Study of chromatographic parameters for glutathione S transferases on an high-performance liquid chromatography affinity stationary phase

Abstract

The chromatographic parameters were examined for recombinant glutathione S-transferases (GSTs) on a new HPLC affinity packing containing the immobilized ligand S-octylglutathione. The k′ values of both rA1-1 and rP1-1 were determined under isocratic conditions with increasing concentrations of the mobile phase ligand S-butyl glutathione. Plots of 1/ k′ vs. S-butylglutathione concentration were non-linear which is consistent with a bivalent model for the association of these dimeric enzymes 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. Association 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 useful 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 and rM1a-1a was made using the GST competitive inhibitors S-hexylglutathione, S-butylglutathione and γ-glutamyl-(S-hexyl)cysteinyl-phenylglycine as mobile phase modifiers. The association constants determined in solution did not always predict the elution order of the recombinant GSTs (rGSTs) using the mobile phase inhibitors. Yields of active rGSTs 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 in placental and liver cytosols.