Table of Contents Author Guidelines Submit a Manuscript
Enzyme Research
Volume 2010, Article ID 165878, 10 pages
http://dx.doi.org/10.4061/2010/165878
Research Article

Purification, Characterization, and Effect of Thiol Compounds on Activity of the Erwinia carotovora L-Asparaginase

Biotechnology Research Laboratory, School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded 431606, India

Received 22 May 2009; Accepted 31 July 2009

Academic Editor: Roberto Fernandez-Lafuente

Copyright © 2010 Suchita C. Warangkar and Chandrahas N. Khobragade. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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 3 7 ยฑ 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 ๐œ‡ M) showed an increase in V m a x values (2000, 2223, 2380, 2500, and control 1666.7  ๐œ‡ moles m g โˆ’ 1 m i n โˆ’ 1 ) and a decrease in K ๐‘š 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 m a x / K ๐‘š 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).