A procedure for the isolation and determination of deoxyribose derivatives

Abstract

A method of determining deoxyribose derivatives in biological material is described. It has high sensitivity, and is particularly useful in that it can be applied to a large range of tissues for which the other available assays are unsuitable. This is because the method is applicable to complex mixtures of nucleotides in which such substances as ribonucleotides are present in very large excess over deoxyribonucleotides, and it is not necessary to equilibrate the nucleotide-precursor pool with radioactive phosphate. The method has mainly been developed with the object of determining deoxyribonucleoside triphosphates, but it can be used to assay ribonucleoside triphosphates, as well as the mono- and diphosphates of both types of nucleoside. The procedure used involves three basic techniques: (1) periodate oxidation and methylamine-induced cleavage of the sugar ring to destroy 2′- and 3′-unsubstituted ribonucleosides; (2) column chromatography to separate the deoxyribonucleotides from each other and from other substances, such as the products of the periodate oxidation; (3) fluorimetric determination of deoxyribose after labilization of the pyrimidine–glycosidic bond by bromination of the heterocyclic ring. Each of these three procedures can be used independently, in conjunction with other analytical procedures.