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Compatibility of osmolytes with Gibbs energy of stabilization of proteins
Biochimica et Biophysica Acta 1476 (2000) 75^84 www.elsevier.com/locate/bba
Compatibility of osmolytes with Gibbs energy of stabilization of proteins Farah Anjum, Vikas Rishi, Faizan Ahmad * Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110 025, India Received 5 July 1999; received in revised form 15 September 1999; accepted 28 September 1999
Abstract This study led to the conclusion that naturally occurring osmolytes which are known to protect proteins against denaturing stresses, do not perturb the Gibbs energy of stabilization of proteins at 25³C (vGD ³) which has been shown to control the in vivo rate of degradative protein turnover (Pace et al., Acta Biol. Med. Germ 40 (1981) 1385^1392). This conclusion has been reached from our studies of heat-induced denaturation of lysozyme, ribonuclease A, cytochrome c and myoglobin in the presence of different concentrations of osmolytes, namely, glycine, proline, sarcosine and glycine-betaine. At a fixed concentration of osmolyte a heat-induced denaturation curve measured by following changes in the molar absorption coefficient of the protein, was analyzed for Tm , the midpoint of the denaturation and vHm , the enthalpy change of denaturation at Tm . Values of vGD ³ were determined with Gibbs^Helmoltz equation using known values of Tm , vHm and vCp , the constant-pressure heat capacity change. It has been observed that Tm increases with the osmolyte concentration, whereas vGD ³ remains unaffected in the presence of the osmolyte. This observation on vGD ³ in the presence of osmolytes has been considered in the physiological context. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Osmolyte; Protein stability ; Lysozyme; Ribonuclease A; Cytochrome c; Myoglobin
1. Introduction Many microorganisms, ¢shes, plants and animals have adapted to harsh environmental conditions, such as water, salts, cold and heat stresses. It is well known that these organisms have adapted one common strategy in protecting their cellular proteins against these harsh environmental stresses [1,2]. This involves accumulation of low molecular weight organic compounds, collectively called osmolytes which fall into one of the three classes, namely, amino acids and their derivatives, polyols and methylamines [2^ 4]. These osmolytes are further classi¢ed as `compat-
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ible' or `counteracting' based on their e¡ect on the functional activity of proteins [2,3,5]. Compatible osmolytes are those which protect proteins against inactivation and denaturation without perturbing the protein functional activity near room temperature [5^ 8]. Counteracting osmolytes are those which are built up by organisms to cope with deleterious e¡ect of urea on proteins' functional activity and stability [9^11]. It is noteworthy that accumulation of compatible osmolytes (amino acids and their derivatives and polyols) occurs when organisms are under stresses such as water, extremes of temperature and salinity, and accumulation of counteracting osmolytes (methylamines) occurs when urea concentration in the cells of organisms is as high as 0.4^4 M [12]. A large body of data suggests that both compatible and counteracting osmolytes enhance thermal
0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 2 1 5 - 0
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stability of proteins [2,13^17]. The main factor responsible for this stabilizing e¡ect was disclosed by Timashe¡ and co-workers [13,14] and is related to the fact that these osmolytes are preferentially excluded from the protein domain, that is, they cause preferential hydration of the protein; this e¡ect should favor the protein state with the lower exposed surface, thus displacing the denaturation equilibrium, native (N) conformationHdenatured (D) conformation, towards the N state and bring about a higher stabilization of the N conformation in the presence of osmolytes. It is interesting to note that almost all studies used Tm (denaturation transition temperature) as an index of conformational stability (vGD ³, the Gibbs energy change of denaturation at 25³C) in the presence of an osmolyte. One potentially interesting aspect of this mechanism that has received little attention is the possible e¡ect of osmolytes on the denaturation Gibbs energy (vGD ³). The interest of probing this possible e¡ect is suggested by the two following facts. (a) Tm is not a measure of vGD ³. For example, lysozyme and cytochrome c have the same Tm of 80³C, but vGD ³ values are 16 and 8 kcal mol31 , respectively [18]. (b) There exists an inverse relation between vGD ³ and susceptibility to protein degradation [19,20]. If an osmolyte perturbs vGD ³, it then means that the degradative protein turnover rate will also be perturbed. This scenario will pose enormous problems for the organisms. In order to investigate the e¡ect of osmolytes on vGD ³, we have been carrying out systematic studies of denaturation of several proteins in the presence of di¡erent concentrations of both compatible and counteracting osmolytes. In this communication, we report an interesting observation that osmolytes do not signi¢cantly perturb vGD ³ values of proteins near room temperature suggesting that they do not alter the rate of degradative protein turnover. That is, osmolytes are compatible with protein stability (vGD ³). 2. Materials and methods Hen egg-white lysozyme (Lzm), ribonuclease A (RNase A), horse heart cytochrome c (cyt c) and horse heart myoglobin (Mb) were purchased from the Sigma. Electrophoretic homogeneity of each pro-
tein was checked on SDS-polyacrylamide gel electrophoresis according to the procedure described by Laemmli [21]. Since all proteins gave a single band, they were used without further puri¢cations. Osmolytes, namely, glycine (Gly), proline (Pro), sarcosine (Sar) and glycine-betaine (GB) were purchased from the Aldrich. Ultrapure guanidine hydrochloride (GdnHCl) was purchased from Schwarz/Mann Biotech. These and other chemicals were of analytical grade and were used without further puri¢cation. Stock solutions of Lzm, RNase A and those of oxidized Mb and cyt c were prepared in 0.1 M KCl (pH 7.0) as described earlier [22,23]. Concentrations of proteins were determined using molar absorption coe¤cient (M31 cm31 ) values of 39 000 at 280 nm for Lzm [24], 9800 at 277.5 nm for RNase A [25], 171 000 at 409 nm for Mb [26] and 10 700 at 530 nm for cyt c [27]. Concentration of GdnHCl stock solution was determined by refractometric measurements [28]. All solutions for optical measurements were prepared in degassed bu¡er containing 0.1 M KCl. Thermal denaturation studies were carried out in Jasco V-560 UV/Vis spectrophotometer with a heating rate of 1³C min31 using its peltier accessory (model ETC-505 temperature controller). The change in absorbance on heating was followed at 292 nm for Lzm, 287 nm for RNase A, 409 nm for Mb, and 530 nm for cyt c. About 350 data points were collected. Reversibility of each denaturation curve was checked as described earlier [29]. Lzm, Mb and cyt c near neutral pH have their Tm values near 80³C. In order to bring the heat-induced transition to a measurable temperature range, Lzm and Mb denaturation was studied in the presence of 1.60 M and 0.60 M GdnHCl, respectively, whereas the thermal denaturation of cyt c was studied at pH 3.0 at which it is already 25% acid denatured at 25³C. Each thermal transition curve was measured at least in triplicate, and analyzed for vGD (T), the Gibbs energy of denaturation at temperature T for a two-state denaturation, using the relation, y T3yN T v G D T 3RT ln 1 yD T3y T where y(T) is the observed optical property at temperature T, and yN and yD are, respectively, the optical properties of the native and denatured protein
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molecules at the same temperature at which y has been measured. The plot of vGD (T) values in the range 31.3 9 vGD (T), kcal mol31 9 1.3 versus corresponding T at constant concentration of an osmolyte was used to determine the values of Tm and vHm , the enthalpy change of denaturation at Tm according to the procedure reported earlier [16]. vGD ³, the value of vGD at T = 298.15 K was determined using the measured values of vHm , Tm , and vCp in the Gibbs^Helmoltz equation, T m 3T T 3v C p T m 3T T ln v GD v H m Tm Tm 2
3. Results and discussion In order to understand the question whether osmolytes are compatible with protein stability (vGD ³), de¢ned as the decrease in Gibbs energy of the denatured polypeptide when it folds to give the native functional protein under physiological conditions, usually taken as neutral dilute bu¡er and 25³C, we have been measuring the thermal denaturation curves of several proteins in the presence and absence of di¡erent osmolytes. Here we present and discuss the results of four proteins, namely, Lzm, RNase A, Mb and cyt c in the presence of di¡erent concentrations of two compatible osmolytes (Gly and Pro) and two counteracting osmolytes (Sar and GB). Panel A of Figs. 1^4 represents the typical denaturation behavior of proteins in the presence and absence of the osmolytes. The optical transition curves of each protein in the presence of di¡erent concentrations of an osmolyte were converted into stability curve [30] using Eq. 1 (e.g. see panel B of Figs. 1^4). Stability curves of a protein were analyzed for vHm and Tm values using the procedure developed earlier [16]. Values of these parameters of all proteins in di¡erent concentrations of osmolytes are given in Table 1. In order to determine vGD ³ values of proteins in the presence and absence of di¡erent concentrations of various osmolytes using Eq. 2, their vCp values at di¡erent concentrations of each osmolytes should also be known. The recommended procedure for the experimental determination of vCp involves the
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measurements of vHm and Tm from the heat-induced denaturation of the protein at several pH values, using di¡erential scanning calorimetry [18] or optical techniques [30]. We have measured thermal denaturation curves of one protein, namely, Mb in the presence of di¡erent ¢xed concentrations of each osmolyte at four di¡erent pH values. For each concentration of an osmolyte, vHm versus corresponding Tm plot was constructed. Such plots of Mb in the presence of Gly are shown in Fig. 1D (plots for other osmolytes not shown). Assuming that vCp is independent of temperature and pH between 20 and 80³C [17,18,30], values of vCp ( = DvHm /DTm ) were obtained from the least-squares analysis. These results are given in Table 2. It is seen in this table that vCp value remains, within experimental errors, unchanged in the presence of osmolytes in the concentration range 0^1 M. However, it has been assumed that vCp values of other proteins are independent of the presence of low osmolyte concentrations. The assumption that the low concentrations of osmolytes do not a¡ect the vCp of RNase A, Lzm and cyt c is based on the following observations. Di¡erential scanning calorimetric measurements of RNase A [17], Lzm [31] and cyt c (Ahmad and Pfeil, unpublished results) in the presence of up to 1 M of various osmolytes suggested that vCp of the protein is not signi¢cantly altered. Using vHm and Tm values given in Table 1 and vCp (kcal mol31 K31 ) values of 1.23 þ 0.12 for RNase A [18], 1.56 þ 0.20 for Lzm [18], 2.74 þ 0.16 for Mb ([18], this study) and 0.71 þ 0.03 for cyt c [16] measured in the absence of the osmolyte, we have calculated vGD ³ values of proteins using Eq. 2, which are given in Table 1. It is seen in Eq. 2 that, assuming no error in temperature, the error in the estimate of vGD ³ depends on the experimental errors in the vHm and vCp . For example, the value of vGD ³ of RNase A is 11.0 kcal mol31 . On incorporating errors in the vHm and vCp the error in vGD ³ are þ 0.4 and þ 0.5, respectively. It should be noted that the errors in vGD ³ of proteins given in Table 1, are due to those in vCp only. The e¡ect of osmolytes on the thermodynamic parameters of proteins is summarized in Table 1. It is seen in this table that Tm of each protein increases with an increase in the concentration of each osmolyte. This ¢nding that proteins are stabilized in terms
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Fig. 1. Thermal stability of Mb at pH 6.1. (A) Transition curves of the protein in the presence of di¡erent concentrations of Gly. For the sake of clarity, all data points are not shown, and for the same reason, transition curves at 0.25 and 0.75 M have been omitted. (B) Plot of vGD (T) versus temperature. Symbols have the same meaning as in A. (C) Stability curves of Mb drawn using Eq. 2 with vHm , Tm and vCp values given in Tables 1 and 2. The error bars represent errors in vGD (T) values due to an error of þ 0.16 kcal mol31 K31 in vCp . (D) Plot of vHm versus Tm of the protein at di¡erent concentrations of Gly.
of Tm agrees with those reported in the literature [2,13^17]. If it is assumed that Tm is an index of stability of proteins [13,15,17], this observation on Tm can be understood in the light of two recent mechanisms of stabilization by naturally occurring osmolytes proposed by Timashe¡ and coworkers [14] and by Bolen and coworkers [32^34]. According to Timashe¡, the main factor for the stabilizing e¡ect of the osmolyte on protein is related to the fact that
these osmolytes are preferentially excluded from the protein surface, i.e. they cause preferential hydration of the protein; this e¡ect should favor protein state with lower exposed surface, thus displacing the denaturation equilibrium, N conformationHD conformation, towards the N state, bringing about a stabilization of the native protein in the presence of osmolytes. Bolen and coworkers [32^34] have interpreted the e¡ect of osmolytes on proteins in terms of
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Fig. 2. Thermal stability of RNase A at pH 6.0. (A) Heat denaturation curves of the protein in the presence of di¡erent concentrations of Sar. All data points and transition curves have not been shown for the same reason given in Fig. 1A. (B) Plot of vGD (T) versus temperature. Symbols have the same meaning as in A. (C) Stability curves of the protein drawn using Eq. 2 with vHm and Tm values given in Table 1 and vCp = 1.23 þ 0.12 kcal mol31 K31 . The error bars have the same meaning as in Fig. 1C.
transfer-free energy of protein groups from water to osmolyte solutions, and in terms of dimensions of the native and denatured proteins in osmolyte solutions. They concluded that in addition to raising the overall vGtr , the Gibbs energy of transfer (vGtr = vGtr; D 3vGtr; N ), the unfavorable interaction of the backbone with osmolyte causes a collateral e¡ect that results in the contraction of the denatured molecules; this e¡ect should decrease the entropy of
the denatured molecule, i.e. osmolyte should promote N state, thus displacing the denatured equilibrium towards N state and bringing about a stabilization of the N conformation in the presence of osmolytes. In fact, both the mechanisms of stabilization of proteins, mentioned in the preceding paragraph, predict that the Gibbs energy change on denaturation (vGD ) should increase in the presence of osmolytes.
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Fig. 3. Thermal stability of Lzm at pH 4.8. (A) Thermal denaturation curves of the protein in the presence of various concentrations of Pro. All data points and transition curves have not been shown for the same reason given in Fig. 1A. (B) Plot of vGD (T) versus temperature. Symbols have the same meaning as in A. (C) Stability curves of Lzm drawn using Eq. 2 with vHm and Tm values given in Table 1 and vCp = 1.56 þ 0.20 kcal mol31 K31 . The error bars have the same meaning as in Fig. 1C.
It is seen in Table 1 that, on the contrary, vGD ³ values of proteins remain, within experimental errors, unchanged in the presence of osmolytes. In order to see how osmolytes a¡ect vGD at other temperatures, we constructed stability curves of proteins using Eq. 2, the Gibbs^Helmoltz equation derived on the assumption that vCp is independent of temperature in the range 20^80³C [17,18,30]. Panel C of Figs. 1^4 shows the typical stability curves of pro-
teins. It is seen in these ¢gures that all proteins are stabilized in the presence of osmolytes around their Tm values, i.e. in the temperature range 40^80³C. We have constructed stability curves of each protein in other three osmolytes, and observed that all osmolytes stabilize proteins in terms of vGD in the temperature range 40^80³C (curves not shown). Thus, our observation in this temperature range is consistent with the prediction that the Gibbs energy change
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Fig. 4. Thermal stability of cyt c at pH 3.0. (A) Transition curves of the protein in the presence of di¡erent concentrations of GB. All data points and transition curves have not been shown for the same reason given in Fig. 1A. (B) Plot of vGD (T) versus temperature. Symbols have the same meaning as in A. (C) Stability curves of cyt c drawn using Eq. 2 with vHm and Tm values given in Table 1 and vCp = 0.71 þ 0.03 kcal mol31 K31 . The error bars have the same meaning as in Fig. 1C.
on denaturation should increase in the presence of osmolytes [14,23,24]. RNase A, Lzm and Mb exist predominantly in their N conformations at 25³C (see Figs. 1^3). Their vGD ³ values are not perturbed in the presence of osmolytes (see Table 1, and also Figs. 1C, 2C and 3C). It should be noted that vGD ³ values of these proteins are obtained from long extrapolation of (vGD , T) data obtained at higher temperatures, i.e.
in the transition region. Thus vGD ³ values of proteins have larger error components than the experimentally determined vGD values in the transition region. In order to see whether our conclusion that osmolytes do not a¡ect the Gibbs energy change near room temperature is valid, we studied the e¡ect of osmolytes on cyt c at pH 3.0 where the protein is not only in equilibrium between N and D states [16], but the vGD value can also be experimentally deter-
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Table 1 Stability parameters of proteins in presence of osmolytesa [Osmolyte] (M)
Mb (pH 6.1)
RNase A (pH 6.0)
Lzm (pH 4.8)
Cyt c (pH 3.0)
Tm (³C)
vHm (kcal mol31 )
vGD ³ (kcal mol31 )
Tm (³C)
vHm (kcal mol31 )
vGD ³ (kcal mol31 )
Tm (³C)
vHm (kcal mol31 )
vGD ³ (kcal Tm (³C) mol31 )
vHm (kcal mol31 )
vGD ³ (kcal mol31 )
Gly 0.00 0.25 0.50 0.75 1.00
67.4 69.1 70.5 71.2 72.6
82 þ 4 83 þ 3 84 þ 2 85 þ 3 88 þ 3
2.7 þ 0.4 2.6 þ 0.4 2.7 þ 0.5 2.7 þ 0.6 2.8 þ 0.6
65.5 66.1 68.0 68.3 69.4
118 þ 2 119 þ 2 119 þ 4 120 þ 3 121 þ 4
11.0 þ 0.3 11.2 þ 0.3 11.5 þ 0.3 11.7 þ 0.4 12.0 þ 0.4
62.7 64.1 65.4 66.8 68.4
89 þ 5 91 þ 3 90 þ 3 92 þ 3 93 þ 3
6.6 þ 0.6 6.9 þ 0.3 6.8 þ 0.4 7.1 þ 0.4 7.3 þ 0.4
45.0 45.3 46.0 46.3 48.3
16 þ 1 13 þ 1 12 þ 1 11 þ 1 11 þ 1
0.6 þ 0.07 0.4 þ 0.06 0.3 þ 0.03 0.2 þ 0.02 0.2 þ 0.04
Pro 0.25 0.50 0.75 1.00
68.1 68.2 67.8 68.0
85 þ 3 85 þ 2 82 þ 3 83 þ 3
2.6 þ 0.4 2.6 þ 0.3 2.8 þ 0.2 2.4 þ 0.3
65.9 66.4 66.5 66.7
115 þ 2 119 þ 2 120 þ 4 120 þ 2
10.8 þ 0.4 11.3 þ 0.3 11.4 þ 0.3 11.4 þ 0.3
63.9 64.5 64.8 65.1
90 þ 4 92 þ 3 92 þ 3 87 þ 3
6.7 þ 0.5 7.0 þ 0.5 7.0 þ 0.5 6.5 þ 0.5
45.8 46.5 47.0 49.3
16 þ 1 16 þ 1 14 þ 1 14 þ 1
0.6 þ 0.02 0.6 þ 0.02 0.4 þ 0.02 0.4 þ 0.03
Sar 0.25 0.50 0.75 1.00
68.5 69.1 70.5 72.1
85 þ 2 87 þ 3 86 þ 3 88 þ 3
2.8 þ 0.5 2.9 þ 0.5 2.8 þ 0.6 3.1 þ 0.5
65.9 67.5 67.8 70.1
118 þ 4 119 þ 4 119 þ 3 119 þ 4
11.1 þ 0.3 11.4 þ 0.3 11.5 þ 0.3 11.8 þ 0.3
64.0 65.1 66.4 67.9
88 þ 4 89 þ 5 90 þ 3 91 þ 3
6.5 þ 0.5 6.7 þ 0.5 6.9 þ 0.5 7.1 þ 0.6
46.8 48.4 50.1 51.1
16 þ 1 16 þ 1 15 þ 1 16 þ 1
0.6 þ 0.02 0.6 þ 0.03 0.5 þ 0.03 0.5 þ 0.03
GB 0.25 0.50 0.75 1.00
68.0 68.8 69.5 70.2
85 þ 2 85 þ 3 86 þ 2 87 þ 3
2.9 þ 0.6 2.8 þ 0.5 2.9 þ 0.6 3.1 þ 0.4
66.1 66.3 67.0 68.1
122 þ 4 120 þ 3 122 þ 2 120 þ 3
11.6 þ 0.3 11.4 þ 0.3 11.7 þ 0.3 11.7 þ 0.4
63.1 64.9 65.0 65.8
89 þ 5 90 þ 3 91 þ 3 89 þ 4
6.6 þ 0.5 6.8 þ 0.5 6.9 þ 0.5 6.7 þ 0.5
45.8 46.5 47.5 49.7
16 þ 2 16 þ 1 15 þ 1 16 þ 1
0.6 þ 0.02 0.6 þ 0.02 0.5 þ 0.03 0.5 þ 0.03
A ` þ ' with a value of vHm represents the standard deviation of the ¢t. The average error from several independent measurements are within errors of the least-squares ¢t. Values of vGD ³ were calculated with Eq. 2 using values of vCp given in the text.
