• Journal of Life Science
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• v.15 no.6 s.73
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• pp.935-940
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• 2005

Fluorescence change of coumarin labeled phosphate binding protein (PBP-MDCC) was monitored to measure the amount of released inorganic phosphate ($P_{i}$) during nucleoside triphosphate (NTP) hydrolysis reaction. After purification of PBP-MDCC, fluorescence emission spectra showed that fluorescence responded linearly to $P_{i}$ up to about 0.7 molar ratio of $P_{i}$ to protein. The correlation of fluorescence signal and $P_{i}$ standard was measured to obtain [$P_{i}$] - 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 $P_{i}$ by the $P_{i}$ standard curve. $P_{i}$ release results showed that single-stranded Ml3 DNA stimulated dTTP hydrolysis reaction several folds by T7 helicase. Instead of end point assay in NTP hydrolysis reaction, real time $P_{i}$-release assay by PBP-MDCC was proven to be very easy and convenient to measure released $P_{i}$.