P8125

Peroxidase from horseradish

Type I, essentially salt-free, lyophilized powder, ≥50 units/mg solid (using pyrogallol)

• Synonym(s): Donor:hydrogen-peroxide oxidoreductase, Horseradish peroxidase
• CAS Number: 9003-99-0
• Enzyme Commission number: 1.11.1.7 (BRENDA, IUBMB)
• EC Number: 232-668-6
• MDL number: MFCD00071339
• UNSPSC Code: 12352204
• eCl@ss: 32160410
• NACRES: NA.54

Properties

• type: Type I
• form: essentially salt-free, lyophilized powder
• specific activity: ≥50 units/mg solid (using pyrogallol)
• mol wt: ~44 kDa
• solubility: 0.1 M phosphate buffer: soluble (pH 6.0); H₂O: soluble
• absorbance ratio: RZ ≥1.0
• storage temp.: 2-8°C
• InChI: 1S/H2O3/c1-3-2/h1-2H
• InChI key: JSPLKZUTYZBBKA-UHFFFAOYSA-N

Description

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.

Application

Peroxidase from horseradish has been used:

• in fecal starch analysis
• to measure plasma glucose levels by Trinder method
• to incubate feruloylated arabinoxylan fractions and also to evaluate oxidative coupling
• in the detection of glycoproteins

Biochem/physiol Actions

HRP readily combines with hydrogen peroxide (H₂O₂) and the resultant [HRP-H₂O₂] complex can oxidize a wide variety of hydrogen donors. The optimal pH is 6.0-6.5 and the enzyme is most stable in the pH range of 5.0-9.0. HRP can be conjugated to antibodies by several different methods including glutaraldehyde, periodate oxidation, through disulfide bonds, and also via amino and thiol directed cross-linkers. It is smaller and more stable than the enzyme labels β-galactosidase and alkaline phosphatase.Hence, it is the most desired label. Also, its glycosylation leads to lower non-specific binding. It is also used for the determination of glucose and peroxides in solution. Sodium azide, cyanide, L-cystine, dichromate, ethylenethiourea, hydroxylamine, sulfide, vanadate, p-aminobenzoic acid, and Cd²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mn²⁺, Ni²⁺, Pb²⁺ ions are known to inhibit the enzyme activity.

When incubated with a substrate, horseradish peroxidase produces a coloured, fluorimetric, or luminescent derivative of the labeled molecule, allowing quantification.

Unit Definition

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

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.

Other Notes

View more information on peroxidase at www.sigma-aldrich.com/enzymeexplorer.

Safety Information

• Pictograms: GHS08
• Signal Word: Danger
• Hazard Statements: H334
• Precautionary Statements: P261 - P284 - P501
• Hazard Classifications: Resp. Sens. 1
• Storage Class Code: 11 - Combustible Solids
• WGK: WGK 1
• Personal Protective Equipment: dust mask type N95 (US), Eyeshields, Gloves