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KMID : 0613820050150060935
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Journal of Life Science
2005 Volume.15 No. 6 p.935 ~ p.940
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Real Time Scale Measurement of Inorganic Phosphate Release by Fluorophore Labeled Phosphate Binding Protein
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Jeong Yong-Joo
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Abstract
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Fluorescence change of coumarin labeled phosphate binding protein (PBP-MDCC) was monitored to measure the amount of released inorganic phosphate (Pi) during nucleoside triphosphate (NTP) hydrolysis reaction. After purification of PBP-MDCC, fluorescence emission spectra showed that fluorescence responded linearly to Pi up to about 0.7 molar ratio of Pi to protein. The correlation of fluorescence signal and Pi standard was measured to obtain [Pi] - fluorescence intensity standard curve on the stopped-flow instrument. When T7 bacteriophage helicase, double-stranded DNA unwinding enzyme using dTTP hydrolysis as an energy source, reacted with dTTP, the change of fluorescence was able to be converted to the amount of released Pi by the Pi standard curve. Pi release results showed that single-stranded M13 DNA stimulated dTTP hydrolysis reaction several folds by T7 helicase. Instead of end point assay in NTP hydrolysis reaction, real time Pi-release assay by PBP-MDCC was proven to be very easy and convenient to measure released Pi.
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