Báo cáo khoa học: Relationship between functional activity and protein stability in the presence of all classes of stabilizing osmolytes ppt

Relationship between functional activity and protein stability in the presence of all classes of stabilizing osmolytes Shazia Jamal*, Nitesh K. Poddar*, Laishram R. Singh*,, Tanveer A. Dar*,à, Vikas Rishi§ and Faizan Ahmad Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India Introduction Both prokaryotic and eukaryotic cells, when subjected to harsh environmental conditions such as water, salts, cold and heat stresses, adopt a common strategy in protecting their proteins by producing low molecular weight organic substances called osmolytes [1,2]. Chemically stabilizing osmolytes (low molecular mass organic compounds that raise the midpoint of thermal denaturation) are divided into three classes: amino Keywords catalytic efficiency; denaturation equilibrium; enzyme activity; osmolytes, protein stability Correspondence F. Ahmad, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India 110025 Fax: +91 11 2698 3409 Tel: +91 11 2698 1733 E-mail: faizan_ahmad@yahoo.com *These authors contributed equally to this work Present addresses Division of Population Science, Fox Chase Cancer Center, Philadelphia, PA, USA àDepartment of Chemistry Biochemistry, University of Montana, Missoula, MT, USA §National Cancer Institute, NIH, Bethesda, MD, USA (Received 29 May 2009, revised 10 August 2009, accepted 19 August 2009) doi:10.1111/j.1742-4658.2009.07317.x We report the effects of stabilizing osmolytes (low molecular mass organic compounds that raise the midpoint of thermal denaturation) on the stabil- ity and function of RNase-A under physiological conditions (pH 6.0 and 25 °C). Measurements of Gibbs free energy change at 25 °C(DG D °) and kinetic parameters, Michaelis constant (K m ) and catalytic constant (k cat )of the enzyme mediated hydrolysis of cytidine monophosphate, enabled us to classify stabilizing osmolytes into three different classes based on their effects on kinetic parameters and protein stability. (a) Polyhydric alcohols and amino acids and their derivatives do not have significant effects on DG D ° and functional activity (K m and k cat ). (b) Methylamines increase DG D ° and k cat , but decrease K m . (c) Sugars increase DG D °, but decrease both K m and k cat . These findings suggest that, among the stabilizing osmo- lytes, (a) polyols, amino acids and amino acid derivatives are compatible solutes in terms of both stability and function, (b) methylamines are the best refolders (stabilizers), and (c) sugar osmolytes stabilize the protein, but they apparently do not yield functionally active folded molecules. Abbreviations DG D °, Gibbs free energy change at 25 °C; DC p , constant pressure heat capacity change; T m , midpoint of thermal denaturation; DH m , enthalpy change at T m ; K m , Michaelis constant; k cat , catalytic constant; k cat ⁄ K m , overall enzyme efficiency. 6024 FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS acids and their derivatives, polyhydric alcohols and sugars, and methyl ammonium derivatives [1]. These osmolytes are known not only to stabilize proteins [3,4], but they also induce refolding of misfolded proteins [5–8] and remove protein aggregation [9–12]. Mechanisms of protein osmolyte interactions, the effect of osmolytes on protein stability, and how osmo- lytes correct protein misfolding defects and remove protein aggregation have been widely investigated. It has been demonstrated that the unfavourable interaction between the peptide backbone and the osmolytes leading to the preferential hydration of the protein domain is the driving force of protein stabiliza- tion or folding [3,4]. Furthermore, the effect of osmolytes on the functional activity of an enzyme has also been investigated on a number of enzymes. Conse- quently, this has led to the classification of osmolytes into two classes: compatible or counteracting. Compat- ible osmolytes increase protein stability against denaturation with little or no effect