Purification, Characterization, and Effect of Thiol Compounds on Activity of theErwinia carotovoraL-Asparaginase

Abstract

L-asparaginase was extracted fromErwinia carotovoraand purified by ammonium sulfate fractionation (60–70%), Sephadex G-100, CM cellulose, and DEAE sephadex chromatography. The apparent Mr of enzyme under nondenaturing and denaturing conditions was 150 kDa and  kDa, respectively. L-asparaginase activity was studied in presence of thiols, namely, L-cystine (Cys), L-methionine (Met), N-acetyl cysteine (NAC), and reduced glutathione (GSH). Kinetic parameters in presence of thiols (10–400 M) showed an increase in values (2000, 2223, 2380, 2500, and control 1666.7 moles ) and a decrease in values (0.086, 0.076, 0.062, 0.055 and control 0.098 mM) indicating nonessential mode of activation. values displayed propensity to bind thiols. A decrease in ratio in concentration plots showed inverse relationship between free thiol groups (NAC and GSH) and bound thiol group (Cys and Met). Enzyme activity was enhanced in presence of thiol protecting reagents like dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and GSH, but inhibited by p-chloromercurybenzoate (PCMB) and iodoacetamide (IA).