SEPARATION AND IDENTIFICATION OF PTEROYLPOLYGLUTAMATES BY POLYACRYLAMIDE-GEL CHROMATOGRAPHY

A simplified procedure for the determination of the glutamate chain lengths of labeled and endogenous tissue folate is described. Pteroylpoly-γ-glutamates in tissue extracts were reductively cleaved at the C,9N,10 bond to p-aminobenzoylpolyglutamates, which were converted to azo dyes by coupling their diazonium salts with naphthylethylene diamine. The azo dyes were well resolved, according to glutamate chain length, by gel chromatography on Bio-Gel P4. Unlabeled tissue folates were detected by the absorbance of their azo dye derivatives. The major endogenous pteroylpolyglutamate in rat liver, identified colorimetrically using 0,5 g tissue, was the pentaglutamate. The major labeled folates in Lactobacillus casei and Streptococcus faecalis, after incubating these bacteria with labeled folic acid, were identified as the octa- and tetraglutamates, respectively. Reductive cleavage of 10-formylfolate and 5.10-methenyltetrahydrofolate resulted in a mixture of N-substituted and unsubstituted p-amino-benzoylpolyglutamates. Methods are described for the complete cleavage of these formyl derivatives to unsubstituted p-aminobenzoylpolyglutamates. © 1979.