Nonenzymatic exchange of tritium from [5- 3H] deoxyuridylate in the thymidylate synthetase assay

Abstract

When 2′-[5- 3H] deoxyuridine-5′-phosphate and 2-mercaptoethanol are used to assay thymidylate synthetase activity, the tritium of 2′-[5- 3H] deoxyuridine-5′-phosphate exchanged nonenzymatically with water. The rate of the isotope exchange depended on the concentrations both 2-m2-mercaptoethanol and amine buffer and the pH of the assay mixture. Such an exchange is a possible source of serious error in the assay when low activity of enzyme is assayed with highly radioactive 2′-deoxyuridine-5′-phosphate but can be minimized by maintaining a low concentration of 2-mercaptoethanol and unprotonated amine buffer in the assay mixture.