on their function under native conditions [1,13,14]. Representatives of this class include certain amino acids (e.g. proline and glycine) and polyols (e.g. trehalose, sucrose and sorbitol). Counteracting osmolytes consist of the methylamine class of osmolytes, which are believed to have the special ability to protect intracellular proteins against the inactivation ⁄ destabilization by urea [14–17]. In contrast to compatible osmolytes, counteracting osmolytes are believed to cause changes in protein function that are opposite to the effects that urea has on protein function [16–19]. Despite significant advances in understanding the effect of osmolytes on protein stability, folding and the activity of proteins and enzymes, the relationship between protein stabilization by osmolytes and its con- sequent effects on the activity of enzymes has not been examined. It is not yet understood how well protein stability and activity are coupled in the presence of an osmolyte. This study was undertaken to investigate the relationship between protein stability and activity changes in the presence of a wide range of osmolytes. For this we evaluated the protein stability (DG D °, Gibbs free energy change at 25 °C) of RNase-A and its activity parameters ( K m , Michaelis constant; k cat , catalytic constant) in the presence and absence of almost all naturally occurring osmolytes. We report here that protein stability and activity are not largely coupled in the presence of osmolytes. However, protein stability and activity have a linear correlation in the presence of methylamines and sugar osmolytes. This study, in fact, has led to the classification of osmolytes into three different classes based on their effects on stability and activity parameters of RNase-A. Results and Discussion Protein stability and enzyme activity have a well-corre- lated function. However, we do not know how this relationship is maintained in the presence of stabilizing osmolytes accumulated under stressed conditions. Because stabilizing osmolytes do not have a direct interaction with the protein domains per se,itis expected that an increase in protein stability (DG D °)by an osmolyte due to the shift in the denaturation equi- librium, native state M denatured state, towards the left, must increase the catalytic efficiency of the enzyme and vice versa. The reason for saying this is that urea, which decreases DG D °, is known to decrease the cata- lytic efficiency of osmolytes [20, references therein]. Thus, it will be interesting to investigate how kinetic parameters of the enzyme-catalyzed reaction change upon modulation of protein stability (DG D °) by osmo- lytes. To investigate the protein stability–activity rela- tionship in the presence of osmolytes, we intentionally chose two different groups of osmolytes. The first group consists of polyols, amino acids and amino acid derivatives, which have been reported to have no effect on DG D ° associated with the protein denaturation equilibrium, native state M denatured state, under physiological conditions. The second group consists of methylamines and sugars, which are shown to increase DG D ° of proteins associated with the denaturation equilibrium, native state M denatured state. The observed effects of polyols, sugars and methylamines and some amino acids on DG D ° of RNase-A have been reported previously [21–25], and DG D ° values in the presence of these osmolytes are given in Table 1. How- ever, DG D ° values of RNase-A in the presence of alanine, serine, lysine, b-alanine, taurine and dimethyl- glycine have not been published elsewhere. We have therefore measured the thermodynamic parameters of RNase-A in the presence of these amino acids and amino acid derivatives, and values of DG D °, measured in triplicate, are given in Table 1. The effect of polyols on the kinetic parameters (K m and k cat ) of the RNase-A mediated hydrolysis of cyti- dine 2¢-3¢ cyclic monophosphate has been previously reported [22]. Values of the kinetic parameters of this protein in the presence of all