A method for the enzymatic synthesis and purification of [alpha-32P] nucleoside triphosphates.
A simplified method is described for the enzymatic synthesis and purification of [alpha-32P]ribo- and deoxyribonucleoside triphosphates. The products are obtained at greater than 97% radiochemical purity with yields of 50--70% (relative to 32Pi) by a two-step elution from DEAE-Sephadex. All reactions are done in one vessel as there is no need for intermediate product purifications. This method is therefore suitable for the synthesis of these radioactive compounds on a relatively large scale. The sequential steps of the method involve first the synthesis of [gamma-32P]ATP and the subsequent phosphorylation of nucleoside 3' monophosphate with T4 polynucleotide kinase to yield nucleoside 3', [5'-32P]diphosphate. Hexokinase is used after the T4 reaction to remove any remaining [gamma-32P]ATP. Nucleoside 3',[5'-32P]diphosphate is treated with nuclease P-1 to produce the nucleoside [5'-32P]monophosphate which is phosphorylated to the [alpha-32P]nucleoside triphosphate with pyruvate kinase and nucleoside monophosphate kinase. Adenosine triphosphate used as the phosphate donor for [alpha-32P]deoxynucleoside triphosphate syntheses is readily removed in a second purification step involving affinity chromatography on boronate-polyacrylamide. [alpha-32P]Ribonucleoside triphosphates can be similarly purified when deoxyadenosine triphosphate is used as the phosphate donor.
D000818:Animals; D002417:Cattle; D003090:Coliphages / Q000201:enzymology; D003854:Deoxyribonucleotides / Q000096:biosynthesis; D007553:Isotope Labeling / Q000379:methods; D008099:Liver / Q000201:enzymology; D009703:Nucleoside-Phosphate Kinase; D010761:Phosphorus Radioisotopes; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D012265:Ribonucleotides / Q000096:biosynthesis* / Q000302:isolation & purification; D013379:Substrate Specificity
D003854:Deoxyribonucleotides; D010761:Phosphorus Radioisotopes; D012265:Ribonucleotides; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D009703:Nucleoside-Phosphate Kinase