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 O-2(-center dot) 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)-O-center dot scavenger could not attain to the complete diminishment of the CL response. It has been reported that luminol monoanion reacts with (OH)-O-center dot to form luminol radical, and then resultant luminol radical reacts with O-2(-center dot) to elicit CL response. Complete scavenging for (OH)-O-center dot 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 (k(2)) between L-012 and O-2(-center dot) 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 O-2(-center dot) even under the conditions of the absence of (OH)-O-center dot. From these, we postulate that the existence of a reactive intermediate oxygen species in the HPX-XOD system.