P6782

Peroxydase from horseradish

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

• Synonyme(s) : Donneur : peroxyde d'hydrogène oxydoréductase, Peroxydase de raifort
• Numéro CAS: 9003-99-0
• Numéro de classification (Commission des enzymes): 1.11.1.7 (BRENDA, IUBMB)
• Numéro CE : 232-668-6
• Numéro MDL: MFCD00071339
• Code UNSPSC : 12352204
• eCl@ss : 32160410
• Nomenclature NACRES : NA.54

Propriétés

• Type: Type VI-A
• Niveau de qualité: 300
• Forme: essentially salt-free, lyophilized powder
• Activité spécifique: ≥250 units/mg solid (using pyrogallol); 950-2000 units/mg solid (using ABTS)
• Poids mol.: ~44 kDa
• Solubilité: 0.1 M phosphate buffer: soluble 10 mg/mL, clear, orange to red (pH 6.0); H₂O: soluble
• Application(s): diagnostic assay manufacturing
• Température de stockage: 2-8°C
• InChI: 1S/H2O3/c1-3-2/h1-2H
• Clé InChI: JSPLKZUTYZBBKA-UHFFFAOYSA-N

Description

Description générale

La peroxydase de raifort (HRP, de l′anglais horseradish peroxidase) est isolée des racines de raifort (Armoracia rusticana) et appartient au groupe des peroxydases de type ferroprotoporphyrine. La HRP est un polypeptide à une seule chaîne qui comporte quatre ponts disulfure. Il s′agit d′une glycoprotéine contenant 18 % de glucides. Selon l′isozyme, les glucides peuvent inclure les constituants suivants : galactose, arabinose, xylose, fucose, mannose, mannosamine et galactosamine. Le poids moléculaire de la peroxydase de raifort (env. 44 kDa) inclut la chaîne polypeptidique (33,890 Daltons), l′hémine plus Ca²⁺ (env. 700 Daltons) et les glucides (env. 9,400 Daltons). Au moins sept isozymes existent. Le point isoélectrique des isozymes de peroxydase de raifort se situe dans une plage de 3,0 à 0,9.

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

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.

Actions biochimiques/physiologiques

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.

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.

Conditionnement

Packaged in mg solid

Liaison

Similar to P8375

Définition de l'unité

Une unité de pyrogallol forme 1,0 mg de purpurogalline à partir de pyrogallol en 20 sec à pH 6,0 et 20 °C.

One ABTS unit will oxidize 1 μmole of ABTS per minute at 25 °C at pH 5.0

Notes préparatoires

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.

Remarque sur l'analyse

La valeur RZ (Reinheitszahl) est le rapport d′absorbance A₄₀₃/A₂₇₅ mesuré à 0,5-1,0 mg/ml dans de l′eau désionisée. C′est une mesure de la teneur en hémine, plutôt que de l′activité enzymatique. Il est possible que des préparations présentant une valeur RZ élevée aient une faible activité enzymatique.

This product is assayed using ABTS for easy comparison to other suppliers′ unit: approx. 1,000 units per mg solid

Autres remarques

Pour en savoir plus sur la peroxydase, rendez-vous sur www.sigma-aldrich.com/enzymeexplorer.

Informations sur la sécurité

• Pictogrammes: GHS08
• Mention d'avertissement: Danger
• Mentions de danger: H334
• Conseils de prudence: P261 - P284 - P501
• Classification des risques: Resp. Sens. 1
• Code de la classe de stockage: 11 - Combustible Solids
• Classe de danger pour l'eau (WGK): WGK 1
• Équipement de protection individuelle: Eyeshields, Gloves, type N95 (US)