title: Conformational stability of alpha-amylase from malted sorghum (Sorghum bicolor): reversible unfolding by denaturants.
abstract: Alpha-amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50mM Hepes containing 13.6mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27 degrees C is characterized by two state transition with DeltaG (H(2)O) of 16.5 kJ mol(-1) and 22 kJ mol(-1), respectively, at pH 4.8 and pH 7.0 for GuHCl and DeltaG (H(2)O) of 25.2 kJ mol(-1) at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (DeltaC(p)), the unfolding enthalpy (H(g)) and the temperature at DeltaG=0 (T(g)) 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+/-4.8K 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 DeltaG (H(2)O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.
authors.affiliation: Department of Protein Chemistry and Technology, Central Food Technological Research Institute, CSIR, Princess Road, Mysore, Karnataka 570 020, India.
authors.forename: R Siva
authors.initials: RS
authors.lastname: Sai Kumar
authors.forename: Sridevi Annapurna
authors.initials: SA
authors.lastname: Singh
authors.forename: A G Appu
authors.initials: AG
authors.lastname: Rao
mesh_terms: D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry; D010944:Plants; D017510:Protein Folding / Q000187:drug effects; D045868:Sorghum / Q000201:enzymology*; D013696:Temperature; D014508:Urea / Q000737:chemistry; D000516:alpha-Amylases / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D014508:Urea; D000516:alpha-Amylases; D019791:Guanidine