STUDY OF CHROMATOGRAPHIC PARAMETERS FOR GLUTATHIONE S-TRANSFERASES ONAN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AFFINITY STATIONARY-PHASE

The chromatographic parameters were examined for recombinant glutathio ne S-transferases (GSTs) on a new HPLC affinity packing containing the immobilized ligand S-octylglutathione. The k' values of both rA1-1 an d rP1-1 were determined under isocratic conditions with increasing con centrations of the mobile phase ligand S-butylglutathione. Plots of 1/ k' vs. S-butylglutathione concentration were non-linear which is consi stent with a bivalent model for the association of these dimeric enzym es and the stationary phase. Low flow-rates were found to be decisive in obtaining good resolution of the isoenzymes, and at 0.10 ml/min it was possible to obtain baseline resolution of rP1-1, rA1-1 and rM1a-1a using shallow, linear gradients of GST competitive inhibitors. Associ ation constants were determined from solution phase kinetics assuming a rapid equilibrium random Bi Bi mechanism. Solution phase association constants provide an approximate guide for the selection of ligands u seful in this affinity phase HPLC separation of GST isoenzymes. A good fit (r(2) = 0.998) for the rA1-1 binding data was obtained using the solution phase binding constant but this was not the case for rP1-1. A comparison of the selectivities for the separation of rP1-1, rA1-1 an d rM1a-1a was made using the GST competitive inhibitors S-hexylglutath ione, S-butylglutathione and gamma-glutamyl-(S-hexyl)cysteinyl-phenylg lycine as mobile phase modifiers. The association constants determined in solution did not always predict the elution order of the recombina nt GSTs (rGSTs) using the mobile phase inhibitors. Yields of active rG STs from the column were 90%, 88% and 61% for rP1-1, rA1-1 and rM1a-1a , respectively. This technique was used in the fractionation of GSTs i n placental and liver cytosols.