other osmolytes were determined and are presented in Table 1. It should be noted that the value for each kinetic parameter repre- sents the mean of three independent measurements together with the mean error. These kinetic parameters in the absence of the osmolytes, shown in Table 1, are in excellent agreement with those reported previously [26–28]. These agreements led us to believe that our measurements of the enzyme-catalyzed reactions and S. Jamal et al. Functional stability and activity by osmolytes FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS 6025 Table 1. Stability and activity parameters of RNase-A in the presence of different classes of osmolytes at physiological conditions. Values are from triplicate measurements. DG D ° values were taken from [21] for class III, from [22,23] for class I and from [24,25] for class II. Class III Class I Class II [Sugars] M DG D ° (kcalÆmol )1 ) k cat (s )1 ) K m (mM) [Polyols] M DG D ° (kcalÆmol )1 ) k cat (s )1 ) K m (mM) [Amino acids and derivatives] M DG D ° (kcalÆmol )1 ) k cat (s )1 ) K m (mM) [Methylamines] M DG D ° (kcalÆmol )1 ) k cat (s )1 ) K m (mM) 0.00 9.83 3.10 ± 0.08 1.05 ± 0.04 0.00 10.60 3.22 ± 0.35 1.33 ± 0.15 0.00 9.83 3.10 ± 0.08 1.05 ± 0.04 0.00 9.83 3.10 ± 0.08 1.05 ± 0.04 Glucose Sorbitol Alanine Sarcosine 0.50 10.31 3.07 ± 0.07 1.03 ± 0.02 0.55 10.67 3.17 ± 0.23 1.36 ± 0.24 0.25 9.92 3.07 ± 0.08 1.05 ± 0.11 0.25 10.25 3.39 ± 0.12 0.92 ± 0.02 1.00 10.79 2.62 ± 0.08 0.83 ± 0.04 1.10 10.72 3.28 ± 0.12 1.33 ± 0.14 0.50 9.96 2.97 ± 0.12 1.02 ± 0.15 0.50 10.70 3.71 ± 0.11 0.80 ± 0.05 1.50 11.32 2.39 ± 0.07 0.71 ± 0.03 1.65 10.57 3.25 ± 0.18 1.38 ± 0.18 0.75 11.20 4.03 ± 0.11 0.71 ± 0.04 2.00 11.77 2.00 ± 0.06 0.53 ± 0.03 2.20 10.65 3.22 ± 0.30 1.30 ± 0.21 1.00 11.60 4.21 ± 0.14 0.58 ± 0.03 Fructose Glycerol Proline Dimethylglycine 1.00 10.84 2.47 ± 0.11 0.81 ± 0.03 1.09 10.50 3.25 ± 0.22 1.25 ± 0.17 0.25 9.83 3.07 ± 0.07 1.03 ± 0.08 0.25 10.06 3.29 ± 0.10 0.99 ± 0.04 1.50 11.39 2.24 ± 0.09 0.69 ± 0.02 2.17 10.67 3.17 ± 0.17 1.34 ± 0.12 0.50 9.70 2.98 ± 0.10 1.01 ± 0.13 0.50 10.31 3.41 ± 0.09 0.95 ± 0.07 2.00 11.79 1.93 ± 0.07 0.58 ± 0.02 3.26 10.56 3.30 ± 0.28 1.31 ± 0.15 1.00 9.77 3.25 ± 0.09 1.11 ± 0.07 0.75 10.58 3.65 ± 0.12 0.88 ± 0.03 2.50 12.18 1.61 ± 0.05 0.42 ± 0.03 4.35 10.53 3.48 ± 0.42 1.43 ± 0.20 1.50 9.80 3.29 ± 0.09 1.07 ± 0.06 1.00 10.93 3.92 ± 0.12 0.79 ± 0.05 Galactose Xylitol Serine Betaine 0.50 10.31 3.05 ± 0.08 1.02 ± 0.03 0.25 10.49 3.15 ± 0.20 1.41 ± 0.16 0.25 9.74 2.91 ± 0.12 1.00 ± 0.15 0.25 9.96 3.22 ± 0.11 1.02 ± 0.03 0.75 10.55 2.87 ± 0.07 0.91 ± 0.04 0.50 10.57 3.32 ± 0.17 1.32 ± 0.19 0.50 9.84 3.02 ± 0.08 1.05 ± 0.10 0.50 10.19 3.37 ± 0.11 0.99 ± 0.04 1.00 10.74 2.68 ± 0.06 0.81 ± 0.03 0.75 10.61 3.22 ± 0.12 1.35 ± 0.08 0.75 10.40 3.46 ± 0.10 0.92 ± 0.03 1.00 10.67 3.25 ± 0.40 1.39 ± 0.19 1.00 10.81 3.62 ± 0.12 0.83 ± 0.06 Sucrose Adonitol Lysine Trimethylamine N-oxide 0.50 10.55 3.01 ± 0.10 1.00 ± 0.04 0.25 10.41 3.12 ± 0.20 1.41 ± 0.13 0.25 9.82 3.05 ± 0.15 1.06 ± 0.18 0.25 10.07 3.34 ± 0.09 0.96 ± 0.03 1.00 11.17 2.50 ± 0.11 0.78 ± 0.02 0.50 10.68 3.18 ± 0.30 1.29 ± 0.16 0.50 9.87 3.08 ± 0.10 1.04 ± 0.12 0.50 10.54 3.61 ± 0.12 0.87 ± 0.04 1.50 11.94 2.10 ± 0.06 0.61 ± 0.03 0.75 10.64 3.33 ± 0.17 1.33 ± 0.11 0.75 10.95 3.83 ± 0.09 0.76 ± 0.03 1.00 10.75 3.15 ± 0.33 1.32 ± 0.09 1.00 11.48 4.13 ± 0.14 0.65 ± 0.05 Raffinose Mannitol Glycine 0.10 10.00 3.07 ± 0.04 1.03 ± 0.02 0.25 10.51 3.25 ± 0.20 1.36 ± 0.13 0.25 9.89 3.17 ± 0.07 1.04 ± 0.05 0.20 10.19 2.94 ± 0.03 0.93 ± 0.02 0.50 10.54 3.18 ± 0.15 1.30 ± 0.12 0.50 10.03 3.11 ± 0.10 1.05 ± 0.04 0.30 10.38 2.85 ± 0.05 0.87 ± 0.03 0.75 10.64 3.25 ± 0.20 1.37 ± 0.16 1.00 10.17 3.05 ± 0.10 1.07 ± 0.07 0.40 10.50 – – 1.00 10.62 3.23 ± 0.32 1.33 ± 0.08 Stachyose b-Alanine 0.25 10.38 2.81 ± 0.07 0.91 ± 0.03 0.25 9.85 3.14 ± 0.08 1.07 ± 0.06 0.50 10.86 2.61 ± 0.08 0.76 ± 0.04 0.50 9.87 3.10 ± 0.07 1.04 ± 0.05 0.75 11.41 – – 1.00 9.90 3.05 ± 0.09 1.05 ± 0.05 Taurine 0.25 9.80 3.18 ± 0.08 1.08 ± 0.05 0.50 9.86 3.09 ± 0.10 1.03 ± 0.06 Functional stability and