Merck
All Photos(4)

P6782

Sigma-Aldrich

Peroxidase from horseradish

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

Synonym(s):
Donor:hydrogen-peroxide oxidoreductase, Horseradish peroxidase
CAS Number:
Enzyme Commission number:
EC Number:
MDL number:
eCl@ss:
32160410
NACRES:
NA.54

type

Type VI-A

Quality Level

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

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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

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.
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 and to study in vitro lipid deposition on hydrogel and silicone hydrogel contact lenses.

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

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.
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. 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.

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

Pictograms

Health hazard

Signal Word

Danger

Hazard Statements

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

Certificate of Analysis

Enter Lot Number to search for Certificate of Analysis (COA).

Certificate of Origin

Enter Lot Number to search for Certificate of Origin (COO).

Technical Information

More documents

Quotes and Ordering

Rapid and sensitive galactose oxidase-peroxidase biosensor for galactose detection with prolonged stability
Tkac J, et al.
Biotechnol. Tech., 13(12), 931-936 (1999)
Stabilization effect of polyvinyl alcohol on horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase.
Boyd S, et al.
Biotechnol. Tech., 10(9), 693- 698 (1996)
From Concept to Product Development
Enzyme Immunoassay, 169-171 (1996)
A Felten et al.
Journal of clinical microbiology, 37(3), 729-733 (1999-02-13)
Lactobacilli recovered from the blood, cerebrospinal fluid, respiratory tract, and gut of 20 hospitalized immunocompromised septic patients were analyzed. Biochemical carbohydrate fermentation and total soluble cell protein profiles were used to identify the species. Hydrogen peroxide production was measured. Susceptibility
Chiara Marabelli et al.
Cell reports, 27(2), 387-399 (2019-04-11)
LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM)

Protocols

Spectrophotometric Determination of Reinheitszahl (RZ) for Peroxidase (EC 1.11.1.7)

Reinheitszahl (RZ) is the ratio of absorbance due to hemin (A403, Soret region) to absorbance due to protein (A275).

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