mined at 25³C (e.g. see Fig. 4). These results also led to the conclusion that: (1) vGD ³ of cyt c is not affected by the presence of osmolytes (see Table 1); and (2) osmolytes stabilize the protein at higher temperatures (e.g. see Fig. 4) There seems to exist a paradox that the osmolyte stabilizes proteins in terms of Gibbs energy at higher temperatures, but it has no signi¢cant e¡ect near room temperature. One possible explanation for these di¡erent e¡ects of the osmolytes on the Gibbs energy of stabilization at di¡erent temperatures may stem from the di¡erent natures of the denatured protein in the presence and absence of the osmolyte. This view is supported by the recent ¢ndings that osmolyte^protein interaction results in the contraction of the denatured ensembles (i.e. it promotes the formation of native molecules), and has no e¡ect
on the dimensions of the native molecules [32^34]. That is, the action of osmolytes is on the denatured ensembles and not on the native state [33]. If we considered our vGD ³ results (Table 1) in the physiological context, the important role played by osmolytes in governing protein stability^function relationship may be appreciated. It is well known that the adaptation of organisms to denaturing stresses is enabled by the accumulation of osmolytes that protect proteins and other cellular components from deleterious e¡ects of denaturing stresses [2]. However, this adaptive pattern should not signi¢cantly a¡ect protein stability in terms of vGD ³, for there exists an inverse relation between vGD ³ and in vivo rate of degradative protein turnover [19,20]. For instance, if vGD ³ values of proteins are increased in the presence of osmolytes, it then means that osmolytes will
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Table 2 Values of temperature-dependence of the enthalpy change of Mb in presence of di¡erent concentrations of osmolytesa [Osmolyte] (M) 0.00 0.25 0.50 0.75 1.00 a
vCp (kcal mol31 K31 ) Gly
Sar
GB
Pro
2.74 þ 0.16 2.71 þ 0.12 2.74 þ 0.15 2.68 þ 0.17 2.72 þ 0.16
2.74 þ 0.16 2.78 þ 0.17 2.81 þ 0.18 2.71 þ 0.21 2.63 þ 0.20
2.74 þ 0.16 2.77 þ 0.20 2.77 þ 0.18 2.73 þ 0.19 2.68 þ 0.14
2.74 þ 0.16 2.76 þ 0.15 2.75 þ 0.12 2.69 þ 0.07 2.71 þ 0.09
A ` þ ' with a value of vCp represents the standard deviation of the ¢t.
decrease the protein turnover rates of all proteins including the metabolic regulatory enzymes which have a very high turnover rate [35]. This scenario will pose enormous problems for the organisms, because now it would be energetically di¤cult to degrade proteins [19,20] and to maintain a steady-state condition [35]. It is noteworthy that Burg [36] has shown that in vivo rate of degradation of aldose reductase remained unchanged during the accumulation of sorbitol which is a compatible osmolyte. His study provides support to our ¢nding that vGD ³ of proteins is not a¡ected by osmolytes. In summary, we conclude that: (1) osmolytes protect proteins against denaturing stresses by working on thermodynamics variables in such a way that the denaturation temperature (Tm ) is raised without perturbing vGD values near physiological temperatures; and (2) that osmolytes are compatible with protein stability (vGD ³) as well. Acknowledgements This work has been supported by grants from the University Grants Commission, Council of Scienti¢c and Industrial Research, and Department of Science and Technology. We thank Dr. Peter McPhie (NIDDK, NIH, Bethesda, MD) for his valuable comments. F.A. is very grateful to Professor D. Wayne Bolen (Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Texas, USA) for enlightening discussions while the author was a visiting Professor in his laboratory, and for the ¢nancial support from the John Sealy Memorial Endowment Fund for Biomedical Research (Grant 2512-98R).
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[12] R.E. MacMillen, A.K. Lee, Australian desert mice: independence of exogenous water, Science 158 (1967) 383^385. [13] T. Arakawa, S.N. Timashe¡, The stabilization of proteins by osmolytes, Biophys. J. 47 (1985) 411^414. [14] S.N. Timashe¡, The physicochemical basis for the selection of osmolytes by nature, in: G.N. Somero, C.B. Osmond, C.L. Bolis (Eds.), Water and Life, Springer-Verlag, Berlin, 1992, pp. 70^84. [15] M.M. Santoro, Y. Liu, S.M.A. Khan, L.-X. Hou, D.W. Bolen, Increased thermal stability of proteins in the presence of naturally occurring osmolytes, Biochemistry 31 (1992) 5278^5283. [16] S. Taneja, F. Ahmad, Increased thermal stability of proteins in presence of amino acids, Biochem. J. 303 (1994) 147^153. [17] I.M. Plaza del Pino, J.M. Sanchez-Ruiz, An osmolyte e¡ect on the heat capacity change for protein folding, Biochemistry 34 (1995) 8621^8630. [18] P.L. Privalov, Stability of proteins: small globular proteins, Adv. Protein Chem. 33 (1979) 167^241. [19] C.N. Pace, L.M. Fisher, J.E. Cupo, Globular protein stability: aspect of interest in protein turnover, Acta Biol. Med. Germ. 40 (1981) 1385^1392. [20] G. Mclendon, A correlation between myoglobin thermodynamic stabilities and species metabolic rates, Biochem. Biophys. Res. Commun. 77 (1977) 959^966. [21] U.K. Laemmli, Cleavage of structural proteins during assembly of the head of bacteriophage T4, Nature 227 (1970) 680^685. [22] R. Gupta, F. Ahmad, Protein stability: functional dependence of denaturational Gibbs energy on urea concentration, Biochemistry 38 (1999) 2471^2479. [23] R. Gupta, S. Yadav, F. Ahmad, Protein stability: urea-induced versus guanidine-induced unfolding of metmyoglobin, Biochemistry 35 (1996) 11925^11930. [24] K. Hamaguchi, A. Kurono, Structure of muramidase (lysozyme) I: the e¡ect of guanidine hydrochloride on muramidase, J. Biochem. 54 (1963) 111^122.