activity by osmolytes S. Jamal et al. 6026 FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS the analysis of the progress curves for kinetic parame- ters are accurate. It can be seen in Fig. 1 (see also Table 1) that sugars and methylamines affect both the thermodynamic (DG D °) and the kinetic (K m and k cat ) properties, whereas polyols, amino acids and amino acid derivatives do not have any significant effect on these parameters. In fact, based on the effects that the osmolytes have on both DG D ° and the catalytic prop- erties of RNase-A (Table 1), we can distinctly classify osmolytes into three different classes. (a) Class I includes polyhydric alcohols (sorbitol, glycerol, xylitol, adonitol, mannitol) and amino acids and derivatives (glycine, alanine, proline, serine, lysine, b-alanine and taurine) that have no significant effects on both DG D ° and k cat . (b) Class II represents methylamines (sarco- sine, dimethylglycine, betaine, trimethylamine N-oxide) that increase both DG D ° and k cat , but decrease K m . (c) Sugars (glucose, fructose, galactose, sucrose, raffinose, stachyose) that increase DG D °, but decrease both K m and k cat belong to class III. k cat alone does not absolutely define the overall cata- lytic activity of an enzyme, as it is a first-order rate constant that refers to the properties and reactions of the enzyme–substrate, enzyme–intermediate and enzyme–product complexes [29]. On the other hand, k cat ⁄ K m is an apparent second-order rate constant that refers to the properties and the reaction of the free enzyme and free substrate [29]. We have therefore esti- mated k cat ⁄ K m values of all the reactions in the pres- ence and absence of all classes of osmolytes. It can be Fig. 1. Effect of osmolytes on enzyme kinetic parameters. Plot of Dk cat of RNase-A versus [osmolyte] (left panels) and DK m of RNase-A versus [osmolyte] (right panels). S. Jamal et al. Functional stability and activity by osmolytes FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS 6027 seen in Fig. 1 that class I osmolytes (polyhydric alco- hols, amino acids and amino acid derivatives) do not significantly perturb kinetic parameters (K m and k cat ) and, hence, the overall catalytic efficiency (k cat ⁄ K m )of RNase-A. This observation on the effect of polyols and amino acids on RNase-A is in agreement with that on other enzymes (lactate dehydrogenase, lysozyme, pyruvate kinases) reported previously [13,22,30]. It has been argued that these compatible osmolytes affect the association of the substrate with the enzyme in any one of several ways, e.g. through solvation effects on substrates or enzyme active sites and through their effects on the thermodynamic activity of substrates and enzymes [13,30,31]. Thus, a lack of effect on both enzymatic parameters (K m and k cat ) of RNase-A sug- gests that polyols, amino acids and amino acid deriva- tives have little or no effect on the solvation properties of the substrate and the enzyme active sites or on their thermodynamic activities. Another explanation for these observations comes from our DG D ° measure- ments. Because of perfect enthalpy–entropy compensa- tion, DG D ° is unperturbed in the presence of class I osmolytes (see Table 1), i.e. the denaturation equilib- rium, native state M denatured state, of RNase-A is unperturbed and, hence, no change in the functional activity of the enzyme in the presence of such osmo- lytes (see Fig. 1). If our explanation is correct, an increase in protein stability (DG D °) by osmolytes must result in an increase in the number of N molecules due to a shift in the denaturation equilibrium, native state M dena- tured state, towards the left. Consequently, both k cat and k cat ⁄ K m are expected to increase in the presence of such osmolytes, as k cat ⁄ K m refers to the reaction of free (active) enzyme [29]. Data presented in Table 1 and Fig. 2 for the effect of methylamines (class II) on DG D ° and kinetic parameters show that this is indeed true. It is noteworthy that our observation of the effect of