Improved methods for the preparation of [ 3H]folate polyglutamates: biosynthesis with Lactobacillus casei and enzymatic synthesis with Escherichia coli folylpolyglutamate synthetase

Abstract

Tritiated forms of polyglutamyl folates are not commercially available but are often needed for experimental uses in folate biochemistry. Thus, considerable interest exists in the preparation of polyglutamyl [ 3H]folates from the commercial monoglutamyl [ 3H]folates. However, refinement of established enzymatic and biological synthesis methods is still needed. To address this need we developed improved procedures for the conversion of monoglutamyl [ 3H]folates to various polyglutamyl forms. In the bacterial synthesis, Lactobacillus casei was grown in the presence of 1 ng/ml (2.27 nM) [ 3H]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10 mM β-mercaptoethanol and heated to release the folates. The extracted [ 3H]folates were purified on a folate-binding protein affinity column and then applied to a Sephadex G-10 column to separate the eluted poly- from the monoglutamyl folate species. High performance liquid chromatography with multichannel electrochemical detection indicated that the bacterial synthesis yielded predominantly polyglutamates of [ 3H]5-methyltetrahydrofolate and [ 3H]5-formyltetrahydrofolate (di- through heptaglutamates). The alternative method consisted of enzymatic polyglutamylation of [ 3H]folic acid catalyzed by recombinant Escherichia coli folylpolyglutamate synthetase. This enzymatic synthesis yielded predominantly tri-, tetra-, and pentaglutamyl species for the [ 3H]folate substrate.