RAPID DETERMINATION OF NUCLEOSIDE KINASE AND NUCLEOTIDASE ACTIVITIES WITH TRITIUM-LABELED SUBSTRATES

Methods suitable for the assay of ribonucleoside and deoxyribonucleoside kinases and of any enzyme that hydrolyzes nucleoside monophosphates have been described. Nucleotides are retained on disks of anion-exchange paper, while nucleosides are washed off. The technique of eluting the nucleotide from the disk within a liquid scintillation vial, followed by solution of the eluate in a suitable scintillation solvent, permits the use of tritium- as well as 14C-labeled substrates. It has been demonstrated that tritium-labeled nucleotides may be counted as reliably as 14C-labeled compounds if the samples are first eluted into the liquid phase. The abilities of diethylaminoethyl (DEAE) substituted paper and of Amberlite impregnated paper (SB-2) to retain nucleotides were compared as the salt concentration of the sample was increased. Under the conditions normally used for enzyme assays both papers retained 100% of the labeled nucleotide, while the percentage of nucleotide retained decreased as the salt concentration of the sample was increased. SB-2 paper was found to be substantially more retentive than DEAE paper, but both papers retained more than 50% of the labeled dCMP or dTMP from samples containing 1 M KCl. © 1969.