Determination of some kinetic and characteristic properties of glutathione S-transferase from bovine erythrocytes

GUVERCIN S., ERAT M., Sakiroglu H.

PROTEIN AND PEPTIDE LETTERS, cilt.15, sa.1, ss.6-12, 2008 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 15 Sayı: 1
  • Basım Tarihi: 2008
  • Doi Numarası: 10.2174/092986608783330332
  • Dergi Adı: PROTEIN AND PEPTIDE LETTERS
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.6-12
  • Anahtar Kelimeler: glutathione, S-transferase, enzyme purification, enzyme kinetics, bovine erythrocytes, MOUSE-LIVER, PURIFICATION, PEROXIDASE, PROTEINS, AFFINITY, BINDING, LUNG, IDENTIFICATION, ISOENZYMES, ISOZYMES
  • Atatürk Üniversitesi Adresli: Evet

Özet

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 degrees 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. V-max, K-m, and k(cat) 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].