Purification and Characterization of Glutathione S-transferase π from Human Placental Tissues

Abstract

Human placental glutathione S-transferase(GST - π ) was purified to the apparent homgeneity through salting-out with ammonium sulfate and the consecutive chromatography on carboxymethyI(CM)-, diethylaminoethyI(DEAE)-cellulose and S-hexylglutathione sepharose 6B affinity column. For the characterization of the apparently purified enzyme, sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS PAGE), kinetic studies, neuraminidase digestion, and isoelectrofocusing were performed. The yield of the enzyme was 11 percent with the 1107 fold purification and the respective specific activity to l-chloro-2,4-dinitrobenzene(CDNB), 1,2-dichloro-4-nitrobenzene(DCNB) and p-nitrophenyl chloride was 62 JU/mg, 0.12 IU/mg, and almost non-detectable. The Km of the enzyme for reduced glutathione(GSH) was 0.085 mM at the concentration of 2 mM CDNB, while its Km for CDNB was 0.46 mM at the fixed concentration of 5 mM GSH. Calcium, magnesium, zinc, ethylenediamintertraacetic acid(EDTA) and ethylenedioxydiethylenedinitrilotetraacetic acid (EGTA) did not show any significant effect on enzyme activity. The subunit of the enzyme with a molecular weight of 25,000 did not reveal the molecular weight change after neuraminidase treatment. Isoelectrofocusing of the enzyme showed two bands, of which the pi of the major band was 4.48, while that of the minor band, 4.55. The specific antibody, raised against the purified GST- π in the rabbit serum indicated the immunologic cross-reactivity to the acidic GST from the human granulocyte.