A continuous spectrophotometric assay for thymidine and deoxycytidine kinases

We have developed a continuous spectrophotometric assay for thymidine and deoxycytidine kinase activities by coupling nucleoside 5'-monophosphate formation to a methylation reaction which generates a product absorbing at 340 nm. With thymidine kinase, we used the alternate substrate deoxyuridine and coupled the reaction to thymidylate synthase. For deoxycytidine kinase, we coupled the reaction to a thymidylate synthase mutant which converts the product 2'-deoxycytidine-5'-monophosphate (dCMP) to m5dCMP. In both cases, the methylation reactions are accompanied by conversion of 5,10-methylene-5,6,7,8-tedrahydrofolate to 7,8-dihydrofolate and can be continuously monitored by the increase of absorbance at 340 nm. The assay should be particularly useful for kinetic studies, and for the purification of these enzymes from various sources.