Abstract
An ultrasensitive chromogenic system for horseradish peroxidase (HRP) is described. The chromogenic substrate system consists of 3-methyl-2-benzothiazolinone hydrazone (MBTH) andN-phenyldiethanolamine (PDEA). The oxidative coupling of MBTH and PDEA using H2O2 and HRP, respectively, as the oxidant and catalyst, yields a blue chromophore that most likely is an indamine dye. The chromophore has a rather broad absorption band, with an absorption maximum at 602 nm. In 0.1M phosphate, 0.1M citrate buffer, pH 7.5, the optimal concentrations of MBTH, PDEA, and H2O2 are, respectively, 0.5, 25, and 8 mM. Using this assay system, HRP can be determined in lower picomolar levels by either a rate or fixed-time method.
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