P8250

Peroxidase from horseradish

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

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

Tulajdonságok

• biológiai forrás: horseradish
• Minőségi szint: 300
• típus: Type II
• form: essentially salt-free, lyophilized powder
• specifikus aktivitás: 150-250 units/mg solid (using pyrogallol)
• molekulatömeg: ~44 kDa
• oldhatóság: 0.1 M phosphate buffer: soluble (pH 6.0); H₂O: soluble
• abszorbancia arány: RZ ≥1.8
• alkalmazás(ok): diagnostic assay manufacturing
• tárolási hőmérséklet: 2-8°C
• InChI: 1S/H2O3/c1-3-2/h1-2H
• Nemzetközi kémiai azonosító kulcs: JSPLKZUTYZBBKA-UHFFFAOYSA-N

Leírás

Általános leírás

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.

Alkalmazás

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.

Biokémiai/fiziológiai hatások

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.

Minőség

Preliminary work shows it to contain at least five isoenzymes.

Egység definíció

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

Analízis megjegyzés

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.

Egyéb megjegyzések

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

Biztonsági adatok

• Piktogramok: GHS08
• Figyelmeztetés: Danger
• Figyelmeztető mondatok: H334
• Óvintézkedésre vonatkozó mondatok: P261 - P284 - P501
• Veszélyességi osztályok: Resp. Sens. 1
• Tárolási osztály kódja: 13 - Non Combustible Solids
• WGK: WGK 1
• Egyéni védőeszköz: Eyeshields, Gloves, type N95 (US)