Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method |
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| Authors: | Shin-ichi Sakasegawa Junji Hayashi Yoshiaki Ikura Shigeru Ueda Shigeyuki Imamura Toshihiko Kumazawa Atsuhisa Nishimura Toshihisa Ohshima Haruhiko Sakuraba |
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| Affiliation: | aAsahi Kasei Pharma Corporation, Shizuoka 410-4321, Japan;bDepartment of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan;cIchibiki Corporation, Toyohashi Aichi 441-8019, Japan;dMicrobial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan;eDepartment of Applied Biological Science, Faculty of Agriculture, University of Kagawa, 2392 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan |
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| Abstract: | Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) from the hyperthermophile Thermotoga maritima was biochemically characterized with the aim of establishing a colorimetric assay for inorganic pyrophosphate (PPi). When heterologously expressed in Escherichia coli, T. maritima PPDK (TmPPDK) was far more stable any other PPDK reported so far: it retained >90% of its activity after incubation for 1 h at 80 °C, and >80% of its activity after incubation for 20 min at pHs ranging from 6.5 to 10.5 (50 °C). In contrast to PPDKs from protozoa and plants, this TmPPDK showed very long-term stability at low temperature: full activity was retained even after storage for at least 2 years at 4 °C. TmPPDK was successfully applied to a novel colorimetric PPi assay, which employed (i) a PPi cycling reaction using TmPPDK and nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) from Saccharomyces cerevisiae and (ii) a NAD cycling reaction to accumulate reduced nitroblue tetrazolium (diformazan). This enabled detection of 0.2 μM PPi, making this method applicable for preliminary measurement of PPi levels in PCR products in an automatic clinical analyzer. |
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| Keywords: | Pyruvate phosphate dikinase Thermotoga maritima Inorganic pyrophosphate assay Enzymatic cycling PCR |
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