N-Benzoyl leucomethylene blue as a novel substrate for the assays of horseradish peroxidase by spectrophotometry and capillary electrophoresis-laser-induced fluorometry

Horseradish peroxidase (HRP) is an enzyme that is frequently employed in various assays because HRP catalyzes the oxidation reactions of chromogenic and fluorogenic compounds to produce chromophores and fluorophores, respectively. The results of this study show that N-benzoyl leucomethylene blue (BLMB) is an excellent substrate for enzyme assay using HRP. In the presence of hydrogen peroxide (H2O2), HRP catalyzed an oxidation reaction of BLMB that produced methylene blue with a deep blue color. Thus, absorption spectrophotometry and capillary electrophoresis-laser-induced fluorometry (CE-LIF) could be used to easily determine the produced methylene blue. Under the optimum conditions, absorption spectrophotometry showed a linear calibration curve that ranged from 25 to 500 µg mL-1. The reaction conditions were also applicable to CE-LIF, showing a linear range of from 25 to 500 µg mL-1 with limits of detection and quantification at 2 and 6 µg mL-1, respectively.