[25] C.C. Bigelow, Di¡erence spectra of ribonuclease and two ribonuclease derivatives, C. R. Trav. Lab. Carlsberg 31 (1960) 305^310. [26] D. Puett, The equilibrium unfolding parameters of horse and sperm whale myoglobin, J. Biol. Chem. 248 (1973) 4623^ 4634. [27] E. Margoliash, N. Frohwitz, Spectrum of horse-heart cytochrome-c, Biochem. J. 71 (1959) 570^572. [28] C.N. Pace, Determination and analysis of urea and guanidine hydrochloride denaturation curves, Methods Enzymol. 131 (1986) 266^280. [29] V. Rishi, F. Anjum, F. Ahmad, Role of non-compatible osmolytes in the stabilization of proteins during heat stress, Biochem. J. 329 (1998) 137^143. [30] W.J. Becktle, J.A. Schellman, Protein stability curves, Biopolymers 26 (1987) 1859^1877. [31] B. Sabulal, N. Kishore, Di¡erential scanning calorimetric study of the interactions of some stabilizing amino acids and oligopeptides with hen egg white lysozyme, J. Chem. Soc. Faraday Trans. 91 (1995) 2101^2106. [32] Y. Liu, D.W. Bolen, The peptide backbone plays a dominant role in protein stabilization by naturally occurring osmolytes, Biochemistry 34 (1995) 12884^12891. [33] Y. Qu, C.L. Bolen, D.W. Bolen, Osmolyte-driven contraction of a random coil protein, Proc. Natl. Acad. Sci. USA 95 (1998) 9268^9273. [34] I. Baskakov, D.W. Bolen, Forcing thermodynamically unfolded proteins to fold, J. Biol. Chem. 273 (1998) 4831^4834. [35] M. Rechsteiner, S. Rogers, K. Rote, Protein structure and intracellular stability, Trends Biochem. Sci. 12 (1987) 390^ 394. [36] M.B. Burg, Molecular basis for accumulation of compatible osmolytes in mammalian cells, in: G.N. Somero, C.B. Osmond, C.L. Bolis (Eds.), Water and Life, Springer-Verlag, Berlin, 1992 pp. 33^51.
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Compatibility of osmolytes with Gibbs energy of stabilization of proteins Farah Anjum, Vikas Rishi, Faizan Ahmad * Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110 025, India Received 5 July 1999; received in revised form 15 September 1999; accepted 28 September 1999
Abstract This study led to the conclusion that naturally occurring osmolytes which are known to protect proteins against denaturing stresses, do not perturb the Gibbs energy of stabilization of proteins at 25³C (vGD ³) which has been shown to control the in vivo rate of degradative protein turnover (Pace et al., Acta Biol. Med. Germ 40 (1981) 1385^1392). This conclusion has been reached from our studies of heat-induced denaturation of lysozyme, ribonuclease A, cytochrome c and myoglobin in the presence of different concentrations of osmolytes, namely, glycine, proline, sarcosine and glycine-betaine. At a fixed concentration of osmolyte a heat-induced denaturation curve measured by following changes in the molar absorption coefficient of the protein, was analyzed for Tm , the midpoint of the denaturation and vHm , the enthalpy change of denaturation at Tm . Values of vGD ³ were determined with Gibbs^Helmoltz equation using known values of Tm , vHm and vCp , the constant-pressure heat capacity change. It has been observed that Tm increases with the osmolyte concentration, whereas vGD ³ remains unaffected in the presence of the osmolyte. This observation on vGD ³ in the presence of osmolytes has been considered in the physiological context. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Osmolyte; Protein stability ; Lysozyme; Ribonuclease A; Cytochrome c; Myoglobin
1. Introduction Many microorganisms, ¢shes, plants and animals have adapted to harsh environmental conditions, such as water, salts, cold and heat stresses. It is well known that these organisms have adapted one common strategy in protecting their cellular proteins against these harsh environmental stresses [1,2]. This involves accumulation of low molecular weight organic compounds, collectively called osmolytes which fall into one of the three classes, namely, amino acids and their derivatives, polyols and methylamines [2^ 4]. These osmolytes are further classi¢ed as `compat-
* Corresponding author. Fax: +91-11-579-1351; E-mail: [email protected]
ible' or `counteracting' based on their e¡ect on the functional activity of proteins [2,3,5]. Compatible osmolytes are those which protect proteins against inactivation and denaturation without perturbing the protein functional activity near room temperature [5^ 8]. Counteracting osmolytes are those which are built up by organisms to cope with deleterious e¡ect of urea on proteins' functional activity and stability [9^11]. It is noteworthy that accumulation of compatible osmolytes (amino acids and their derivatives and polyols) occurs when organisms are under stresses such as water, extremes of temperature and salinity, and accumulation of counteracting osmolytes (methylamines) occurs when urea concentration in the cells of organisms is as high as 0.4^4 M [12]. A large body of data suggests that both compatible and counteracting osmolytes enhance thermal
0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 2 1 5 - 0
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stability of proteins [2,13^17]. The main factor responsible for this stabilizing e¡ect was disclosed by Timashe¡ and co-workers [13,14] and is related to the fact that these osmolytes are preferentially excluded from the protein domain, that is, they cause preferential hydration of the protein; this e¡ect should favor the protein state with the lower exposed surface, thus displacing the denaturation equilibrium, native (N) conformationHdenatured (D) conformation, towards the N state and bring about a higher stabilization of the N conformation in the presence of osmolytes. It is interesting to note that almost all studies used Tm (denaturation transition temperature) as an index of conformational stability (vGD ³, the Gibbs energy change of denaturation at 25³C) in the presence of an osmolyte. One potentially interesting aspect of this mechanism that has received little attention is the possible e¡ect of osmolytes on the denaturation Gibbs energy (vGD ³). The interest of probing this possible e¡ect is suggested by the two following facts. (a) Tm is not a measure of vGD ³. For example, lysozyme and cytochrome c have the same Tm of 80³C, but vGD ³ values are 16 and 8 kcal mol31 , respectively [18]. (b) There exists an inverse relation between vGD ³ and susceptibility to protein degradation [19,20]. If an osmolyte perturbs vGD ³, it then means that the degradative protein turnover rate will also be perturbed. This scenario will pose enormous problems for the organisms. In order to investigate the e¡ect of osmolytes on vGD ³, we have been carrying out systematic studies of denaturation of several proteins in the presence of di¡erent concentrations of both compatible and counteracting osmolytes. In this communication, we report an interesting observation that osmolytes do not signi¢cantly perturb vGD ³ values of proteins near room temperature suggesting that they do not alter the rate of degradative protein turnover. That is, osmolytes are compatible with protein stability (vGD ³). 2. Materials and methods Hen egg-white lysozyme (Lzm), ribonuclease A (RNase A), horse heart cytochrome c (cyt c) and horse heart myoglobin (Mb) were purchased from the Sigma. Electrophoretic homogeneity of each pro-
