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Folate antagonists covalently linked to carbohydrates: synthesis, properties, and use in the purification of dihydrofolate reductases
Authors:J M Whiteley  R C Jackson  G P Mell  J H Drais  F M Huennekens
Affiliation:Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037 USA
Abstract:The folate antagonists, aminopterin and amethopterin, have been linked covalently via their carboxyl groups to various high molecular-weight water-soluble (starch, dextrans) and water-insoluble (cellulose, agarose) carbohydrates. The amount of folate compound incorporated into these polymers was determined by absorbance measurements and by radioactivity when 3H-labeled precursors were used. Optimal yields (ca. 150 mg/g) were obtained by using carbodiimides to effect amide linkages between the folate compounds and aminoalkyl derivatives of the carbohydrates. The soluble antagonist-carbohydrate complexes were characterized with respect to absorbance spectra, molecular weight, and ability to inhibit dihydrofolate reductase.Folate antagonists bound to carbohydrates retained an appreciable affinity for dihydrofolate reductases. This property has provided the basis for two different procedures for purification of these enzymes: (1) complex formation with amethopterin-aminoethyl-soluble starch and isolation of this complex via Sephadex G-100; and (2) affinity chromatography on amethopterin-aminoalkyl-Sepharose. Both methods were facilitated by the ability of dihydrofolate reductases to bind tightly to the antagonist at acidic pH values and the tendency for such complexes to dissociate at alkaline pH values, especially in the presence of dihydrofolate or folate.
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