Abstract
A rapid enzyme assay technique is described in which a labeled anionic product is determined as the radioactivity remaining on anion-exchange paper as measured by direct liquid scintillation counting of the paper after elution of the labeled nonionic substrate from the paper with water by one of three methods: (A) descending chromatography, (B) water extraction, or (C) direct elution employing a Millipore filtration apparatus. Reactions catalyzed by galactokinase and hexokinase were employed as examples. The technique generally should be applicable to reactions catalyzed by enzymes converting uncharged materials into anionic materials. The technique should also prove useful in enzymic reactions in which the product can be separated readily from the reactant on anion-exchange paper by means of a suitable solvent or by electrophoretic means. Benzoic acid-7-C 14, galactose-1-C 14, and galactose-1-P, C 14 were counted with an efficiency of about 58% on DEAE-cellulose paper by liquid scintillation counting. Windowless gas-flow Geiger counting of radioactivity of these compounds on DEAE-cellulose paper gave satisfactory results but the efficiency of counting was only about 18%.
Published Version
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