tein was checked on SDS-polyacrylamide gel electrophoresis according to the procedure described by Laemmli [21]. Since all proteins gave a single band, they were used without further puri¢cations. Osmolytes, namely, glycine (Gly), proline (Pro), sarcosine (Sar) and glycine-betaine (GB) were purchased from the Aldrich. Ultrapure guanidine hydrochloride (GdnHCl) was purchased from Schwarz/Mann Biotech. These and other chemicals were of analytical grade and were used without further puri¢cation. Stock solutions of Lzm, RNase A and those of oxidized Mb and cyt c were prepared in 0.1 M KCl (pH 7.0) as described earlier [22,23]. Concentrations of proteins were determined using molar absorption coe¤cient (M31 cm31 ) values of 39 000 at 280 nm for Lzm [24], 9800 at 277.5 nm for RNase A [25], 171 000 at 409 nm for Mb [26] and 10 700 at 530 nm for cyt c [27]. Concentration of GdnHCl stock solution was determined by refractometric measurements [28]. All solutions for optical measurements were prepared in degassed bu¡er containing 0.1 M KCl. Thermal denaturation studies were carried out in Jasco V-560 UV/Vis spectrophotometer with a heating rate of 1³C min31 using its peltier accessory (model ETC-505 temperature controller). The change in absorbance on heating was followed at 292 nm for Lzm, 287 nm for RNase A, 409 nm for Mb, and 530 nm for cyt c. About 350 data points were collected. Reversibility of each denaturation curve was checked as described earlier [29]. Lzm, Mb and cyt c near neutral pH have their Tm values near 80³C. In order to bring the heat-induced transition to a measurable temperature range, Lzm and Mb denaturation was studied in the presence of 1.60 M and 0.60 M GdnHCl, respectively, whereas the thermal denaturation of cyt c was studied at pH 3.0 at which it is already 25% acid denatured at 25³C. Each thermal transition curve was measured at least in triplicate, and analyzed for vGD (T), the Gibbs energy of denaturation at temperature T for a two-state denaturation, using the relation, y T3yN T v G D T 3RT ln 1 yD T3y T where y(T) is the observed optical property at temperature T, and yN and yD are, respectively, the optical properties of the native and denatured protein
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molecules at the same temperature at which y has been measured. The plot of vGD (T) values in the range 31.3 9 vGD (T), kcal mol31 9 1.3 versus corresponding T at constant concentration of an osmolyte was used to determine the values of Tm and vHm , the enthalpy change of denaturation at Tm according to the procedure reported earlier [16]. vGD ³, the value of vGD at T = 298.15 K was determined using the measured values of vHm , Tm , and vCp in the Gibbs^Helmoltz equation, T m 3T T 3v C p T m 3T T ln v GD v H m Tm Tm 2
3. Results and discussion In order to understand the question whether osmolytes are compatible with protein stability (vGD ³), de¢ned as the decrease in Gibbs energy of the denatured polypeptide when it folds to give the native functional protein under physiological conditions, usually taken as neutral dilute bu¡er and 25³C, we have been measuring the thermal denaturation curves of several proteins in the presence and absence of di¡erent osmolytes. Here we present and discuss the results of four proteins, namely, Lzm, RNase A, Mb and cyt c in the presence of di¡erent concentrations of two compatible osmolytes (Gly and Pro) and two counteracting osmolytes (Sar and GB). Panel A of Figs. 1^4 represents the typical denaturation behavior of proteins in the presence and absence of the osmolytes. The optical transition curves of each protein in the presence of di¡erent concentrations of an osmolyte were converted into stability curve [30] using Eq. 1 (e.g. see panel B of Figs. 1^4). Stability curves of a protein were analyzed for vHm and Tm values using the procedure developed earlier [16]. Values of these parameters of all proteins in di¡erent concentrations of osmolytes are given in Table 1. In order to determine vGD ³ values of proteins in the presence and absence of di¡erent concentrations of various osmolytes using Eq. 2, their vCp values at di¡erent concentrations of each osmolytes should also be known. The recommended procedure for the experimental determination of vCp involves the
77
measurements of vHm and Tm from the heat-induced denaturation of the protein at several pH values, using di¡erential scanning calorimetry [18] or optical techniques [30]. We have measured thermal denaturation curves of one protein, namely, Mb in the presence of di¡erent ¢xed concentrations of each osmolyte at four di¡erent pH values. For each concentration of an osmolyte, vHm versus corresponding Tm plot was constructed. Such plots of Mb in the presence of Gly are shown in Fig. 1D (plots for other osmolytes not shown). Assuming that vCp is independent of temperature and pH between 20 and 80³C [17,18,30], values of vCp ( = DvHm /DTm ) were obtained from the least-squares analysis. These results are given in Table 2. It is seen in this table that vCp value remains, within experimental errors, unchanged in the presence of osmolytes in the concentration range 0^1 M. However, it has been assumed that vCp values of other proteins are independent of the presence of low osmolyte concentrations. The assumption that the low concentrations of osmolytes do not a¡ect the vCp of RNase A, Lzm and cyt c is based on the following observations. Di¡erential scanning calorimetric measurements of RNase A [17], Lzm [31] and cyt c (Ahmad and Pfeil, unpublished results) in the presence of up to 1 M of various osmolytes suggested that vCp of the protein is not signi¢cantly altered. Using vHm and Tm values given in Table 1 and vCp (kcal mol31 K31 ) values of 1.23 þ 0.12 for RNase A [18], 1.56 þ 0.20 for Lzm [18], 2.74 þ 0.16 for Mb ([18], this study) and 0.71 þ 0.03 for cyt c [16] measured in the absence of the osmolyte, we have calculated vGD ³ values of proteins using Eq. 2, which are given in Table 1. It is seen in Eq. 2 that, assuming no error in temperature, the error in the estimate of vGD ³ depends on the experimental errors in the vHm and vCp . For example, the value of vGD ³ of RNase A is 11.0 kcal mol31 . On incorporating errors in the vHm and vCp the error in vGD ³ are þ 0.4 and þ 0.5, respectively. It should be noted that the errors in vGD ³ of proteins given in Table 1, are due to those in vCp only. The e¡ect of osmolytes on the thermodynamic parameters of proteins is summarized in Table 1. It is seen in this table that Tm of each protein increases with an increase in the concentration of each osmolyte. This ¢nding that proteins are stabilized in terms
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Fig. 1. Thermal stability of Mb at pH 6.1. (A) Transition curves of the protein in the presence of di¡erent concentrations of Gly. For the sake of clarity, all data points are not shown, and for the same reason, transition curves at 0.25 and 0.75 M have been omitted. (B) Plot of vGD (T) versus temperature. Symbols have the same meaning as in A. (C) Stability curves of Mb drawn using Eq. 2 with vHm , Tm and vCp values given in Tables 1 and 2. The error bars represent errors in vGD (T) values due to an error of þ 0.16 kcal mol31 K31 in vCp . (D) Plot of vHm versus Tm of the protein at di¡erent concentrations of Gly.
