Isolation and Characterization of the Multiple Glutathione &Transferases from Human Liver

Thirteen forms of glutathione S-transferase were isolated from human liver in high yields by glutathione-affinity chromatography and chromatofocusing. Apparent isoelectric points ranged from 4.9 to 8.9 and included neutral forms. All 13 forms appeared to be identical immunochemically in a quantitative enzymelinked immunosorbent assay. These forms were immunochemically distinct from the major acidic glutathione S-transferase found in placenta and erythrocyte and were immunochemically distinct from two forms of higher molecular weight glutathione S-transferase found in some but not all liver samples. The 13 forms exhibited similar activities with l-chloro-2,4-dinitrobenzene as substrate, specific activities of 33-94 pmol/ min/mg. Likewise, these forms all exhibited glutathione peroxidase activity with cumene hydroperoxide, specific activities of 1.5-8.3 pmol/min/mg. All 13 forms bound bilirubin with subsequent conformational changes leading to states devoid of transferase activity, a process prevented by the presence of foreign proteins. As hematin-binding proteins, however, these multiple transferases exhibited a very broad range of binding extending from nonbinding to high-affinity binding (KO lo-’ M). Hematin binding was noncompetitive with transferase activity and did not involve the bilirubin-binding site, suggesting the existence of unique heme-binding sites on these proteins. The two forms of the immunochemically distinct glutathione Stransferases found in some liver samples also exhibited both transferase and peroxidase activities. In addition, they also have separate sites for binding bilirubin and hematin.