Determination of Some Kinetic and Characteristic Properties of Glutathione S-transferase from Bovine Erythrocytes

Mustafa      Erat; Serdar      Guvercin; Halis      Sakiroglu

doi:10.2174/092986608783330332

Abstract

Glutathione S-transferase was purified from bovine erythrocytes and some kinetic and characteristic properties of the enzyme were investigated. The purification procedure was composed of preparation of homogenate and Glutathione- Agarose affinity chromatography. Thanks to the procedure, the enzyme was purified 6,800 fold with 97% yield and a specific activity of 136 EU/mg proteins. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), one band with a mass of 27 kDa was found. The native molecular weight of the enzyme was found to be approximately 53 kDa by Sephadex G-100 gel filtration chromatography. Optimum pH, stable pH, optimum temperature, and optimum ionic strength were determined as 7.0, 6.5 in K-phosphate buffer, 20 °C, 0.1 M K-phosphate, respectively. The best activity was obtained with 1-chloro-2,4-dinitrobenzene (CDNB) in a study performed with different substrates. Vmax, Km, and kcat values were calculated as 402.63 ± 4.99 EU/mg proteins, 0.7447 ± 0.0007 mM, and 11436 min-1 for CDNB, and 88.00 ± 2.30 EU/mg proteins, 0.3257 ± 0.0012 mM, and 477 min-1 for GSH, respectively, by using Lineweaver-Burk graphs obtained from 1/V versus 1/[CDNB] and 1/[GSH].

Keywords: Glutathione S-transferase, Enzyme purification, Enzyme kinetics, Bovine erythrocytes

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