of Tm agrees with those reported in the literature [2,13^17]. If it is assumed that Tm is an index of stability of proteins [13,15,17], this observation on Tm can be understood in the light of two recent mechanisms of stabilization by naturally occurring osmolytes proposed by Timashe¡ and coworkers [14] and by Bolen and coworkers [32^34]. According to Timashe¡, the main factor for the stabilizing e¡ect of the osmolyte on protein is related to the fact that
these osmolytes are preferentially excluded from the protein surface, i.e. they cause preferential hydration of the protein; this e¡ect should favor protein state with lower exposed surface, thus displacing the denaturation equilibrium, N conformationHD conformation, towards the N state, bringing about a stabilization of the native protein in the presence of osmolytes. Bolen and coworkers [32^34] have interpreted the e¡ect of osmolytes on proteins in terms of
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Fig. 2. Thermal stability of RNase A at pH 6.0. (A) Heat denaturation curves of the protein in the presence of di¡erent concentrations of Sar. All data points and transition curves have not been shown for the same reason given in Fig. 1A. (B) Plot of vGD (T) versus temperature. Symbols have the same meaning as in A. (C) Stability curves of the protein drawn using Eq. 2 with vHm and Tm values given in Table 1 and vCp = 1.23 þ 0.12 kcal mol31 K31 . The error bars have the same meaning as in Fig. 1C.
transfer-free energy of protein groups from water to osmolyte solutions, and in terms of dimensions of the native and denatured proteins in osmolyte solutions. They concluded that in addition to raising the overall vGtr , the Gibbs energy of transfer (vGtr = vGtr; D 3vGtr; N ), the unfavorable interaction of the backbone with osmolyte causes a collateral e¡ect that results in the contraction of the denatured molecules; this e¡ect should decrease the entropy of
the denatured molecule, i.e. osmolyte should promote N state, thus displacing the denatured equilibrium towards N state and bringing about a stabilization of the N conformation in the presence of osmolytes. In fact, both the mechanisms of stabilization of proteins, mentioned in the preceding paragraph, predict that the Gibbs energy change on denaturation (vGD ) should increase in the presence of osmolytes.
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Fig. 3. Thermal stability of Lzm at pH 4.8. (A) Thermal denaturation curves of the protein in the presence of various concentrations of Pro. All data points and transition curves have not been shown for the same reason given in Fig. 1A. (B) Plot of vGD (T) versus temperature. Symbols have the same meaning as in A. (C) Stability curves of Lzm drawn using Eq. 2 with vHm and Tm values given in Table 1 and vCp = 1.56 þ 0.20 kcal mol31 K31 . The error bars have the same meaning as in Fig. 1C.
It is seen in Table 1 that, on the contrary, vGD ³ values of proteins remain, within experimental errors, unchanged in the presence of osmolytes. In order to see how osmolytes a¡ect vGD at other temperatures, we constructed stability curves of proteins using Eq. 2, the Gibbs^Helmoltz equation derived on the assumption that vCp is independent of temperature in the range 20^80³C [17,18,30]. Panel C of Figs. 1^4 shows the typical stability curves of pro-
teins. It is seen in these ¢gures that all proteins are stabilized in the presence of osmolytes around their Tm values, i.e. in the temperature range 40^80³C. We have constructed stability curves of each protein in other three osmolytes, and observed that all osmolytes stabilize proteins in terms of vGD in the temperature range 40^80³C (curves not shown). Thus, our observation in this temperature range is consistent with the prediction that the Gibbs energy change
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Fig. 4. Thermal stability of cyt c at pH 3.0. (A) Transition curves of the protein in the presence of di¡erent concentrations of GB. All data points and transition curves have not been shown for the same reason given in Fig. 1A. (B) Plot of vGD (T) versus temperature. Symbols have the same meaning as in A. (C) Stability curves of cyt c drawn using Eq. 2 with vHm and Tm values given in Table 1 and vCp = 0.71 þ 0.03 kcal mol31 K31 . The error bars have the same meaning as in Fig. 1C.
on denaturation should increase in the presence of osmolytes [14,23,24]. RNase A, Lzm and Mb exist predominantly in their N conformations at 25³C (see Figs. 1^3). Their vGD ³ values are not perturbed in the presence of osmolytes (see Table 1, and also Figs. 1C, 2C and 3C). It should be noted that vGD ³ values of these proteins are obtained from long extrapolation of (vGD , T) data obtained at higher temperatures, i.e.
