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Despite the several models given above, the mechanism of the color change has not yet been unambiguously clarified [9, 10]. We have already identified several reaction characteristics through kinetic analysis in the millisecond time range of in vitro firefly bioluminescence using photolytic reactions of caged adenosine triphosphate (cATP) [11]. However, for the ordinary method of in vitro firefly bioluminescence initiated by manually injecting an ATP solution into a solution of Luc and Ln containing Mg2+, there is very little data on bioluminescence intensity changes in the millisecond range. Accordingly, we developed a time course measurement system that accurately records the origin of the time scale at the instant of ATP injection. We also measured time-resolved bioluminescence spectra using a multichannel http://www.selleckchem.com/products/dabrafenib-gsk2118436.html photomultiplier system to obtain information about the aforementioned color change. This kind of measurement is not possible using conventional multichannel photodiode arrays because of their low sensitivity. Here, we report real-time progression of the total light intensity and spectrum of in vitro firefly bioluminescence together with their dependence on pH and reactant concentrations. The variation and characteristics of the bioluminescence mechanism are clarified by comparison with results obtained from photolysis of cATP. Measurements were carried out for three different concentration conditions at each of three pH values (6.8, 7.0 and 7.8), as presented in Table?1. Ln (Sigma, St. Louis, MO), Luc (Sigma), http://www.selleck.cn/products/z-vad-fmk.html MgSO4 (Wako, Osaka, Japan), ATP (Calbiochem, La Jolla, CA) and cATP (Dojindo laboratory, Kumamoto, Japan) were each diluted with pH-adjusted 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES). A quantity of 0.25?mL of each Ln, Luc, MgSO4, HEPES buffer solution and ATP or cATP were prepared to give a total of 1.00?mL excluding ATP for measurements by ATP injections or 1.25?mL mixing all the reagent solutions for measurements by cATP photolysis, which was placed in a 10?mm?��?10?mm?��?50?mm quartz cell. As shown in Table?1, concentration conditions 1 and 2 are for measurements by ATP injections and concentration condition 3 is for the measurement by cATP photolysis. To verify the effect of concentration conditions on the bioluminescence spectra and their time evolution, concentration http://www.selleckchem.com/products/sch772984.html conditions 1 and 2 were set with [Ln]?>?[Luc] and with [Ln]?