Quantification of hydrogen peroxide and glucose using 3-methyl-2-benzothiazolinonehydrazone hydrochloride with 10,11-dihydro-5H-benz(b,f)azepine as chromogenic probe

Abstract

A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H2O2) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H-benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H2O2 by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620nm. The linear response for H2O2 is found between 5×10−6 and 45×10−6molL−1 at pH 4.0 and a temperature of 25°C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415×106M−1min−1 and 9.81×10−4min−1, respectively. The catalytic constant (kcat) and specificity constant (kcat/Km) at saturated concentration of the cosubstrates were 163.2min−1 and 4.156×106Lmol−1min−1, respectively. This method can be incorporated into biochemical analysis where H2O2 undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum.