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P8250

Sigma-Aldrich

Peroxidase from horseradish

Type II, essentially salt-free, lyophilized powder, 150-250 units/mg solid (using pyrogallol)

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

biological source

horseradish

Quality Level

type

Type II

form

essentially salt-free, lyophilized powder

specific activity

150-250 units/mg solid (using pyrogallol)

mol wt

~44 kDa

solubility

0.1 M phosphate buffer: soluble (pH 6.0)
H2O: soluble

absorbance ratio

RZ ≥1.8

application(s)

diagnostic assay manufacturing

storage temp.

2-8°C

InChI

1S/H2O3/c1-3-2/h1-2H

InChI key

JSPLKZUTYZBBKA-UHFFFAOYSA-N

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1 of 4

This Item
P6782P8125P2088
vibrant-m

P8250

Peroxidase from horseradish

vibrant-m

P6782

Peroxidase from horseradish

vibrant-m

P8125

Peroxidase from horseradish

vibrant-m

P2088

Peroxidase from horseradish

biological source

horseradish

biological source

-

biological source

-

biological source

horseradish

application(s)

diagnostic assay manufacturing

application(s)

diagnostic assay manufacturing

application(s)

-

application(s)

-

storage temp.

2-8°C

storage temp.

2-8°C

storage temp.

2-8°C

storage temp.

2-8°C

mol wt

~44 kDa

mol wt

~44 kDa

mol wt

~44 kDa

mol wt

~44 kDa

Quality Level

300

Quality Level

300

Quality Level

200

Quality Level

300

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. Horseradish peroxidase, product P8250, has been used to study nonoral antigens in inflamed gingiva and Ebola virus glycoprotein toxicity.
The enzyme has been used as a comparison during the peroxidase assay of extract from mature tall fescue leaf blades. It has also been used to measure H2O2 production.

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 found 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. Horseradish peroxidase has been shown to slightly reduce the level of inhibition in a cydAB mutant.

Quality

Preliminary work shows it to contain at least five isoenzymes.

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.

pictograms

Health hazard

signalword

Danger

Hazard Classifications

Resp. Sens. 1 - Skin Sens. 1

Storage Class

13 - Non Combustible Solids

wgk_germany

WGK 3

ppe

Eyeshields, Gloves, type N95 (US)


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Protocols

To standardize a procedure for the enzymatic assay of Diamine Oxidase.

To measure alcohol oxidase activity, this assay uses 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) and a continuous spectrophotometric rate determination at 405 nm.

This procedure uses peroxidase and o-dianisidine to measure cholesterol oxidase activity at 500 nm. Cholesterol oxidase is an enzyme that acts on the CH-OH group of the donor with oxygen as the acceptor.

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

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