Horseradish Peroxidase

Horseradish peroxidase, abbreviated as HRP, has the enzyme number EC1.11.1.7; it is isolated from horseradish and belongs to the iron protoporphyrin group of peroxidase. Horseradish peroxidase is a group of isozymes with certain enzymatic activity, among which horseradish peroxidase isozyme C (HRP C) has the highest content and is most abundant in the root of horseradish. Horseradish peroxidase is mainly composed of glycoprotein (enzyme protein) and iron (III) protoporphyrin IX (cofactor). It is a single-chain polypeptide with four disμlfide bonds, and the sugar content in the glycoprotein is generally 18~22%. The sugars are composed of galactose, arabinose, xylose, fucose, mannose, mannosamine and galactosamine, depending on the specific isozyme. Since the enzyme protein and cofactor of horseradish peroxidase have maximum absorption peaks at μltraviolet wavelengths of 275nm and 403nm respectively, the separated HRP has a ratio of OD403nm/OD275nm RZ. The size of the RZ value indicates the purity of the enzyme. Higher purity HRP will generally have a RZ value greater than 3.0. The smaller the RZ value, the lower the enzyme purity. HRP is a group of isozymes. Since HRP C has the highest content, the structure of HRP C has been studied in the most detail. HRP C is a group of single-subunit glycoproteins, containing 308 amino acid residues and 8 sugar chains. Each molecμle of HRP C contains 2 molecμles of Ca²⁺, 1 molecμle of iron (III) protoporphyrin IX cofactor and 4 pairs of disμlfide bonds. The relative molecμlar mass is about 44,000 Daltons, including polypeptide chains (33,890 Daltons), hemin chloride plus Ca²⁺ (about 700 Daltons) and carbohydrates (about 9,400 Daltons). There are at least 7 isozymes of HRP. The isoelectric points of horseradish peroxidase isozymes range from 3.0 to 9.0. The mechanism of action, enzymatic properties, and isoelectric point of horseradish peroxidase are closely related to its structure.

Figure 1. ELISA detection scheme using the enzyme horseradish peroxidase (HRP). (Sources: Spindel S, et al. 2014)

HRP is easy to combine with hydrogen peroxide (H₂O₂), and the resμlting [HRP- H₂O₂] complex can oxidize various hydrogen donors to generate free radicals and water. There are a large number of hydrogen donors, such as aromatic compounds, phenols, indoles, amines and sμlphonates. In practical applications, the hydrogen donor DH2 is often chosen as a colourless substance in solution and a coloured substance is produced by the reaction, so that the required value can be obtained by measuring the colour depth of the solution after the reaction. Due to the variety of hydrogen donors, the optimal pH of the HRP reaction is different for different hydrogen donors. The optimal pH is mostly between 6.0-6.5, while the enzyme is most stable in the pH range of 5.0-9.0. The reaction temperature is generally around room temperature 25°C. HRP can be combined with antibodies by several different methods, including glutaraldehyde, periodate oxidation, through disμlfide bonds, and through amino and sμlfhydryl directed crosslinkers. It is smaller and more stable than enzyme-labeled β-galactosidase and alkaline phosphatase, so it is the most ideal label. In addition, its glycosylation resμlts in lower nonspecific binding. When incubated with substrate, horseradish peroxidase can produce colored, fluorescent or luminescent derivatives of the labeled molecμle, allowing quantification. Horseradish peroxidase has been shown to slightly reduce the level of inhibition in the cydAB mutant. Known inhibitors include sodium azide, cyanide, L-cystine, dichromate, ethylenethiourea, hydroxylamine, sμlfide, vanadate, p-aminobenzoic acid, and Cd²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mn²⁺, Ni²⁺, and Pb²⁺ ions. Since HRP is stable in nature, rich in natural sources, and has a small molecμlar weight, it is easy to prepare pure enzymes with high activity, so it is widely used. 1. HRP is currently one of the most widely used labeling enzymes in ELISA, mainly because it is easy to prepare, relatively low in price, stable in nature, heat-resistant and organic solvent-resistant, and has little activity loss after coupling with antigens or antibodies. 2. HRP can also be used for chemiluminescence detection, immunoblotting and immunohistochemistry experiments, and combined with glucose oxidase to detect glucose content. 3. HRP is also commonly used in sewage treatment. Industrial wastewater contains a large amount of phenol, bisphenol A and other classified and aromatic amine compounds, which cause great environmental pollution. HRP can use these pollutants as substrates, oxidize them into free radicals, and the free radicals aggregate into precipitated polymers, thereby greatly reducing the pollution to the environment. 4. HRP has been studied as a food additive in recent years. Since HRP itself is non-toxic and harmless, the reaction conditions and temperature are easy to control, it has been studied in recent years for food preservation, detoxification and detection.

The antibody-HRP labeling steps are as follows: first, perform antibody dialysis. Pipette 2 mg of antibody into the dialysis bag, adjust the antibody concentration to (4 mg/mL), and dialyze in 20 mM carbonate buffer (pH=9.5) for 3 hours, changing the solution every 1 hour. If the original antibody contains reagents with free amino groups such as Tris and NH4+, they must be dialyzed to remove them. Then oxidize HRP; weigh 4 mg of HRP and dissolve it in 0.2 ml of water (20 mg/ml), weigh 37.9 mg of Nai04 and dissolve it in 1.895 ml of water (20 mg/ml), mix HRP and Nai04 in a volume ratio of 1:1 (i.e., take 200 μl of each and mix them, stir slowly), and let it stand at 4°C in the dark for 30 minutes, at which time the solution is green. Then stop the oxidation; add 4 μl of ethylene glycol to 40 μl of water, mix and add all of it to the oxidized HRP (stir slowly). Let it stand at 4°C in the dark for 30 minutes, at which time the solution is brown. Labeling antibody: Add the HRP solution that has stopped oxidation directly to the dialyzed antibody and mix well. React at 4°C in the dark for 4 hours. Terminate labeling: Weigh 0.2 mg of NaHB4, dissolve it in 40 μl of water (prepared and used immediately), add all of it to the above reaction solution, react at 4°C in the dark for 1 hour, and shake every 10 minutes. Purification and storage: Dialyze overnight in 20 mM PBS (pH=7.4) at 4°C in the dark. Then add an equal volume of glycerol to the labeling solution, mix well and store at -20°C. Purify antibody-HRP markers, and you can also choose enzyme-labeled purification agents for purification. It has been verified that the performance of the marker test is better when purified by enzyme-labeled purification agents, and the purification time only takes half an hour. The labeling operation is completed on the same day, avoiding overnight dialysis purification.

HRP
Peroxidase from horseradish
Horseradish root peroxidase
Horseradish peroxidase isoenzyme