Coupled affinity-reversed-phase high-performance liquid chromatography systems for the measurement of glutathione...

HPLC affinity and reversed-phase modes were coupled for the direct measurement of glutathione S-transferases (GSTs) in cytosol extracts. Two coupling designs were examined. In the sequential configuration the affinity column served to extract the isoenzymes which were then eluted directly onto the reversed-phase column as a single fraction. Subsequent separation in the reversed-phase mode provided a GST profile based on the subunit composition of the isoenzymes as a whole. In the second configuration (rapid sampling configuration), gradient elution was performed in the affinity mode resulting in resolution of the intact isoenzymes. The eluate from the affinity separation was sampled in continuous, repetitive intervals and automatically subjected to ongoing reversed-phase analysis. This multidimensional approach provided information on the GST subunit content and also gave information about the distribution of the subunits among individual isoenzymes, thereby forming a basis for the determination of the actual isoenzymatic composition of the GSTs. In both configurations, events were automated and co-ordinated through the use of computer and multiport switching valves. Examples of GST separations from these procedures are shown for human lung and liver tissues. A comparison of the GST subunit analyses from normal and cancer lung tissue excised from the same patient showed substantial elevations of GSTs in the cancer sample. Two-dimensional affinity-reversed-phase analysis of a human liver sample illustrates the utility of the technique for determining the isoenzymatic organization of GST subunits. The criteria for extending two-dimensional analysis to more complex GST mixtures are discussed.