A sensitive and versatile chromogenic assay for peroxidase and peroxidase-coupled reactions
Ngo, T.T.; Lenhoff, H.M.
Analytical Biochemistry 105(2): 389-397
1980
ISSN/ISBN: 0003-2697 PMID: 7457843 Document Number: 334
A sensitive and versatile chromogenic assay for peroxidase and peroxidase-coupled reactions is described. The assay is based on the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino)benzoic acid (DMAB). In the presence of H2O2, MBTH and DMAB peroxidase catalyze the formation of a deep purple compound, most likely an indamine dye, which has a broad absorption band between 575 and 600 nm with a peak at 590 mm. Using this assay system, solutions of peroxidase can be determined in picomolar amounts by either a rate or fixed-time method. The assay was adapted for the measurement of free H2O2 at concentrations of 2-20 .mu.M. By coupling the assay with glucose oxidase, it was possible to measure glucose at levels of 5-25 .mu.M; from the data an operational molar extinction coefficient of 47,600 was calculated. Maltose could be assayed by the glucose oxidase modified system by 1st preincubating with .alpha.-glucosidase; a linear relationship between the absorbance and maltose concentrations in the range of 3 to 13 .mu.M was obtained. Comparison of this assay to others shows it to have many more advantages; in addition to its increased sensitivity and versatility, it employs compounds not shown to be carcinogenic and which are very soluble in water. This assay should offer broad applicability to assays based on peroxidase-coupled reactions such as for glucose determination and in enzyme immunoassays.
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