P8415

Peroxidase

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

• Sinônimo(s): Doador:peróxido de hidrogênio oxidorredutase, Peroxidase de raiz forte
• Número CAS: 9003-99-0
• Número da licença da enzima: 1.11.1.7 (BRENDA, IUBMB)
• Número CE: 232-668-6
• Número MDL: MFCD00071339
• Código UNSPSC: 12352204
• NACRES: NA.54

Propriedades

• tipo: Type XII
• Formulário: essentially salt-free, lyophilized powder
• atividade específica: ≥250 units/mg solid (using pyrogallol)
• peso molecular: ~44 kDa
• solubilidade: 0.1 M phosphate buffer: soluble (pH 6.0); H₂O: soluble
• razão de absorbância: RZ ≥3.0
• temperatura de armazenamento: 2-8°C
• cadeia de caracteres SMILES: [O+H2]O[O-]
• InChI: 1S/H2O3/c1-3-2/h1-2H
• chave InChI: JSPLKZUTYZBBKA-UHFFFAOYSA-N

Descrição

Descrição geral

A peroxidase de raiz forte é isolada da raiz de Amoracia rusticana e pertence ao grupo ferroprotoporfirínico de peroxidases. A HRP é um polipeptídeo de cadeia simples com quatro pontes dissulfeto. É uma glicoproteína que contém 18% de carboidratos. A composição de carboidratos consiste em galactose, arabinose, xilose, fucose, manose, manosamina e galactosamina, dependendo da isoenzima específica. O peso molecular (~44 kDa) inclui a cadeia polipeptídica (33.890 Daltons), hemina mais Ca²⁺ (~700 Daltons) e carboidratos (~9.400 Daltons). Existem no mínimo sete isoenzimas de HRP. O ponto isoelétrico das isoenzimas de peroxidase de raiz forte varia entre 3,0 e 9,0.

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.

Aplicação

Horseradish peroxidase (HRP) is isolated from horseradish roots (Amoracia rusticana) and belongs to the ferroprotoporphyrin group of peroxidases. 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 P8415, type XII, is an essentially salt free lyophilized powder. It is a further purification of product P8375. It is commonly used to determine amounts of glucose and peroxides in solution. It has been used in an aspergillus fumigatus rapid susceptibility assay[1].

Peroxidase from horseradish has been used to initiate peroxidase-coupled assay.[2] It has also been used in the preparation of β-galactosidase (β-gal) stock solution.[3]

The enzyme has been used to determine H₂O₂ production in tobacco BY-2 cells using a spectrofluorimetric method.[4]

Ações bioquímicas/fisiológicas

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 and hence, it is the most desired label. Also, its glycosylation leads to lower non-specific binding.[5] It is also used for the determination of glucose[6] and peroxides[7] in solution.

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. Known inhibitors are sodium azide, cyanide, L-cystine, dichromate, ethylenethiourea, hydroxylamine, sulfide, vanadate, p-aminobenzoic acid, and Cd²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mn²⁺, Ni²⁺, and Pb²⁺ ions.

Outras notas

A further purification of Peroxidase TypeVI (P8375).

Veja mais informações sobre peroxidase em www.sigma-aldrich.com/enzymeexplorer.

Definição da unidade

Uma unidade de pirogalol forma 1,0 mg de purpurogalina a partir do pirogalol em 20 segundos em pH 6,0 a 20 °C.

Nota de preparo

Chromatographically purified

Nota de análise

RZ (Reinheitszahl) é a taxa de absorbância A₄₀₃/A₂₇₅ determinada a 0,5-1,0 mg/ml em água deionizada. É uma medida do teor de hemina, e não da atividade enzimática. Mesmo preparações com RZ alto podem ter atividade enzimática baixa.

Preliminary isoelectric focusing data indicates this is primarily isoenzyme C

Informações de segurança

• Pictogramas: GHS08
• Palavra indicadora: Danger
• Frases de perigo: H334
• Declarações de precaução: P261 - P284 - P501
• Classificações de perigo: Resp. Sens. 1
• Código de classe de armazenamento: 11 - Combustible Solids
• Classe de risco de água (WGK): WGK 1
• Equipamento de proteção individual: Eyeshields, Gloves, type N95 (US)