Nitroxyl Radicals Remarkably Enhanced the Superoxide Anion Radical-Induced Chemiluminescence of Cypridina Luciferin Analogues

Abstract

Measuring the superoxide anion radical (superoxide) with high sensitivity is necessary to clarify the mechanisms of diseases for the development of methods for their prophylaxes, diagnoses, and therapies. The chemiluminescence technique using Cypridina luciferin analogues such as MCLA and CLA is currently the most sensitive method available. Using large concentrations of these reagents, however, leads to increases in background levels due to spontaneous luminescence of the reagent, which is a limitation of this method. This study demonstrated that the superoxide-induced chemiluminescence of MCLA or CLA was markedly enhanced by adding a cyclic nitroxyl radical to the reaction medium. When MCLA was measured spectrophotometrically, the nitroxyl radical was shown to increase the reaction rate of superoxide and MCLA without altering their stoichiometry, whereas consumption of the nitroxyl radical was negligible, as determined by electron paramagnetic resonance (EPR) spectroscopy. These observations indicate that the nitroxyl radical catalytically enhanced the reaction between superoxide and MCLA, resulting in an enhancement in superoxide-dependent MCLA chemiluminescence. This method is applicable to biological systems such as superoxide-generation by neutrophils. The inclusion of the cyclic nitroxyl radical in a sample solution contributed to reductions in the concentration of the chemiluminescence reagent, thereby decreasing background levels. The catalytic mechanism was also discussed.