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
Protein & Peptide Letters
Title: Determination of Some Kinetic and Characteristic Properties of Glutathione S-transferase from Bovine Erythrocytes
Volume: 15 Issue: 1
Author(s): Mustafa Erat, Serdar Guvercin and Halis Sakiroglu
Affiliation:
Keywords: Glutathione S-transferase, Enzyme purification, Enzyme kinetics, Bovine erythrocytes
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].
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Erat Mustafa, Guvercin Serdar and Sakiroglu Halis, Determination of Some Kinetic and Characteristic Properties of Glutathione S-transferase from Bovine Erythrocytes, Protein & Peptide Letters 2008; 15 (1) . https://dx.doi.org/10.2174/092986608783330332
DOI https://dx.doi.org/10.2174/092986608783330332 |
Print ISSN 0929-8665 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5305 |
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