Stopped-flow study of the enhanced chemiluminescence for the oxidation of luminol with hydrogen peroxide catalyzed by microperoxidase 8

The presence of carbonate or Tris causes a dramatic enhancement in the cheminluminescence (CL) for the oxidation of luminol with hydrogen peroxide catalyzed by microperoxidase 8 (MP8). A nearly constant enhancement in CL was observed over a wide range of H2O2 and luminol concentrations. The enhancement in CL is strongly pH-dependent, varying from 1.3 to 22.2 for carbonate and 1.6 to 10.2 for Tris. The CL enhancement is much more prominent at pH 9–10 than at high pH (>10.5) because of the extremely weak CL emission at pH below 10 when no enhancer is present. The CL enhancement is attributed to an accelerated CL cycle and the existence of alternative routes for luminol CL, possibly involving the carbonate, or Tris radicals. The dramatic enhancement in CL of the MP8-luminol-H2O2 system by the readily available reagents, sodium carbonate or Tris, will have general applications for sensitive CL assays. As an example, the presence of antioxidant results in a diminished and delayed CL emission, allowing the determination of its concentration at sub-micromolar level.