methylamines on RNase-A is also in agreement with previous reports on many other enzymes, such as rab- bit muscle lactate dehydrogenase, triose phosphate isomerase, pyruvate kinase, creatine kinase, A4-lactate dehydrogenase, glutamate dehydrogenase, argininosuc- cinate lyase, porcine arginosuccinase [17,19,32–35]. However, it should be noted that both K m , the overall dissociation constant of all enzyme bound species [29], and k cat are decreased in the presence of sugar (class III) osmolytes (see Fig. 1, Table 1). One possible explanation for this observation is that the original native state ensembles and ⁄ or the refolded protein molecules in the presence of sugars undergo a subtle change in conformation, yielding all or some enzyme bound species that are more stable than those in the absence of sugars, i.e. K m is decreased. On the other hand, this change in conformation results in a decrease in k cat , the turnover number of the enzyme in the pres- ence of sugars, i.e. the maximum number of substrate molecules converted to product per active site per unit time is decreased. A subtle change in the enzyme active site that occurs in the presence of sugars may be a pos- sible cause for the observations on K m and k cat of RNase-A in the presence of class III osmolytes. To evaluate if all the refolded protein fractions produced by an osmolyte are in functionally active conformation, we determined the relationship between changes in protein stability (DDG D °) and overall catalytic efficiency (Dlog(k cat ⁄ K m )) in the presence of Fig. 2. Relationship between protein stability and catalytic effi- ciency. Plot of Dlog(k cat ⁄ K m ) versus DDG D ° of RNase-A obtained in the presence of various osmolytes. Functional stability and activity by osmolytes S. Jamal et al. 6028 FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS different concentrations of each osmolyte (Fig. 2). It can be seen in Fig. 2 that for class I osmolytes (poly- ols, amino acids and amino acid derivatives) the slope is nearly 0. This is an expected result, as there is no perturbation of the denaturation equilibrium and, hence, there is no increase in catalytic efficiency in the presence of this group of osmolytes. Interestingly, there is a linear relationship between DDG D ° and Dlog(k cat ⁄ K m ) in the presence of methylamines and sugar. However, the slopes of the plot (Dlog(k cat ⁄ K m ) versus DDG D °) are very different. In fact, the slope in the presence of sugar osmolytes is 10 times less than that in the presence of methylamines. A higher slope in the case of methylamines will mean that the total refolded protein fraction generated by the methylam- ines is more active than those generated by sugars. Taking these observations and k cat values of RNase-A in the presence of class II and III osmolytes, it seems that the refolded protein fraction in the presence of sugars is not as active as the original native molecules, whereas it is opposite in the presence of methylamines. We therefore conclude that equilibrium shift is not the only ultimate step to increase the activity of an enzyme in the presence of osmolytes. In general, two thermodynamic models are used to explain the effect of osmolytes on protein stability [36, references therein]. The binding model claims that an increase in the osmolyte-induced stability arises from the preferential hydration (or exclusion of the osmo- lyte) leading to a shift in the denaturation equilibrium, native state M denatured state, towards the left. The excluded volume model focuses on the fact that osmo- lytes limit the conformational freedom of proteins by driving them to their most compact native state (cata- lytically most efficient form). The decrease in confor- mational freedom arises from steric repulsions between the protein and the osmolyte. The latter model assumes that the native state of a protein consists of inter-converting high (most compact) and low (less compact) activity state ensembles and also demon- strated that the presence of osmolytes shifts the native