Reestimation of Cypridina luciferin analogs (MCLA) as a chemiluminescence probe to detect active oxygen species - Cautionary note for use of MCLA

Cypridina luciferin analogs have been widely used as a specific chemiluminescence probe for the detection of superoxide anion (O ₂^-) and singlet oxygen (¹O₂). However, light emission during the reaction of Cypridina luciferin analogs and other active oxygen species (AOS) has not been reported in detail. Therefore, we re-evaluated 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazine-3-one (MCLA), one of the Cypridina luciferin analogs, as a chemiluminescence probe to detect various AOS. MCLA-dependent chemiluminescence was observed when MCLA was incubated with the following systems; 1) hypoxanthine plus xanthine oxidase (O₂^-), 2) thermolysis of endoperoxide (¹O ₂), 3) hydrogen peroxide plus ferrous ion (hydroxyl radical), 4) ferrous ion, 5) thermolysis of azo compound (alkyl peroxyl radical) and 6) hydrogen peroxide. Superoxide dismutase inhibited MCLA-dependent chemiluminescence observed during ferrous ion-induced decomposition of hydrogen peroxide. Alkyl peroxyl radical reacted with MCLA, but light was not emitted when the concentration of MCLA was high. These results suggest that radicals, except o₂^-, appeared not to be direct inducers of MCLA-dependent light emission. In summary, MCLA-dependent chemiluminescence was induced by various AOS in addition to O₂^-and ¹O₂, but active species must be O₂ ^-and ¹O₂ in many cases. These points should be appreciated when Cypridina luciferin analogs, such as MCLA, are used for the detection of AOS.