[101] Purification of thymidylate synthetase from enzyme-poor sources by affinity chromatography

This chapter discusses the purification of thymidylate synthetase (EC 2.1.1.4.5) from enzyme-poor sources by affinity chromatography. Thymidylate synthetase from Lactobacillus casei and other sources is able to form binary and ternary complexes, with various substrate and cofactor analogs, as demonstrated by difference spectra in the ultraviolet (UV) region, tryptophan fluorescence quenching, circular dichroic spectra, electrophoresis, and other methods. Tetrahydromethotrexate is a quite effective inhibitor of thymidylate synthetase from both bacterial and eukaryotic cells, where it is noncompetitive versus methylenetetrahydrofolate and uncompetitive versus deoxyuridylate (dUMP). Thymidylate synthetase from E. coli B forms on discontinual polyacrylamide gel electrophoresis one sharp zone migrating near to the bromophenol blue marker. After reductive denaturation in the presence of 4 M urea and sodium dodecyl sulfate, one species of molecular weight, approximately 35,000, can be detected by electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is stable in buffer D for several weeks at 0-10°. Freezing and freeze-drying causes rapid inactivation, as well as dialysis against water or dilute buffer solution. In the latter case, the irreversible inactivation is accompanied by the precipitation of the enzyme protein.