Peroxydase from horseradish

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.

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 P6140 has been used to detect low density lipoprotein (LDL).

The enzyme from Sigma has been used while assessing the skin sensitization potential of pro-haptens. It has also been used to show that peroxidase (PO) activity and its heat stability correlate with the availability of free Ca²⁺ ions.

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. 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 its 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.

Crystalline suspension in 3.2 M (NH₄)₂SO₄ solution containing potassium phosphate buffer, pH 6.0

Water may be used to dilute suspension if needed.

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.

Preliminary studies indicate the presence of two basic and no acidic isoenzymes

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

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