Purification, Characterization, and Effect of Thiol Compounds on Activity of the Erwinia carotovora L-Asparaginase.
L-asparaginase was extracted from Erwinia carotovora and 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 37 ± 0.5 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 μM) showed an increase in V(max) values (2000, 2223, 2380, 2500, and control 1666.7 μmoles mg(-1)min(-1)) and a decrease in K(m) values (0.086, 0.076, 0.062, 0.055 and control 0.098 mM) indicating nonessential mode of activation. K(A) values displayed propensity to bind thiols. A decrease in V(max)/K(m) 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).
Biotechnology Research Laboratory, School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded 431606, India.
Suchita C
SC
Warangkar
Chandrahas N
CN
Khobragade