conformational equilibria towards the most compact protein species within native state ensembles [32,37,38]. The variation in the effect of stabilizing osmolytes in modulating the catalytic efficiency of RNase-A in the presence of each class of osmolyte may best be explained by the combination of both thermodynamic models. Our results suggest that: (a) methylamines not only decrease conformational freedom, but also increase preferential hydration, which consequently generates more active protein molecules; (b) sugar osmolytes affect the conformational freedom and preferential hydration in such a way that it produces catalytically less competent species; and (c) class I osmolytes have no significant effects on both the conformational freedom and the preferential hydration of the protein. In agreement with the explanation on methylamines, previous reports on trimethylamine N-oxide indicate that it not only produces more active molecules by shifting the denaturation equilibrium [24,25,36,39], but also affects the native state by con- verting the low activity ensembles to the high activity ensembles [37]. Very interestingly, a recent refolding kinetic study of carbonic anhydrase II in sucrose showed that the sugar significantly accelerates the rate of refolding of the enzyme to the native or compact near-native conformations, but decreases the fraction of catalytically active enzyme recovered [40]. It has already been reported that osmolytes indepen- dently affect proteins and, hence, their effects are algebraically additive [21,41]. Based on our results given in Table 1, one can speculate that: (a) the poly- ols–amino acids (or amino acid derivatives) system is an exclusive mixture that is compatible both with thermodynamic stability ( DG D °) and function, and (b) sugar–methylamine mixtures are attractive candidates to yield amazingly enhanced protein stability and function. Thus, different osmolyte mixtures may serve as post-translational modulators of stability and ⁄ or function of many enzymes. This may perhaps be the main reason why many organisms use multi-osmolyte systems [1,14,15,42–44]. Furthermore, the osmolyte-induced folding of pro- teins is determined by interactions of the osmolyte with all protein groups (peptide backbone and side chains) exposed on denaturation. For various osmo- lytes, Bolen & Baskakov [3] have shown that: (a) the main driving force for the folding is the unfavourable interaction between the osmolyte and the peptide back- bone, and (b) the total contribution of side chains to the stability of the native state, which may interact differently with different osmolytes, is very small. These conclusions are supported by our measurements of DG D ° of RNase-A in the absence and presence of sugars and methylamines. It is seen in Table 1 that, on the molar scale, these osmolytes, which are chemically different, have, within experimental errors, almost identical effects on DG D °. We are confident of three findings: (a) Polyols, amino acids and amino acid derivatives are ideal osmolytes, for they neither perturb the denaturation equilibrium nor affect the functional activity under native conditions. However, they have the ability to protect proteins from denaturing stresses. (b) Methyl- amines not only stabilize proteins, but also refold the denatured protein to a more active state under native S. Jamal et al. Functional stability and activity by osmolytes FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS 6029 conditions. (c) Sugar osmolytes stabilize proteins, but they convert the denatured protein molecule to a less active form under native conditions. These findings make these chemical chaperones aptly suitable for structure–function studies of proteins, as each class of osmolytes (classes I–III) can modulate the stability and ⁄ or function of a protein differently. Experimental procedures Chemicals Commercial lyophilized preparations of RNase-A (type III-A) were purchased from Sigma Chemical Company (St