Improved measurement of thymidylate synthetase activity by a modified tritium-release assay

Accurate quantitation of thymidylate synthetase activity using a tritium-release assay is dependent upon measurement of only that tritium released from deoxy[5-3H]uridine monophosphate ([3H]dUMP) during the biosynthesis of thymidylate. Removal of remaining [3H]dUMP on completion of the assay by charcoal adsorption and correction for the nonenzymatic release of tritium are necessary. Although over 99% of [3H]dUMP is removed immediately following addition of charcoal, these studies demonstrate that sufficient [3H]dUMP can remain to prevent accurate measurement of low levels of thymidylate synthetase activity. By delaying measurement of radioactivity for at least 24 h following addition of charcoal, this problem is minimized. To account for nonenzymatic release of tritium, a blank containing enzyme extract with omission of +/- ,L-5,10-methylenetetrahydrofolate is demonstrated to be more effective than the commonly used blank in which water is substituted for enzyme extract. In samples containing 5-fluoro-2'-deoxyuridine monophosphate (FdUMP), a potent inhibitor of thymidylate synthetase activity, an alternative blank containing a high concentration of FdUMP (approximately 1 mM) is useful in demonstrating a theoretical maximal or complete inhibition of thymidylate synthetase activity.