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Quantification of polyphosphate: different sensitivities to short-chain polyphosphate using enzymatic and colorimetric methods as revealed by ion chromatography
| Title: | Quantification of polyphosphate: different sensitivities to short-chain polyphosphate using enzymatic and colorimetric methods as revealed by ion chromatography |
| Authors: | Ohtomo, Ryo Browse this author | | Sekiguchi, Yoko Browse this author | | Mimura, Tetsuro Browse this author | | Saito, Masanori Browse this author | | Ezawa, Tatsuhiro5 Browse this author |
| Authors(alt): | 江沢, 辰広5 |
| Keywords: | Ion chromatography | | Polyphosphate | | Polyphosphate kinase | | Toluidine blue O |
| Issue Date: | 15-May-2004 |
| Publisher: | Elsevier |
| Journal Title: | Analytical Biochemistry |
| Volume: | 328 |
| Issue: | 2 |
| Start Page: | 139 |
| End Page: | 146 |
| Publisher DOI: | 10.1016/j.ab.2004.03.004 |
| Abstract: | Polyphosphate is ubiquitous and has a variety of biochemical functions. Among polyphosphate quantification methods, an enzymatic assay using Escherichia coli polyphosphate kinase (PPK), in which polyphosphate is converted to adenosine 5′-triphosphate and quantified by luciferase assay, is the most specific and most sensitive. However, chain-length specificity of the assay has not been analyzed in detail so far. Ion chromatography equipped with an on-line hydroxide eluent generator enabled us to analyze polyphosphate up to 50 inorganic phosphate (Pi) residues, and we employed this method to investigate the chain-length specificity of PPK in this study. Several fractions of short-chain polyphosphate were prepared by electrophoresis, and the chain-length distribution was analyzed before and after 1–6 h PPK reaction by ion chromatography. Polyphosphates longer than 23 Pi residues were processed by PPK completely after 1 h incubation, but complete processing of those between 11 and 22 Pi residues required 6 h incubation. Limited processing of polyphosphates of 10 Pi residues or shorter were observed even after 6 h incubation. Metachromasy of Toluidine blue O, an alternative method for polyphosphate quantification, showed broader chain-length specificity although it was not as sensitive as the enzymatic assay. Combination of these two methods would be practically applicable to analysis of polyphosphate dynamics in living organisms. |
| Relation: | http://www.sciencedirect.com/science/journal/00032697 |
| Type: | article (author version) |
| URI: | http://hdl.handle.net/2115/671 |
| Appears in Collections: | 農学院・農学研究院 (Graduate School of Agriculture / Faculty of Agriculture) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
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Submitter: 江澤 辰広
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