in the transition region. Thus vGD ³ values of proteins have larger error components than the experimentally determined vGD values in the transition region. In order to see whether our conclusion that osmolytes do not a¡ect the Gibbs energy change near room temperature is valid, we studied the e¡ect of osmolytes on cyt c at pH 3.0 where the protein is not only in equilibrium between N and D states [16], but the vGD value can also be experimentally deter-
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Table 1 Stability parameters of proteins in presence of osmolytesa [Osmolyte] (M)
Mb (pH 6.1)
RNase A (pH 6.0)
Lzm (pH 4.8)
Cyt c (pH 3.0)
Tm (³C)
vHm (kcal mol31 )
vGD ³ (kcal mol31 )
Tm (³C)
vHm (kcal mol31 )
vGD ³ (kcal mol31 )
Tm (³C)
vHm (kcal mol31 )
vGD ³ (kcal Tm (³C) mol31 )
vHm (kcal mol31 )
vGD ³ (kcal mol31 )
Gly 0.00 0.25 0.50 0.75 1.00
67.4 69.1 70.5 71.2 72.6
82 þ 4 83 þ 3 84 þ 2 85 þ 3 88 þ 3
2.7 þ 0.4 2.6 þ 0.4 2.7 þ 0.5 2.7 þ 0.6 2.8 þ 0.6
65.5 66.1 68.0 68.3 69.4
118 þ 2 119 þ 2 119 þ 4 120 þ 3 121 þ 4
11.0 þ 0.3 11.2 þ 0.3 11.5 þ 0.3 11.7 þ 0.4 12.0 þ 0.4
62.7 64.1 65.4 66.8 68.4
89 þ 5 91 þ 3 90 þ 3 92 þ 3 93 þ 3
6.6 þ 0.6 6.9 þ 0.3 6.8 þ 0.4 7.1 þ 0.4 7.3 þ 0.4
45.0 45.3 46.0 46.3 48.3
16 þ 1 13 þ 1 12 þ 1 11 þ 1 11 þ 1
0.6 þ 0.07 0.4 þ 0.06 0.3 þ 0.03 0.2 þ 0.02 0.2 þ 0.04
Pro 0.25 0.50 0.75 1.00
68.1 68.2 67.8 68.0
85 þ 3 85 þ 2 82 þ 3 83 þ 3
2.6 þ 0.4 2.6 þ 0.3 2.8 þ 0.2 2.4 þ 0.3
65.9 66.4 66.5 66.7
115 þ 2 119 þ 2 120 þ 4 120 þ 2
10.8 þ 0.4 11.3 þ 0.3 11.4 þ 0.3 11.4 þ 0.3
63.9 64.5 64.8 65.1
90 þ 4 92 þ 3 92 þ 3 87 þ 3
6.7 þ 0.5 7.0 þ 0.5 7.0 þ 0.5 6.5 þ 0.5
45.8 46.5 47.0 49.3
16 þ 1 16 þ 1 14 þ 1 14 þ 1
0.6 þ 0.02 0.6 þ 0.02 0.4 þ 0.02 0.4 þ 0.03
Sar 0.25 0.50 0.75 1.00
68.5 69.1 70.5 72.1
85 þ 2 87 þ 3 86 þ 3 88 þ 3
2.8 þ 0.5 2.9 þ 0.5 2.8 þ 0.6 3.1 þ 0.5
65.9 67.5 67.8 70.1
118 þ 4 119 þ 4 119 þ 3 119 þ 4
11.1 þ 0.3 11.4 þ 0.3 11.5 þ 0.3 11.8 þ 0.3
64.0 65.1 66.4 67.9
88 þ 4 89 þ 5 90 þ 3 91 þ 3
6.5 þ 0.5 6.7 þ 0.5 6.9 þ 0.5 7.1 þ 0.6
46.8 48.4 50.1 51.1
16 þ 1 16 þ 1 15 þ 1 16 þ 1
0.6 þ 0.02 0.6 þ 0.03 0.5 þ 0.03 0.5 þ 0.03
GB 0.25 0.50 0.75 1.00
68.0 68.8 69.5 70.2
85 þ 2 85 þ 3 86 þ 2 87 þ 3
2.9 þ 0.6 2.8 þ 0.5 2.9 þ 0.6 3.1 þ 0.4
66.1 66.3 67.0 68.1
122 þ 4 120 þ 3 122 þ 2 120 þ 3
11.6 þ 0.3 11.4 þ 0.3 11.7 þ 0.3 11.7 þ 0.4
63.1 64.9 65.0 65.8
89 þ 5 90 þ 3 91 þ 3 89 þ 4
6.6 þ 0.5 6.8 þ 0.5 6.9 þ 0.5 6.7 þ 0.5
45.8 46.5 47.5 49.7
16 þ 2 16 þ 1 15 þ 1 16 þ 1
0.6 þ 0.02 0.6 þ 0.02 0.5 þ 0.03 0.5 þ 0.03
A ` þ ' with a value of vHm represents the standard deviation of the ¢t. The average error from several independent measurements are within errors of the least-squares ¢t. Values of vGD ³ were calculated with Eq. 2 using values of vCp given in the text.
mined at 25³C (e.g. see Fig. 4). These results also led to the conclusion that: (1) vGD ³ of cyt c is not affected by the presence of osmolytes (see Table 1); and (2) osmolytes stabilize the protein at higher temperatures (e.g. see Fig. 4) There seems to exist a paradox that the osmolyte stabilizes proteins in terms of Gibbs energy at higher temperatures, but it has no signi¢cant e¡ect near room temperature. One possible explanation for these di¡erent e¡ects of the osmolytes on the Gibbs energy of stabilization at di¡erent temperatures may stem from the di¡erent natures of the denatured protein in the presence and absence of the osmolyte. This view is supported by the recent ¢ndings that osmolyte^protein interaction results in the contraction of the denatured ensembles (i.e. it promotes the formation of native molecules), and has no e¡ect
on the dimensions of the native molecules [32^34]. That is, the action of osmolytes is on the denatured ensembles and not on the native state [33]. If we considered our vGD ³ results (Table 1) in the physiological context, the important role played by osmolytes in governing protein stability^function relationship may be appreciated. It is well known that the adaptation of organisms to denaturing stresses is enabled by the accumulation of osmolytes that protect proteins and other cellular components from deleterious e¡ects of denaturing stresses [2]. However, this adaptive pattern should not signi¢cantly a¡ect protein stability in terms of vGD ³, for there exists an inverse relation between vGD ³ and in vivo rate of degradative protein turnover [19,20]. For instance, if vGD ³ values of proteins are increased in the presence of osmolytes, it then means that osmolytes will
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Table 2 Values of temperature-dependence of the enthalpy change of Mb in presence of di¡erent concentrations of osmolytesa [Osmolyte] (M) 0.00 0.25 0.50 0.75 1.00 a
vCp (kcal mol31 K31 ) Gly
Sar
GB
Pro
2.74 þ 0.16 2.71 þ 0.12 2.74 þ 0.15 2.68 þ 0.17 2.72 þ 0.16
2.74 þ 0.16 2.78 þ 0.17 2.81 þ 0.18 2.71 þ 0.21 2.63 þ 0.20
2.74 þ 0.16 2.77 þ 0.20 2.77 þ 0.18 2.73 þ 0.19 2.68 þ 0.14
2.74 þ 0.16 2.76 þ 0.15 2.75 þ 0.12 2.69 þ 0.07 2.71 þ 0.09
A ` þ ' with a value of vCp represents the standard deviation of the ¢t.
decrease the protein turnover rates of all proteins including the metabolic regulatory enzymes which have a very high turnover rate [35]. This scenario will pose enormous problems for the organisms, because now it would be energetically di¤cult to degrade proteins [19,20] and to maintain a steady-state condition [35]. It is noteworthy that Burg [36] has shown that in vivo rate of degradation of aldose reductase remained unchanged during the accumulation of sorbitol which is a compatible osmolyte. His study provides support to our ¢nding that vGD ³ of proteins is not a¡ected by osmolytes. In summary, we conclude that: (1) osmolytes protect proteins against denaturing stresses by working on thermodynamics variables in such a way that the denaturation temperature (Tm ) is raised without perturbing vGD values near physiological temperatures; and (2) that osmolytes are compatible with protein stability (vGD ³) as well. Acknowledgements This work has been supported by grants from the University Grants Commission, Council of Scienti¢c and Industrial Research, and Department of Science and Technology. We thank Dr. Peter McPhie (NIDDK, NIH, Bethesda, MD) for his valuable comments. F.A. is very grateful to Professor D. Wayne Bolen (Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Texas, USA) for enlightening discussions while the author was a visiting Professor in his laboratory, and for the ¢nancial support from the John Sealy Memorial Endowment Fund for Biomedical Research (Grant 2512-98R).
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