Purification and partial characterization of the glutathione S-transferase of rat erythrocytes.

The single glutathione S-transferase (EC 2.5.1.18) present in rat erythrocytes was purified to apparent homogeneity by affinity chromatography on glutathione-Sepharose and hydroxyapatite chromatography. Approx. 1.86 mg enzyme is found in 100 ml packed erythrocytes and accounts for about 0.01% of total soluble protein. The native enzyme (Mr 48,000) displays a pI of 5.9 and appears to possess a homodimeric structure with a subunit of Mr 23,500. Enzyme activities with ethacrynic acid and cumene hydroperoxide were 24 and 3%, respectively, of that with 1-chloro-2,4-dinitrobenzene. The Km values for 1-chloro-2,4-dinitrobenzene and glutathione were 1.0 and 0.142 mM, respectively. The concentrations of certain compounds required to produce 50% inhibition (I50) were as follows: 12 microM bromosulphophthalein, 34 microM S-hexylglutathione, 339 microM oxidized glutathione and 1.5 mM cholate. Bromosulphophthalein was a noncompetitive inhibitor with respect to 1-chloro-2,4-dinitrobenzene (Ki = 8 microM) and glutathione (Kis = 4 microM; Kii = 11.5 microM) while S-hexylglutathione was competitive with glutathione (Ki = 5 microM).