CL : Vol. 38 (2009) , No. 4 p.302

Proposed Mechanisms for HOOOH Formation in Two Typical Enzyme Reactions Responsible for Superoxide Anion Production in Biological Systems

Masahiro Kohno¹⁾, Emiko Sato¹⁾, Noriko Yaekashiwa¹⁾, Takayuki Mokudai¹⁾ and Yoshimi Niwano¹⁾

1) New Industry Creation Hatchery Center, Tohoku University

(Received December 17, 2008)

We investigated the hypoxanthine (HPX)–xanthine oxidase (XOD) reaction by examining the chemiluminescence (CL) response mediated by a luminol analog, 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)-dione sodium salt (L-012). It was found that addition of a high concentration of dimethyl sulfoxide (DMSO), a potent ^•OH scavenger, could not completely reduce the CL response. This result suggests the existence of an unknown reactive oxygen intermediate other than O₂^−• and ^•OH. We further examined the HPX–XOD reaction and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidation reaction by applying an electron spin resonance (ESR) spin-trapping method. In both reaction systems, similar responses were observed. That is, addition of DMSO increased the formation of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) –OOH in a concentration-dependent manner. This indicates that scavenging of ^•OH increases the detected O₂^−• level, further suggesting the existence of an intermediate oxygen species derived from O₂^−• and ^•OH. One candidate for this species is HOOOH, presumably formed in the following way.

O₂^−• + H⁺ + ^•OH →HOOOH

In two typical enzyme reaction systems producing reactive oxygen species, hypoxanthine–xanthine oxidase and nicotinamide adenine dinucleotide phosphate oxidation, we postulate the existence of an intermediate oxygen species derived from O₂^−• and ^•OH, as in the following reaction. O₂^−• + H⁺ + ^•OH → HOOOH