Chemiluminescence of a cypridina luciferin analogue, 2-methyl-6-phenyl-3,7-dihydroimidazo[l,2-A]pyrazin-3-one, in the presence of the xanthine-xanthine oxidase system

Abstract

Synopsis. The title compound chcmilumincsccs in aqueous solutions (pH 5-6.5) in the presence of xanthine-xanthine oxidase system; 02- and not 1Oa was proven to be the active species. Quantitative determination of xanthine and xanthine oxidase as low as 5 nmol and 10-5 unit, respectively, is possible by measurement of the chemilumines- cence light yield and the reaction rate. In the case of the biolumincsccncc of Cypridina system, light is emitted during the oxidation of Cypridina luciferin (LH) with molecular oxygen by catalysis of an enzyme, Cypridina luciferase. It was suggested that the substrate reacts with molecular oxygen (302) when it is taken in a hydrophobic pocket of the enzyme,2) since the luciferin reacts with molecular oxygen and emits light when it is dissolved in aprotic polar solvents such as dimethyl sulfoxide, diethylcne glycol dimethyl ether, etc., but in aqueous solutions no luminescence is observed without the enzyme. The chemi-luminescence reaction in the aprotic solvents is second order with respect to the concentrations of the substrate (LH) and molecular oxygen;3) a suggested mechanism involves a non-chain reaction between the luciferin anion and oxygen giving a hydroperoxide anion through a radical pair intermediate:2).