A new assay of thymidylate synthetase activity based on the release of tritium from deoxyuridylate-5-3-H.

The tritium atom of deoxyuridylate-5-3H is obligatorily displaced in the thymidylate synthetase reaction and equilibrates with water. Accordingly, a new and simple assay for the enzyme has been devised which is based on the measurement of the tritium released. This method shows essentially the same substrate requirements and pH optimum as the spectrophotometric assay. It has the advantage of much greater sensitivity and lower blanks and is applicable to crude extracts without modification. The ratio of tritium released to dihydrofolate formed is less than 1.0 and is considered to be the result of an isotope effect. This paper describes a method for the assay of thymidylate synthetase which is based on the obligatory release of tritium from dUMP-5-3H into water. The principle of the method is apparent from Fig. 1. The methylene carbon (*) of 5,10methylenetetrahydrofolate becomes the 5-methyl group of dTMP