An assay method for hypoxanthine-guanine and adenine phosphoribosyltrans-ferase in hemolysate of human red cells and liver of mice is described.
Reaction was carried out in a microtube using isotope labelled substrate and terminated by the addition of formic acid. An aliquot of the reaction mixtures was applied on cellulose acetate membrane over standard carrier substances and the products were separated electrophoretically using two buffer systems, 0.1 M borate buffer pH 9.0 and 0.1 M Tris-HCI buffer pH 7.5. The areas corresponding to the products were identified by inspection of the strip under ultraviolet light, cut out and counted in a liquid scincillation counter. The enzyme activity was calculated from the recovery of radioactivity associated with the products.
In this assay method, the electrophoretic apparatus commonly used inclinical laboratory was used and the enzyme activities in tissue extracts which contained various enzymes concerning with purine metabolism could be assayed by electrophoresis using two buffer systems.