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A Rapid Microassay for Determination of Peroxidase in Wheat and Flour¹

¹Contribution of the Grain Research Laboratory, Canadian Grain Commission, Winnipeg, MB, Canada. Use of a commercial product does not imply endorsement. ²Research scientist and biochemistry technician, respectively, Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main St., Winnipeg, MB R3C 3G8, Canada. ³Corresponding author. Phone: 204-983-5362. Fax: 204-983-0724. E-mail: dhatcher@grainscanada.gc.ca

A rapid sensitive microplate assay for the determination of peroxidase activity in wheat, flour, or individual wheat kernels has been developed using a commercially prepared 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) solution. The assay derives a six-point calibration curve based on commercially available horseradish peroxidase (r² > 0.990), while simultaneously analyzing eight extracts in triplicate. The coefficients of variation (CV) of triplicate assays of a single extract from any of the three sources investigated were generally <2.0%. Multiple extracts (n = 5) of either whole meal or flour yielded assays with an average CV < 5.0%. Preparation of the calibration standards with commercially available peroxidase stabilizing buffer allowed the standards to be used for five days without any deterioration in the assay's reproducibility. This assay is ideally suited for high-throughput operations such as millstream analysis or plant breeder screening evaluations.