Existence of a new reactive intermediate oxygen species in hypoxanthine and xanthine oxidase reaction

We investigated a hypoxanthine (HPX) and xanthine oxidase (XOD) reaction by using a luminol analog 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012)-mediated chemiluminescence (CL) response. Addition of a high activity of superoxide dismutase (SOD), a potent O2* scavenger, and of a high concentration of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a potent spin trapping agent, diminished completely the CL response. Whereas a high concentration of dimethyl sulfoxide (DMSO), as a potent *OH scavenger could not attain to the complete diminishment of the CL response. It has been reported that luminol monoanion reacts with *OH to form luminol radical, and then resultant luminol radical reacts with O2* to elicit CL response. Complete scavenging for *OH is assumed to result in lack of luminol radical, which in turn induces lack of CL response. However, our results did not support the idea. Furthermore, we examined the effect of L-012 on the DMPO-OOH formation in the presence or absence of DMSO in the HPX-XOD system by applying an electron spin resonance (ESR)-spin trapping method. The DMPO-OOH formation was inhibited even in the presence of DMSO, and the rate constant (k2) between L-012 and O2* obtained in the presence of DMSO was 9.77 x 10(2) M(-1) s(-1) and the constant in the absence of DMSO was 2.97 x 10(3) M(-1) s(-1). The data suggests that L-012 is converted to a radical form that reacts with O2* even under the conditions of the absence of *OH. From these, we postulate that the existence of a reactive intermediate oxygen species in the HPX-XOD system.