Electrophoretic identification of poly-γ-glutamate chain-lengths of 5,10-methylenetetrahydrofolate using thymidylate synthetase complexes

A method has been developed to characterize the poly-γ-glutamates of 5,10-methyl-enetetrahydrofolate. Incorporation of 5,10-methylenetetrahydrofolates into a ternary complex with L. casei thymidylate synthetase and 5-fluoro-2-deoxy³H]uridylate stabilizes the reduced folate against oxidation, loss of the one carbon moiety, and poly-γ-glutamate degradation. The covalent ternary complexes, containing 5,10-methylenetetrahydrofolate polyglutamates, were resolved electrophoretically. Electrophoretic mobility was shown to be a linear function of polyglutamate chain-length. The method can potentially be applied to analysis of chemically prepared folate polyglutamates, the monitoring of enzyme-mediated interconversions of polyglutamates and characterization of tissue extract polyglutamates.