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Quantification of polyphosphate: different sensitivities to short-chain polyphosphate using enzymatic and colorimetric methods as revealed by ion chromatography

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タイトル: Quantification of polyphosphate: different sensitivities to short-chain polyphosphate using enzymatic and colorimetric methods as revealed by ion chromatography
著者: Ohtomo, Ryo 著作を一覧する
Sekiguchi, Yoko 著作を一覧する
Mimura, Tetsuro 著作を一覧する
Saito, Masanori 著作を一覧する
Ezawa, Tatsuhiro 著作を一覧する
キーワード: Ion chromatography
Polyphosphate
Polyphosphate kinase
Toluidine blue O
発行日: 2004年 5月15日
出版者: Elsevier
誌名: Analytical Biochemistry
巻: 328
号: 2
開始ページ: 139
終了ページ: 146
出版社 DOI: 10.1016/j.ab.2004.03.004
抄録: 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 (URI): http://www.sciencedirect.com/science/journal/00032697
資料タイプ: article (author version)
URI: http://hdl.handle.net/2115/671
出現コレクション:雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

提供者: 江澤 辰広

 

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