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

Ryo Ohtomo, Yoko Sekiguchi, Tetsuro Mimura, Masanori Saito, Tatsuhiro Ezawa

Research output: Contribution to journalArticle

22 Citations (Scopus)

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 1-6h PPK reaction by ion chromatography. Polyphosphates longer than 23 Pi residues were processed by PPK completely after 1h incubation, but complete processing of those between 11 and 22 Pi residues required 6h incubation. Limited processing of polyphosphates of 10 Pi residues or shorter were observed even after 6h 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.

Original languageEnglish
Pages (from-to)139-146
Number of pages8
JournalAnalytical Biochemistry
Volume328
Issue number2
DOIs
Publication statusPublished - 2004 May 15

Keywords

  • Ion chromatography
  • Polyphosphate
  • Polyphosphate kinase
  • Toluidine blue O

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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