Peroxidases (EC number 1.11.1.x) catalyze the following oxidationreduction reactions: ROOR'+electron donor (2 e-)+2H+ → ROH+R'OH For many peroxidases the optimal substrate is hydrogen peroxide (H2O2), but others are more active with organic hydroperoxides such as lipid peroxides. In the cell, peroxidases destroy toxic hydroxide radicals that are formed as byproducts during aerobic respiration. The peroxidases represent a large family of enzymes that are found in animals (e.g. myeloperoxidase-like enzymes), plant, fungi and bacteria (cytochrome-c peroxidase like enzymes such a horseradish peroxidase).Simple, direct and automation-ready procedures for determining peroxidase activity find wide Applications: Peroxidase assay uses H2O2 and an electron donor dye that forms a pink color during the peroxidase reaction. The optical density (570nm) or fluorescence intensity (lexc=530nm, lem=590nm) is a direct measure of the enzyme activity.Key Features:Use as little as 10ul samples. Linear detection range: Colorimetric Assay:s 4 to 1000 IU/L, Fluorometric Assay:s 0.8 to 25 IU/L peroxidase.Kit Contents:Assay Buffer (pH 7.0): 20ml Dye Reagent: 60ulStabilized H2O2: 100ul 3% S top Reagent: 12mlCalibrator: 5ml (equivalent to 1000 IU/L)Storage conditions. The kit is shipped on blue ice. Store Assay Buffer and Dye Reagent at -20°C, all other reagents at 4°C. Shelf life: 3 months after receipt.Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.Procedures:Sample Preparation. Samples can be prepared according to established methods [1-3]. It is prudent to test multiple sample dilutions to ensure activity is in the linear range.Reagent Preparation: Bring all reagents to room temperature prior to assay. Dilute 3% H2O2 in Assay Buffer to 0.6% and use within one hour.96-Well Colorimetric Assay: Procedure:. Use clear flat-bottom plates for Colorimetric Assay:s1. Transfer 200ul H2O and 200ul Calibrator into two wells of a clear flat-bottom 96-well plate. Transfer 10ul H2O (sample blank), 10ul sample to separate wells. Prepare fresh Working Reagent for each reaction well by mixing 95ul Assay Buffer, 0.5ul Dye Reagent and 0.5ul freshly diluted 0.6% H2O2. Add 90ul Working Reagent to each sample well. Tap plate to mix and incubate for 10 min at room temperature.2. Add 100ul Stop Reagent to the sample blank and sample wells. Tap plate to mix and read OD570nm.Note: if Sample OD values are higher than that of the Calibrator, dilute sample in Assay Buffer, repeat assay and multiply results by the dilution factor. Peroxidase activity is calculated from the OD values of the sample, sample blank, calibrator and H2O wells.Peroxidase Activity=x 1000 (IU/L)ODSAMPLE–ODBLANKODCALIBRATOR–ODH2OUnit definition: one unit of enzyme will catalyze the oxidization by H2O2 of 1uMole dye reagent per min under the assay conditions.96-Well Fluorometric Assay: Procedure:. Use black flat-bottom plates. If desirable, a peroxidase standard can be run together with the samples.1. Transfer 10ul H2O, 10ul sample to separate wells. Prepare fresh Working Reagent for each sample well by mixing 95ul Assay Buffer, 0.5ul Dye Reagent and 0.5ul freshly diluted 0.6% H2O2. Add 90ul Working Reagent to each well. Tap plate to mix and incubate for 10 min at room temperature.2. Add 100ul Stop Reagent to the sample blank and sample wells. Tap plate to mix and read fluorescence at 590nm (lexc= 530nm).384-Well Fluorometric Assay: Procedure:. Use 5ul H2O, 5ul sample, 35ul Working Reagent prepared from 38ul Assay Buffer, 0.2ul Dye Reagent, 0.2ul 0.6% H2O2. Use 40ul Stop Reagent.General Considerations:This assay is based on a kinetic reaction, the use of a multi-channel pipettor for adding the Working Reagent and Stop Reagent is recommended.Materials Required, But Not Provided:Pipeting devices, centrifuge tubes, clear flat bottom 96/384-well plates for Colorimetric Assay:s, black 96/384-well plate for Fluorometric Assay:s and plate reader.
