P8250
Peroxidase from horseradish
Type II, essentially salt-free, lyophilized powder, 150-250 units/mg solid (using pyrogallol)
Synonym(s):
Donor:hydrogen-peroxide oxidoreductase, Horseradish peroxidase
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About This Item
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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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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.
inhibitor
Product No.
Description
Pricing
substrate
Product No.
Description
Pricing
Signal Word
Danger
Hazard Statements
Precautionary Statements
Hazard Classifications
Resp. Sens. 1
Storage Class Code
13 - Non Combustible Solids
WGK
WGK 1
Personal Protective Equipment
dust mask type N95 (US), Eyeshields, Gloves
Certificates of Analysis (COA)
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