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 [H-3]folates from the commercial monoglutamyl [H-3]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 monoglutamy] [H-3]folates to various polyglutamyl forms. In the bacterial synthesis, Lactobacillus casei was grown in the presence of I ng/ml (2.27 nM) [H-3]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10 mM P-mercaptoethanol and heated to release the folates. The extracted [H-3]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 [H-3]5-methyltetrahydrofolate and [H-3]5-formyltetrahydrofolate (di- through heptaglutamates). The alternative method consisted of enzymatic polyglutamylation of [H-3]folic acid catalyzed by recombinant Escherichia coli folylpolyglutamate synthetase. This enzymatic synthesis yielded predominantly tri-, tetra-, and pentaglutamyl species for the [H-3]folate substrate. (C) 2007 Elsevier Inc. All rights reserved.

Improved methods for the preparation of [3H]folate polyglutamates: biosynthesis with Lactobacillus casei and enzymatic synthesis with Escherichia coli folylpolyglutamate synthetase / Naponelli, Valeria; Hanson, Andrew D; Gregory, Jesse F. - In: ANALYTICAL BIOCHEMISTRY. - ISSN 0003-2697. - 371:2(2007), pp. 127-34-134. [10.1016/j.ab.2007.08.026]

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

Naponelli, Valeria;
2007

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 [H-3]folates from the commercial monoglutamyl [H-3]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 monoglutamy] [H-3]folates to various polyglutamyl forms. In the bacterial synthesis, Lactobacillus casei was grown in the presence of I ng/ml (2.27 nM) [H-3]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10 mM P-mercaptoethanol and heated to release the folates. The extracted [H-3]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 [H-3]5-methyltetrahydrofolate and [H-3]5-formyltetrahydrofolate (di- through heptaglutamates). The alternative method consisted of enzymatic polyglutamylation of [H-3]folic acid catalyzed by recombinant Escherichia coli folylpolyglutamate synthetase. This enzymatic synthesis yielded predominantly tri-, tetra-, and pentaglutamyl species for the [H-3]folate substrate. (C) 2007 Elsevier Inc. All rights reserved.
Improved methods for the preparation of [3H]folate polyglutamates: biosynthesis with Lactobacillus casei and enzymatic synthesis with Escherichia coli folylpolyglutamate synthetase / Naponelli, Valeria; Hanson, Andrew D; Gregory, Jesse F. - In: ANALYTICAL BIOCHEMISTRY. - ISSN 0003-2697. - 371:2(2007), pp. 127-34-134. [10.1016/j.ab.2007.08.026]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/2860002
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