chem. 55, 3700-3706. Hendrickson, J. B. & Kandall, C. (1970). Tetrahedron Letters, 5, 343-344. Hopp, T. P. & Woods, K: 1%. (1981). Proc. Nat. Acad. Sci., U.S.A. 78, 3824-3828. Janin, J. (1979). Nature (London), 277, 491-492. Jolicouer, C. & Boileau, J. {1978). Can. J. Chem. 56, 2707-2713. Jolicoeur, C. & Lacroix, G. (1976). Can. J. Chem. 54, 624-628. Jolicoeur, C., 1%iedl, B., Desrochers, D., Lemelin, L.L., Zamojska, R. & Enea, O. (1986). J. Solution Chem. 15, 109-128. Kisfaludy, L., Low, M., Nyeki, 0., Szirtes, T. & Schon, I. (1973). Lieb. Ann. Chem. 9, 1421-1429. Konicek, J. & WadsS, I. ~I. (1971). Acta Chem. Scand. 25, I541-1551. Kresheck, G. C. & Benjamin, L. (1964). J. Phys. Chem. 68, 2476-2486. Levitt, M. (1976). J. Mol. Biol. 104, 59-107. Makhatadze, G. I. & Privalov, P. L. (1988). J. Chem. Themodynam. 20, 405-412. Makhatadze, G. I. & Privalov, P. L. (1989). J. Solution Chem. 18, 927-937. Makhatadze, G. I., Medvedkin, V. N. & Privalov, P.L. (1990). Biopolymers, in the press. Mananvalan, P. & Ponnuswamy, P. K. (1978). Nature (London), 275, 673-674.
Miller, S., Janin, J., Lesk, A. M. & Chothia, C. (1987). J. Mol. Biol. 195, 641-656. Naghibi, H., Dec, S. F. & Gill, S. J. (1986). J. Phys. Chem. 90, 4621-4623. Maghibi, H., Dec, S. F. & Gill, S.J. (1987a). J. Phys. Chem. 91,245-248. Naghibi, H., Dec, S. F. & Gill, S. J. (1987b). J. Chem. Ing. Data, 32, 422-425. Nichols, N. & WadsS, I. (1975). J. Chem. Thermodynam. 7, 329-336. Nichols, N., ScSld, R., Spink, C., Suurkuusk, J. & WadsS, I. J. (1976). J. Chem. Thermodynam. 8, 1081-1093. Olofsson, G., Oshodj, A. A., Qvarnstr5m, E. & Wads5, I. (1984). J. Chem. Thermodynam. 16, 1041-1052. Parker, J. M. 1%., Guo, D. & Hodges, R.S. (1986). Biochemistry, 25, 5425-5432. Perrin, D. D., Armarego, W. L. F. & Perrin, D. R. (1980). Purification of Laboratory Compounds, Pergamon Press, Oxford, New York, Toronto, Sydney, Paris, Frankfurt. Pfeil, W. & Privalov, P. L. (1976). Biophys. Chem. 4, 33-40. Pievano, M. (1928). Gazz. Chim. Italiana, 58, 247-249. Privalov, P. L. (1979). Advan. Protein Chem. 33,167-241. Privaiov, P. L. & Gill, S. J. (1989). Advan. Protein Chem. 39, 191-234. Privalov, P. L. & Makhatadze, G. I. (1990). d. Mol. Biol. 213, 385-391. Privalov, P. L. & Potekhin, S. A. (1986). Methods Enzymol. 131, 4-51. Privalov, P. L., Tiktopulo, E. I., Venyaminov, S. Yu., Griko, Yu V., Makhatadze, G.I. & Khechinashvili, N. N. (1989). J. Mol. Biol. 205, 737-750. Riedl, B. & Jolicoeur, C. (1984). J. Phys. Chem. 88, 3348-3356. Roux, G., Perron, G. & Desnoyers, J. E. {1978). Can. J. Chem. 56, 2808-2814. Spink, C. H. & WadsS, I. (1975). J. Chem. Thermodynam. 7, 561-572. Stelakatos, G. S., Panganou, A. & Zarvas, L. (1966). J. Chem. Soc. 21, 1191-1199. Sturtevant, J. M. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 2236-2240. Suurkuusk, J. (1974). Acta Chem. Scand. B, 28, 409-417. Tanford, C. (1968). Advan. Protein Chem. 23, 121-275. Weast, 1%. W. (1970). Handbook of Chemistry & Physics, The Chemical 1%ubber Co., Cleveland, U.S.A. Willstatter, 1%. & Waldschmidt-Leitz, E. (1921). Chem. Bet. 54, 113-138.
Edited by R. Huber
