:-i ; ELSEVIER Journal of ChromatographyB, 688 (1997) 239-243 JOURNAL OF CHROMATOGRAPHY B Use of thiopropyl Sepharose for the synthesis of an adsorbent for the affinity chromatography of glutathione S-transferase Z. Glatz a'*, J. Psotovfi a, O. Janiczek a, K. Chroust b, T. Jowet c "Department of Biochemistry, Masaryk University, Kotl6~sk6 2, 61 ! 37 Brno, Czech Republic bDepartment of Genetics, Masaryk University, KothiCsl~ 2, 611 37 Brno, Czech Republic "Department of Biochemistry and Genetics, Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK Received 6 February 1996; revised 29 May 1996; accepted l0 June 1996 Abstract Thiopropyl Sepharose 6B in the 2-thiopyridyl-activated form was used for the reversible immobilisation of reduced glutathione (GSH). The resulting affinity matrix was successfully tested as a sorbent for the partial purification of glutathione S-transferase (GST) from pig kidney. The specific elution of the enzyme was performed with 10 mM GSH in Tris-HCl buffer (pH 7.8), non-specific elution with 20 mM dithiotreitol (DTT) in the same buffer. Keywords: Glutathione S-transferase; Enzymes 1. Introduction The glutathione S-transferases (GST; EC 2.5.1.18) are a group of multifunctional, primarily cytosolic enzymes, that catalyse the nucleophilic attack of the thiol group of glutathione (GSH) on electrophilic centres in a wide variety of endogenous and exogen- ous organic molecules [1-3]. The reaction most frequently results in the covalent linkage of GSH, a tripeptide with the amino acid sequence y-Glu-Cys- Gly on the second substrate, yielding a GSH conju- gate, which is generally less toxic than the parent compound. This process thus leads to the detoxifica- tion of these compounds since resulting adducts are normally metabolised and excreted. GSTs are ubiquitous enzymes, their activity has been detected *Corresponding author. in several species ranging from microorganisms through fungi, plants, animals to man [4]. The GSTs from a variety of sources have been purified by conventional chromatographic procedures [5,6] as well as affinity chromatography [7,8], that has also been used for the examination and the measurement of the GST isoenzyme profile in animal and human tissues [9-11 ]. Because the expression of GST in cancerous and in normal tissues varies widely in both level and type, these techniques are very important in several aspects of cancer therapy. Affinity chromatography of GST has usually been carried out with glutathione or S-glutathione deriva- tives irreversibly immobilised on agarose, cellulose, methacrylate or hydroxymethacrylate supports. In this paper we describe the preparation of an ad- sorbent for affinity chromatography of GST using the 2-thiopyridyl-activated thiopropyl Sepharose for re- versible immobilisation of GSH through thiol-di- 0378-4347/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved Pll S0378-4347(96)00267-8