P6782

Peroxidase from horseradish

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

• 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
• eCl@ss: 32160410
• NACRES: NA.54

Properties

• type: Type VI-A
• Quality Level: 300
• form: essentially salt-free, lyophilized powder
• specific activity: ≥250 units/mg solid (using pyrogallol); 950-2000 units/mg solid (using ABTS)
• mol wt: ~44 kDa
• solubility: 0.1 M phosphate buffer: soluble 10 mg/mL, clear, orange to red (pH 6.0); H₂O: soluble
• application(s): diagnostic assay manufacturing
• storage temp.: 2-8°C

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

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 it is essentially a salt-free, lyophilized powder. It is used to quantify cholesterol and cholesterol esters[1] and to study in vitro lipid deposition on hydrogel and silicone hydrogel contact lenses[2].

The enzyme from Sigma has been used in the development of rapid and sensitive galactose oxidase-peroxidase biosensor for galactose detection with prolonged stability.[3] 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.[4] It has been used to study the stabilization effect of polyvinyl alcohol on horseradish peroxidase.[5] It is also used for the determination of glucose[6] and peroxides[7] in solution.

Packaging

5 mg in glass bottle

10, 25 mg in poly bottle

50, 100 mg in glass bottle

Packaged in mg solid

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 that include the use of glutaraldehyde, periodate oxidation, 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.[8] Sodium azide, cyanide, L-cystine, dichromate, ethylenethiourea, hydroxylamine, sulfide, vanadate, p-aminobenzoic acid, as well as Cd²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mn²⁺, Ni²⁺, and Pb²⁺ ions are found to inhibit the enzyme activity.[9]

When incubated with a substrate, horseradish peroxidase produces a coloured, fluorimetric, or luminescent derivative of the labeled molecule, allowing quantification. Horseradish peroxidase has been shown to slightly reduce the level of inhibition in a cydAB mutant.

Linkage

Similar to P8375

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.

Preparation Note

Solutions of this product at 1 mg/mL in 0.1 M phosphate buffer (pH 6.0) remain active for at least two weeks at room temperature. The solution retains activity after 5 freeze-thaw cycles.

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

Other Notes

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

Safety Information

• Pictograms: GHS08
• Signal Word: Danger
• Hazard Statements: H317 - H334
• Precautionary Statements: P261 - P272 - P280 - P284 - P302 + P352 - P333 + P313
• Hazard Classifications: Resp. Sens. 1 - Skin Sens. 1
• Storage Class Code: 11 - Combustible Solids
• WGK: WGK 3
• Personal Protective Equipment: dust mask type N95 (US), Eyeshields, Gloves