The Chromatographic Separation and Recovery of Reduced and Oxidized Pyridine Nucleotides

Abstract

Despite the availability of many excellent analytical procedures, no convenient method has been described for the separation and isolation of small amounts of the oxidized and reduced pyridine nucleotides from mixtures. This surprising situation became apparent when we were faced with the problem of preparing a micromole of pure tritium-labeled reduced triphosphopyridine nucleotide for use in stereochemical studies related to folate metabolism. This communication describes a method which makes possible the separation and quantitative recovery of diphosphopyridine nucleotide, reduced diphosphopyridine nucleotide, triphosphopyridine nucleotide, and reduced triphosphopyridine nucleotide from diethylaminoethyl-cellulose columns. Quantities of the order of 1 pmole or less can be manipulated with ease. The method has been used for the preparation of enzymatically reduced triphosphopyridine nucleotide labeled with tritium.