Purification of mammalian tumor (L1210) thymidylate synthetase by affinity chromatography on stable biospecific adsorbent. Stabilization of the enzyme with neutral detergents.

Thymidylate synthetase from mouse leukemic L1210 cells was purified to electrophoretic homogeneity with 70% yield as a result of an affinity chromatography procedure based on reversible deoxyuridylate-dependent binding of the enzyme to a stable biospecific adsorbent, 10-formyl-5,8-dideazafolate, immobilized on aminoethyl-Sepharose. The presence of neutral detergents, Triton X-100, or Nonidet P40 stabilized thymidylate synthetase during purification. Analytical electrophoresis of the enzyme treated with an excess of 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate showed the presence of two forms of thymidylate synthetase--5-fluorodeoxyuridylate.5,10-methylenetetrahydrofolate complex, indicating that there are two binding sites for 5-fluorodeoxyuridylate present on the enzyme molecule. Molecular weight of native thymidylate synthetase was found to be 75,000, whereas that for the monomer was 38,500.