P8250

Perossidasi

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

• Sinonimo/i: Donatore: idrogeno-perossido ossidoreduttasi, Perossidasi di rafano
• Numero CAS: 9003-99-0
• eCl@ss: 32160410
• UNSPSC Code: 12352204
• NACRES: NA.54
• EC Number: 232-668-6
• MDL number: MFCD00071339
• Numero CE: 1.11.1.7 (BRENDA, IUBMB)
• Specific activity: 150-250 units/mg solid (using pyrogallol)
• Biological source: horseradish

Proprietà

• biological source: horseradish
• Quality Segment: 300
• 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
• SMILES string: [O+H2]O[O-]
• InChI: 1S/H2O3/c1-3-2/h1-2H
• InChI key: JSPLKZUTYZBBKA-UHFFFAOYSA-N

Descrizione

General description

La perossidasi di rafano si isola dalle radici del rafano (Amoracia rusticana) e appartiene al gruppo delle perossidasi con ferroprotoporfirina. L′HRP è un polipeptide a singola catena che contiene quattro ponti disolfuro. È una glicoproteina che contiene il 18% di carboidrati. La composizione dei carboidrati include galattosio, arabinosio, xilosio, fucosio, mannosio, mannosamina e galattosamina, a seconda del singolo isozima. Il peso molecolare (circa 44 kDa) include la catena polipeptidica (33.890 Dalton), la emina con Ca²⁺ (circa 700 Dalton) e il carboidrato (circa 9.400 Dalton). Esistono almeno sette isoenzimi di HRP. Il punto isoelettrico degli isoenzimi della perossidasi di rafano è compreso fra 3,0 e 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[1] and Ebola virus glycoprotein toxicity[2].

The enzyme has been used as a comparison during the peroxidase assay of extract from mature tall fescue leaf blades.[3] It has also been used to measure H₂O₂ production.[4]

Biochem/physiol Actions

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.

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.

Analysis Note

Il RZ (Reinheitszahl) è il rapporto di assorbanza A₄₀₃/A₂₇₅ determinato a 0,5-1,0 mg/mL in acqua deionizzata. Misura il contenuto di emina, non l′attività enzimatica. Anche preparazioni con un RZ elevato possono avere una bassa attività enzimatica.

Preliminary work shows it to contain at least five isoenzymes.

Other Notes

Per maggiori informazioni sulla perossidasi visitare la pagina www.sigma-aldrich.com/enzymeexplorer.

Una unità di pirogallolo forma 1,0 mg di purpurogallina da pirogallolo in 20 secondi a pH 6,0 e 20 °C.

Informazioni sulla sicurezza

• pictograms: GHS08
• signalword: Danger
• hcodes: H334
• pcodes: P261 - P284 - P501
• Hazard Classifications: Resp. Sens. 1
• Classe di stoccaggio: 13 - Non Combustible Solids
• ppe: Eyeshields, Gloves, type N95 (US)