15] 5-Fluoro-2′-deoxyuridylate-agarose in the affinity-chromatographic purification of thymidylate synthetase

Abstract

This chapter describes the involvement of 5-Fluoro-2'-deoxyuridylate-agarose in the affinity-chromatographic purification of thymidylate synthetase enzyme. 5-Fluoro-2'-deoxyuridine 5'-phosphate is a potent inhibitor of thymidylate synthetase that binds covalently to the enzyme. Replacement of the 5'-phosphate by a 5'- p -aminophenyl phosphate ester group reduces this interaction and additionally provides a suitable substituent for linking the inhibitor to an agarose matrix. Thymidylate synthetase is assayed by measuring the overall increase in absorbance at 340 nm caused by the conversion of 5, 10- methylene-5, 6, 7, 8-tetrahydrofolate to 7, 8-dihydrofolate. Assays are performed at 30° in 0.1 M potassium phosphate buffer (pH 6.8) with 0.2 mM methylene-tetrahydrofolate and 0.1 mM deoxyuridylate. The column effluent should be monitored for enzymic activity during the application of the 0.1 M buffer solution, and upon appearance of activity, the eluant should immediately change on reaching the higher buffer strength. The chapter also presents other affinity-chromatographic procedures for the isolation of thymidylate synthestase.