Louis, MO, USA). d-glucose, d-fructose, d-galactose, d-sucrose, d-raffinose, d-stachyose, glycine, l-alanine, l-proline, l-lysine, l-serine, b-alanine, taurine, sarcosine, dimethylglycine, glycine betaine, trimethylamine N-oxide, and cytidine 2¢-3¢ cyclic monophosphate were also obtained from Sigma. These and other chemicals, which were of analytical grade, were used without further purification. Dialysis and the determination of the concentration of protein An RNase-A solution was dialyzed extensively against 0.1 m KCl solution at  4 °C. Protein stock solutions were filtered using 0.45 lm Millipore filter paper. The protein gave a single band during the native and SDS poly- acrylamide gel electrophoresis. The concentration of the protein stock solution was determined experimentally using a value of 9800 at 277.5 nm for e, the molar absorption coefficient (m )1 Æcm )1 ) [45]. All solutions for activity measurements were prepared in 0.05 m cacodylic acid buffer containing 0.1 m KCl. Because the pH of the protein solu- tion may change on the addition of the osmolytes, the pH of each solution was also measured after each measurement. It was observed that the change in pH was not significant. Activity measurements In order to see the effect of an osmolyte on the kinetic parameters (K m and k cat ) of RNase-A, the substrate and the enzyme were preincubated in a given concentration of each osmolyte. Following the procedure described previ- ously [22], RNase-A activity using cytidine 2¢-3¢ cyclic monophosphate as a substrate was measured. The progress curve for RNase-A mediated hydrolysis of cytidine 2¢-3¢ cyclic monophosphate in the concentration range 0.05– 0.50 mgÆmL )1 in the absence and presence of a given concentration of each osmolyte was followed by measuring the change in absorbance at 292 nm for 20 min in a Jasco V-560 UV ⁄ Vis spectrophotometer (Hachioji, Tokyo, Japan). Sample and reference cells were maintained at 25.0 ± 0.1 °C. From each progress curve at a given sub- strate concentration and in the absence and presence of a fixed osmolyte concentration, initial velocity (m) was deter- mined from the linear portion of the progress curve, usually 30 s. The plot of initial velocity (m) versus [S] (in mm)at each osmolyte concentration was analysed for K m and k cat using Eqn (1). v ¼ k cat ½S=ðK m þ½SÞ ð1Þ Thermal denaturation measurements Thermal denaturation studies were carried out in a Jasco V-560 UV ⁄ Vis spectrophotometer equipped with a Peltier- type temperature controller (ETC-505T), with a heating rate of 1 °CÆmin )1 . The change in absorbance with increas- ing temperature was followed at 287 nm for RNase-A. Approximately 650 data points of each transition curve were collected. The raw absorbance data were converted into (De 287 ), the difference molar absorption coefficient (m )1 Æcm )1 ). Each heat-induced transition curve (plot of De 287 versus temperature; not shown) was analysed for T m (midpoint of denaturation) and DH m (enthalpy change at T m ) using a nonlinear least squares analysis according to the relationship described earlier (see equation (1) in [25]). Using a value of 1.24 kcalÆmol )1 ÆK )1 for DC p (the constant pressure heat capacity change in RNase-A; [39]), DG D (T), the value of DG D at any temperature T was estimated using the Gibbs–Helmholtz equation with known values of T m , DH m and DC p using the relationship described previously (see equation (2) in [25]). Acknowledgements FA is grateful to the Department of Science and Technology (India) and the Council of Scientific and Industrial Research (India) for financial support. References 1 Yancey PH, Clark ME, Hand SC, Bowlus RD & Somero GN (1982) Living with water stress: evolution of osmolyte system. Science 217, 1212–1222. 2 Borowitzka LJ (1985) Glycerol and other carbohydrate osmotic effectors. In Transport Processes, Iono- and Osmoregulation (Gilles R & Gilles-Baillen M eds) pp. 437–453. Springer, Berlin. 3 Bolen DW & Baskakov IV (2001) The osmophobic effect: natural selection of a thermodynamic force in protein folding. 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