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

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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: Bovine erythrocytes; Enzyme kinetics; Enzyme purification; Glutathione S-transferase

Document Type: Research Article

DOI: http://dx.doi.org/10.2174/092986608783330332

Publication date: January 1, 2008

More about this publication?
  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
ben/ppl/2008/00000015/00000001/art00002
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