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ABSTRACT: The intra- and extracellular urea concentration in many organisms is sufficiently high to destabilize (inhibit) many proteins, yet organisms survive and function. The generally accepted explanation is the counteracting hypothesis, which holds that methylamines stabilize proteins and oppose the deleterious effect of urea. The two osmolytes are typically found at 2:1 concentration ratio (urea:methylamine) under physiological conditions. Does this mean that this ratio holds for all proteins in a cell? The present study tests the counteracting hypothesis by determining the effects of urea and methylamines (trimethylamine N-oxide and sarcosine), singly and in combination at a concentration ratio of 2:1 (urea:methylamine) on the thermal denaturation equilibrium, native state<-->denatured state of three different proteins (alpha-lactalbumin, lysozyme and Ribonuclease-A). We show here that the molar concentration of a methylamine required to offset the denaturing effect of urea at a given concentration is different for different proteins.
Biochim Biophys Acta. 12/2007; 1774:1555-62.
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ABSTRACT: We have recently concluded from the heat-induced denaturation studies that polyols do not affect deltaG(D) degrees (the Gibbs free energy change (deltaG(D)) at 25 degrees C) of ribonuclease-A and lysozyme at physiological pH and temperature, and their stabilizing effect increases with decrease in pH. Since the estimation of deltaG(D) degrees of proteins from heat-induced denaturation curves requires a large extrapolation, the reliability of this procedure for the estimation of deltaG(D) degrees is always questionable, and so are conclusions drawn from such studies. This led us to measure deltaG(D) degrees of ribonuclease-A and lysozyme using a more accurate method, i.e., from their isothermal (25 degrees C) guanidinium chloride (GdmCl)-induced denaturations. We show that our earlier conclusions drawn from heat-induced denaturation studies are correct. Since the extent of unfolding of heat- and GdmCl-induced denatured states of these proteins is not identical, the extent of stabilization of the proteins by polyols against heat and GdmCl denaturations may also differ. We report that in spite of the differences in the structural nature of the heat- and GdmCl-denatured states of each protein, the extent of stabilization by a polyol is same. We also report that the functional dependence of deltaG(D) of proteins in the presence of polyols on denaturant concentration is linear through the full denaturant concentration range. Furthermore, polyols do not affect the secondary and tertiary structures of the native and GdmCl-denatured states.
Biophys Chem. 02/2006; 119:224-33.
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ABSTRACT: Thermal denaturation curves of lysozyme and ribonuclease-A were determined by measuring their far-UV circular dichroism (CD) spectra in the presence of different concentrations of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at various pH values in the range 7.0--1.9. The denaturation curve at each polyol concentration and pH was analysed to obtain values of T(m) (midpoint of denaturation) and DeltaH(m) (enthalpy change at T(m)), and these DeltaH(m) and T(m) values obtained at different pH values were used to obtain DeltaC(p) (constant-pressure heat capacity change) at each polyol concentration. Using values of DeltaH(m), T(m) and DeltaC(p) in the Gibbs-Helmholtz equation, DeltaG(D) degrees (Gibbs energy change at 25 degrees C) was determined at a given pH and polyol concentration. Main conclusions of this study are that polyols have no significant effect on DeltaG(D) degrees at pH 7.0, and they stabilise proteins in terms of DeltaG(D) degrees against heat denaturation at lower pH values. Other conclusions of this study are: (i) T(m) at each pH increases with increasing polyol concentration, (ii) DeltaH(m) remains, within experimental error, unperturbed in the presence of polyols, and (iii) DeltaC(p) depends on polyol concentration. Furthermore, measurements of the far- and near-UV CD spectra suggested that secondary and tertiary structures of both proteins in their native and denatured states are not perturbed on the addition of polyols.
Biophys Chem. 08/2005; 117:1-12.
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ABSTRACT: It is generally believed that compatible osmolytes stabilize proteins by shifting the denaturation equilibrium, native state <--> denatured state toward the left. We show here that if osmolytes are compatible with the functional activity of the protein at a given pH and temperature, they should not significantly perturb this denaturation equilibrium under the same experimental conditions. This conclusion was reached from the measurements of the activity parameters (K(m) and k(cat)) and guanidinium chloride-induced denaturations of lysozyme and ribonuclease-A in the presence of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at pH 7.0 and 25 degrees C.
FEBS Lett. 07/2005; 579:3891-8.
