We have developed a continuous spectrophotometric assay for thymidine and d
eoxycytidine kinase activities by coupling nucleoside 5'-monophosphate form
ation 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 p
roduct 2'-deoxycytidine-5'-monophosphate (dCMP) to m(5)dCMP. 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 monit
ored by the increase of absorbance at 340 nm. The assay should be particula
rly useful for kinetic studies, and for the purification of these enzymes f
rom various sources. (C) 1998 Academic Press.