Conformational stability of α-amylase from malted sorghum (Sorghum bicolor): Reversible unfolding by denaturants

α-Amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50 mM Hepes containing 13.6 mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27 °C is characterized by two state transition with ΔG (H2O) of 16.5 kJ mol−1 and 22 kJ mol−1, respectively, at pH 4.8 and pH 7.0 for GuHCl and ΔG (H2O) of 25.2 kJ mol−1 at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (ΔCp), the unfolding enthalpy (Hg) and the temperature at ΔG = 0 (Tg) are 17.9 ± 0.7 kJ mol−1 K−1, 501.2 ± 18.2 kJ mol−1 and 337.3 ± 6.9 K at pH 4.8 and 14.3 ± 0.5 kJ mol−1 K−1, 509.3 ± 21.7 kJ mol−1 and 345.4 ± 4.8 K at pH 7.0, respectively. The reactivity of the conserved cysteine residues, during unfolding, indicates that unfolding starts from the 'B' domain of the enzyme. The oxidation of cysteine residues, during unfolding, can be prevented by the addition of DTT. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. The pH dependent stability described by ΔG (H2O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.