Fluorimetric Determination of Peroxynitrite Based on an Enzymatic Reaction

Abstract

A novel fluorimetric method for the determination of peroxynitrite (ONOO^-) using hemoglobin (Hb) as a catalyst is described. The method employs the reaction of ONOO^- with thiamine (TM), a colorless, non-fluorescent reagent in a glycine-NaCl-NaOH buffer solution (pH 12.7), to generate a highly fluorescent product, thiochrome (TC). The fluorescent product was monitored by fluorimetry. A linear calibration graph was obtained over an ONOO^- concentration range from 4.95 × 10^-7 mol L^-1 to 2.97 × 10^-5 mol L^-1, with a detection limit of 9.78 × 10^-9 mol L^-1 ONOO^-. The relative standard deviation at an ONOO^- concentration of 2.11 × 10^-6 mol L^-1 was 4.15% (n = 9).