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

A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H(2)O(2)) 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 H(2)O(2) by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H(2)O(2) is found between 5x10(-6) and 45x10(-6) mol L(-1) at pH 4.0 and a temperature of 25 degrees C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415x10(6) M(-1) min(-1) and 9.81x10(-4) min(-1), respectively. The catalytic constant (k(cat)) and specificity constant (k(cat)/K(m)) at saturated concentration of the cosubstrates were 163.2 min(-1) and 4.156x10(6) L mol(-1) min(-1), respectively. This method can be incorporated into biochemical analysis where H(2)O(2) undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum.