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Hydrogen peroxide in the presence of horseradish peroxidase effects the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone with its formaldehyde azine to form a tetraazapentamethine dye. The blue chromophore, when formed at pH 3.5 and quenched with acetone or 1 N hydrochloric acid, has an extinction coefficient of 69 +/- 2 or 55 +/- 2 mM-1 cm-1, respectively. This chromogen system has been adapted for enzymatic determinations of hydrogen peroxide and of glucose in the 10- to 45-nmol range and of choline in the 5- to 20-nmol range.
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