HRP is a plant glycohemoprotein that belongs to the ferroprotoporphyrin group of peroxidases. HRP is composed of seven isozymes. All isozymes contain photohemin IX as prosthetic group. Neutral and amino sugars account for approximately 18% of the enzyme. The iron-containing hemin group is covalently bound to the glycoprotein apoenzyme. The enzymatic activity of HRP is due to the cyclic reduction and oxidation of the iron atom in the hematin group. The active site of HRP involves both the apoprotein and the heme group.
Horseradish peroxidase is used in biochemistry applications such as western blots, ELISA and Immunohistochemistry. It is used to amplify a weak signal and increase detectability of a target molecule, such as a protein. HRP labeled immunoglobulins are used as probes for the demonstration of tissue antigens and in enzyme immunoassay (EIA) systems for determination of soluble and insoluble antigens. HRP is the most desired label for antibodies since it is the smallest and most stable of the three most popular enzyme labels (HRP, alkaline phosphatase, and B–galactosidase) and its glycosylation leads to lower non–specific binding. It is also useful for tracing neural connections. These include motor and sensory innervation of peripheral organs, and connections of peripheral nerves, ganglia and individual dorsal roots. 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.
When incubated with a substrate, horseradish peroxidase produces a coloured, fluorimetric, or luminescent derivative of the labeled molecule, allowing quantification. HRP reacts with hydrogen peroxide to form a [HRP-H2O2] complex which can oxidize a wide variety of hydrogen donors.
RZ Definition: The ratio of the absorbance at 403 nm to the absorbance at 275 nm. This value is an expression of the ratio of hemin to protein content.
That amount of enzyme which catalyses the production of one milligram of purpurogallin in 20 seconds at 20°C. at a pH of 6.0.
For assay of rate of decomposition hydrogen peroxidide by peroxidase with pyrogallol: 0.50% (w/w) Hydrogen peroxide solution (H2O2); Prepare 50 mL in deionized water using a 30% Hydrogen peroxide solution. RZ Determination=0.5 mg/mL solution of horseradish peroxidase.
Specific Activity: ≥250 u/mg solid
Key Applications: ELISA, Immunoblotting, Immunohistochemistry
Application Areas: Molecular biology
Product Type: Proteins, Enzymes & Peptides
Extinction Coefficient (E1%): 100 (403 nm)(Lit.)
Presentation: Reddish Brown Crystalline Powder
Format: Crystalline Powder
Isoelectric point (pI): 7.2(Lit.); Isozymes range from 3.0–9.0(Lit)
pH: Optimum pH: 7.0(Lit.)
NOTES:
Inhibitors: HRP is reversibly inhibited by cyanide and sulfide at a concentration of 10-5 M. The following can also inhibit HRP: p-Aminobenzoic acid, azide, cyclopropanone, L-cystine, dichromate, ethylenethiourea, hydroxylamine, sulfite and vanadate and the divalent metals Cd, Co, Cu, Fe, Mn, Ni, Pb. Various substrate system use with enztme =2,2'-Azino-bis(3-ethylbenz- thiazoline-6-sulfonic acid) (ABTS); o-Phenylenediamine (OPD); 3,3',5,5'-Tetramethylbenzidine (TMB); o-Dianisidine; 5-Aminosalicylic Acid (5AS); 3,3'-Diaminobenzidine (DAB); 3-Amino-9-Ethylcarbazole (AEC); 4-Chloro-1-naphthol (4C1N). RZ value=≥3.0.
Specificity: Activity is observed with H2O2, MeOOH and EtOOH.
Solubility: Soluble in distilled water. Soluble in 0.1 M potassium phosphate buffer, pH 6.0 (monobasic potassium phosphate adjusted to pH 6.0 with 1.0 M potassium hydroxide) (10 mg/mL yields a clear, red-brown solution).
Storage & Handling: Store at -20°C.