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http://dx.doi.org/10.5352/JLS.2005.15.6.935

Real Time Scale Measurement of Inorganic Phosphate Release by Fluorophore Labeled Phosphate Binding Protein  

Jeong Yong-Joo (Department of Bio and Nanochemistry, Kookmin University)
Publication Information
Journal of Life Science / v.15, no.6, 2005 , pp. 935-940 More about this Journal
Abstract
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 $P_{i}$.
Keywords
phosphate binding protein; inorganic phosphate; T7 bacteriophage helicase; stopped-flow instrument;
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