TY - JOUR
T1 - Quantification of polyphosphate
T2 - Different sensitivities to short-chain polyphosphate using enzymatic and colorimetric methods as revealed by ion chromatography
AU - Ohtomo, Ryo
AU - Sekiguchi, Yoko
AU - Mimura, Tetsuro
AU - Saito, Masanori
AU - Ezawa, Tatsuhiro
N1 - Funding Information:
This study was supported in part by Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of the Bio-oriented Technology Research Advancement Institution, Japan (R.O. and M.S.), the Core Research for Evolutional Science and Technology of the Japan Science and Technology Corp. (Y.S. and T.M.), and the Japan Society for the Promotion of Science (No. 13760050) (T.E.).
PY - 2004/5/15
Y1 - 2004/5/15
N2 - 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.
AB - 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.
KW - Ion chromatography
KW - Polyphosphate
KW - Polyphosphate kinase
KW - Toluidine blue O
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U2 - 10.1016/j.ab.2004.03.004
DO - 10.1016/j.ab.2004.03.004
M3 - Article
C2 - 15113689
AN - SCOPUS:2142758630
VL - 328
SP - 139
EP - 146
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -