Peroxidase from horseradish Type VI-A, essentially salt-free, lyophilized powder, 250-330 units/mg solid (using pyrogallol), 950-2000 units/mg solid (using ABTS)

Synonym: Donor:hydrogen-peroxide oxidoreductase, Horseradish peroxidase

CAS Number: 9003-99-0  
EC Number: 232-668-6  
Enzyme Commission (EC) Number: 1.11.1.7
MDL number: MFCD00071339

Analysis Note
The RZ (Reinheitszahl) is the absorbance ratio A₄₀₃/A₂₇₅ determined at 0.5-1.0 mg/ml in deionized water. It is a measure of hemin content, not enzymatic activity. Even preparations with high RZ may have low enzymatic activity.

This product is assayed using ABTS for easy comparison to other suppliers′ unit: approx. 1,000 units per mg solid

Packaging
Packaged in mg solid

Preparation Note
The product is a 1 mg/mL solution in 0.1 M phosphate buffer(pH 6.0) and remains active for at least two weeks at room temperature. The solution retains activity after 5 freeze-thaw cycles.

Unit Definition
One ABTS unit will oxidize 1 μmole of ABTS per minute at 25 °C at pH 5.0

One pyrogallol unit will form 1.0 mg purpurogallin from pyrogallol in 20 sec at pH 6.0 at 20 °C.

Application
Horseradish peroxidase (HRP) is isolated from horseradish roots (Amoracia rusticana). It is used in biochemistry applications such as western blots, ELISA and Immunohistochemistry. Horseradish peroxidase is used to amplify a weak signal and increase detectability of a target molecule, such as a protein. Product P6782 is type VI-A and premium quailty. It is essentially a salt-free, lyophilized powder. It is used to quantify cholesterol and cholesterol esters and to study in vitro lipid deposition on hydrogel and silicone hydrogel contact lenses.

The enzyme from Sigma has been used in the development of rapid and sensitive galactose oxidase-peroxidase biosensor for galactose detection with prolonged stability. It has been used as a component of a media (MRS agar) plate containing 3,3′,5,5′-tetramethylbenzidine (TMB) for the growth of Lactobacillus sp. TMB acts as a chromogenic substrate of peroxidase. Peroxidase generates O₂ from H₂O₂ produced by the lactobacilli, and the TMB stains the colonies blue in the presence of O₂. Thus, after 48 hours of incubation under 5% CO₂ in air, the colonies that produce H₂O₂ on MRS agar appear dark blue. H₂O₂ nonproducers are colorless. It has been used to study the stabilization effect of polyvinyl alcohol on horseradish peroxidase. It is also used for the determination of glucose and peroxides in solution.

General description
Horseradish peroxidase is isolated from horseradish roots (Amoracia rusticana) and belongs to the ferroprotoporphyrin group of peroxidases. HRP is a single chain polypeptide containing four disulfide bridges. It is a glycoprotein containing 18% carbohydrate. The carbohydrate composition consists of galactose, arabinose, xylose, fucose, mannose, mannosamine, and galactosamine depending upon the specific isozyme. Its molecular weight (~44 kDa) includes the polypeptide chain (33,890 Daltons), hemin plus Ca²⁺ (~700 Daltons), and carbohydrate (~9,400 Daltons). At least seven isozymes of HRP exist. The isoelectric point for horseradish peroxidase isozymes ranges from 3.0 - 9.0.