Abstract
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.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Glynn I. M., Chappell J. B. A simple method for the preparation of 32-P-labelled adenosine triphosphate of high specific activity. Biochem J. 1964 Jan;90(1):147–149. doi: 10.1042/bj0900147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kihara K., Nomiyama H., Yukuhiro M., Mukai J. I. Enzymatic synthesis of [alpha32P]ATP of high specific activity. Anal Biochem. 1976 Oct;75(2):672–673. doi: 10.1016/0003-2697(76)90128-7. [DOI] [PubMed] [Google Scholar]
- Lillehaug J. R., Kleppe K. Kinetics and specificity of T4 polynucleotide kinase. Biochemistry. 1975 Mar 25;14(6):1221–1225. doi: 10.1021/bi00677a020. [DOI] [PubMed] [Google Scholar]
- Richardson C. C. Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected Escherichia coli. Proc Natl Acad Sci U S A. 1965 Jul;54(1):158–165. doi: 10.1073/pnas.54.1.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
- STROMINGER J. L., HEPPEL L. A., MAXWELL E. S. Nucleoside monophosphate kinases. I. Transphosphorylation between adenosine triphosphate and nucleoside monophosphates. Biochim Biophys Acta. 1959 Apr;32:412–421. doi: 10.1016/0006-3002(59)90614-6. [DOI] [PubMed] [Google Scholar]
- Schott H., Rudloff E., Schmidt P., Roychoudhury R., Kössel H. A dihydroxyboryl-substituted methacrylic polymer for the column chromatographic separation of mononucleotides, oligonucleotides, and transfer ribonucleic acid. Biochemistry. 1973 Feb 27;12(5):932–938. doi: 10.1021/bi00729a022. [DOI] [PubMed] [Google Scholar]
- Symons R. H. Practical methods for the routine chemical synthesis of 32P-labelled nucleoside di- and triphosphates. Biochim Biophys Acta. 1970;209(2):296–305. doi: 10.1016/0005-2787(70)90728-8. [DOI] [PubMed] [Google Scholar]
- Symons R. H. Synthesis of (alpha-32P) ribo- and deoxyribonucleoside 5'-triphosphates. Methods Enzymol. 1974;29:102–115. [PubMed] [Google Scholar]
- Symons R. H. The rapid, simple and improved preparation of high specific activity alpha-[32P]dATP and alpha-[32P]ATP. Nucleic Acids Res. 1977 Dec;4(12):4347–4355. doi: 10.1093/nar/4.12.4347. [DOI] [PMC free article] [PubMed] [Google Scholar]