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Abstract: Thermal denaturation curves of ribonuclease-A were measured by monitoring changes in the far-UV circular dichroism (CD) spectra in the presence of different concentrations of six sugars (glucose, fructose, galactose, sucrose, raffinose and stachyose) and mixture of monosaccharide constituents of each oligosaccharide at various pH values in the range of 6.0-2.0. These measurements gave values of T m (midpoint of denaturation), ΔH m (enthalpy change at T m), ΔC p (constant-pressure heat capacity change) under a given solvent condition. Using these values of ΔH m, T m and ΔC p in appropriate thermodynamic relations, thermodynamic parameters at 25 °C, namely, ΔG D o (Gibbs energy change), ΔH D o (enthalpy change), and ΔS D o (entropy change) were determined at a given pH and concentration of each sugar (including its mixture of monosaccharide constituents). Our main conclusions are: (i) each sugar stabilizes the protein in terms of T m and ΔG D o, and this stabilization is under enthalpic control, (ii) the protein stabilization by the oligosaccharide is significantly less than that by the equimolar concentration of the constituent monosaccharides, and (iii) the stabilization by monosaccharides in a mixture is fully additive. Furthermore, measurements of the far- and near-UV CD spectra suggested that secondary and tertiary structures of protein in their native and denatured states are not perturbed on the addition of sugars. [Copyright &y& Elsevier]

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 ΔH m (enthalpy change at T m), and these ΔH m and T m values obtained at different pH values were used to obtain ΔC p (constant-pressure heat capacity change) at each polyol concentration. Using values of ΔH m, T m and ΔC p in the Gibbs–Helmholtz equation, ΔG D° (Gibbs energy change at 25 °C) was determined at a given pH and polyol concentration. Main conclusions of this study are that polyols have no significant effect on ΔG D° at pH 7.0, and they stabilise proteins in terms of ΔG D° 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) ΔH m remains, within experimental error, unperturbed in the presence of polyols, and (iii) ΔC 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. [Copyright &y& Elsevier]

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.