CHEMILUMINESCENCE OF CIPRIDINA LUCIFERIN ANALOGS .2. KINETIC-STUDIES ON THE REACTION OF 2-METHYL-6-PHENYLIMIDAZO[1,2-A]PYRAZIN-3(7H)-ONE (CLA) WITH SUPEROXIDE - HYDROPEROXYL RADICAL IS AN ACTUAL ACTIVE SPECIES USED TO INITIATE THE REACTION

Using the xanthine-xanthine oxidase-O-2 system as a continuous superoxide radical anion supplying system, kinetic studies of the chemiluminescence reaction of the Cypridina luciferin analogue (CLA), 2-methyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, with superoxide have been carried out. One mole of CLA appeared to consume two moles of superoxide to emit light in aqueous solution in a pH range 5.5-10.0 under aerobic conditions. Only when the supply of superoxide is very small while the concentration of molecular oxygen is high, was a little contribution of autoxidation of CLA by a chain reaction process observed. A substantial lag in the emission of light after the disappearance of CLA was observed, indicating that the initial reaction of CLA with superoxide is not solely the rate-determining step. Subsequent reactions of intermediates that lead to the final light emitter also contribute to the rate-determining step in this chemiluminescence reaction. The decay of CLA was found to be first order with respect to the concentration of CLA and superoxide from competitive kinetics with superoxide dismutase. From the effect of pH on the apparent second-order rate constant for the decay of CLA, the active species of superoxide that initiates the reaction was found to be the hydroperoxyl radical. The second-order rate constants have been determined to be(7.03 +/- 0.59) x 10(5) dm(3) mol(-1) s(-1) for the reaction with CLA and (2.56 +/- 0.40) x 10(8) dm(3) mol(-1) s(-1) for the reaction with the conjugate base of CLA at 20.0 degrees C.