EXAMINATION OF GLUTATHIONE-S-TRANSFERASE ISOENZYME PROFILES IN HUMAN LIVER USING HIGH-PERFORMANCE AFFINITY-CHROMATOGRAPHY

'A method for the examination of the glutathione S-transferase isoenzy me profiles in human liver using a new HPLC affinity support is descri bed. Liver cytosol was injected directly onto an HPLC column (5 x 0.46 cm) containing a support with a covalently bound affinity ligand (S-o ctylglutathione) specific for the isoenzymes. Contaminating cytosolic proteins were removed in a washing step. The isoenzymes were eluted wi th a linear gradient of a different affinity ligand in the mobile phas e. Coinciding with the affinity ligand gradient, a salt gradient (0-20 0 mM sodium chloride) was applied. In this manner the isoenzymes were fractionated into the enzymatically active homodimers and heterodimers . The classes of the affinity fractionated isoenzymes were determined by SDS-PAGE and ELISA while the subunit content was determined by reve rsed-phase chromatography. For one liver three Alpha class isoenzyme s ubunits, forming three heterodimers and two homodimers, were detected. Five livers were examined, and the homodimer A1-1 was found to be the predominant glutathione S-transferase isoenzyme. Minor amounts of Pi and Mu class isoenzymes were also detected. This non-denaturing high-p erformance affinity chromatography method reduced analysis time by a f actor of ten when compared to other affinity analysis methods for the glutathione S-transferases.