P6140

Peroxidasa from horseradish

Type X, ammonium sulfate suspension

• Sinónimos: Donante:peróxido de hidrógeno oxidorreductasa, Peroxidasa de rábano picante
• Número de CAS: 9003-99-0
• Comisión internacional de enzimas: 1.11.1.7 (BRENDA, IUBMB)
• EC Number: 232-668-6
• MDL number: MFCD00071339
• UNSPSC Code: 12352204
• eCl@ss: 32160410
• NACRES: NA.54

Propiedades

• biological source: horseradish
• type: Type X
• form: ammonium sulfate suspension
• specific activity: ≥225 units/mg protein (biuret, using pyrogallol)
• mol wt: ~44 kDa
• concentration: ≥1.0 mg/mL
• absorbance ratio: RZ 2.5-3.5
• storage temp.: 2-8°C
• InChI: 1S/H2O3/c1-3-2/h1-2H
• InChI key: JSPLKZUTYZBBKA-UHFFFAOYSA-N

Descripción

General description

La peroxidasa de rábano picante se aísla de las raíces de rábano picante (Amoracia rusticana) y pertenece al grupo de ferroprotoporfirina de las peroxidasas. La HRP es un polipéptido monocatenario que contiene cuatro puentes bisulfuro. Es una glucoproteína que contiene un 18 % de carbohidratos. La composición de los carbohidratos consiste en galactosa, arabinosa, xilosa, fucosa, manosa, manosamina y galactosamina dependiendo de la isoenzima específica. Su peso molecular (~44 kDa) incluye la cadena polipeptídica (33 890 dalton), la hemina más Ca²⁺ (~700 dalton) y los carbohidratos (~9 400 dalton). Existen al menos siete isoenzimas de la HRP. El punto isoeléctrico de las isoenzimas de la peroxidasa de rábano picante oscila entre 3,0 y 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. 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.

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 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.

Unit Definition

Una unidad de pirogalol formará 1,0 mg de purpurogalina a partir de pirogalol en 20 segundos a pH 6,0 a 20 °C.

Physical form

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

Preparation Note

Water may be used to dilute suspension if needed.

Analysis Note

La RZ (Reinheitszahl) es la relación de absorbancia A₄₀₃/A₂₇₅ determinada a 0,5-1,0 mg/ml en agua desionizada. Es una medida del contenido de hemina, no de la actividad enzimática. Incluso preparaciones con RZ elevada pueden tener una actividad enzimática baja.

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

Other Notes

Véase más información sobre la peroxidasa en www.sigma-aldrich.com/enzymeexplorer.

Información de seguridad

• pictograms: GHS08
• signalword: Danger
• hcodes: H334
• pcodes: P261 - P284 - P501
• Hazard Classifications: Resp. Sens. 1
• Storage Class: 11 - Combustible Solids
• wgk_germany: WGK 1
• ppe: Eyeshields, Gloves, type N95 (US)