High Performance Liquid Chromatographic Assay of Adenine Phosphoribosyltransferase and Hypoxanthine-Guanine Phosphoribosyltransferase Activities in Human Erythrocytes

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A sensitive and rapid nonradiometric determination of human erythrocyte adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) activities is described. The procedure was based on a reversedphase high performance liquid chromatographic (HPLC) separation of purine bases, nucleosides, and nucleotides, after the enzyme reaction. APRT activity was assayed with adenine as a substrate and the AMP and AMP metabolites which were produced were converted to inosine by the enzymes alkaline phosphatase (EC, 3.1.3.1) and adenosine deaminase (EC, 3.5.4.4). The inosine was determined by the HPLC. HGPRT activity was assayed with hypoxanthine or guanine as a substrate and the IMP or GMP was determined by the HPLC. The activities of APRT, HPRT, and GPRT assayed by this method in erythrocytes from thirty normal adults were 0.35±0.14, 2.02±0.42, and 3.22±0.56μmol/min/gHb (mean±2SD), respectively.
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