Chemiluminescence and Enzyme Immunoassay-HRP Labeling

Chemiluminescence and Enzyme Immunoassay-HRP Labeling

Introduction to HRP

Horseradish peroxidase, referred to as HRP, enzymatic number EC1.11.1.7; is isolated from horseradish and belongs to the iron protoporphyrin group of peroxidase. Horseradish peroxidase is a group of isoenzymes with certain enzymatic activity. Among them, horseradish peroxidase isoenzyme C (HRP C) has the highest content and is the most abundant in the roots of horseradish.

HRP Structure

Horseradish peroxidase is mainly composed of glycoprotein (enzyme protein) and iron (III) protoporphyrin IX (prosthetic group). It is a single-chain polypeptide containing four disulfide bonds. The sugar content in glycoprotein is generally 18~22%. Sugars are composed of galactose, arabinose, xylose, fucose, mannose, mannosamine, and galactosamine, depending on the specific isoenzyme. Since the enzyme protein and prosthetic group of horseradish peroxidase have maximum absorption peaks at UV wavelengths of 275nm and 403nm respectively, the separated HRP has a ratio RZ of OD403nm/OD275nm. The RZ value indicates the purity of the enzyme. The RZ value of HRP with higher purity is generally above 3.0. The smaller the RZ value, the lower the purity of the enzyme.

Principle of the chemiluminescence enzyme.Figure 1. Principle of the chemiluminescence enzyme. (Wang X, et al.; 2007)

HRP is a group of isoenzymes. 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 molecule of HRP C contains 2 molecules of Ca2+, 1 molecule of iron (III) protoporphyrin IX prosthetic group and 4 pairs of disulfide bond, with a relative molecular mass of about 44,000 Daltons, including polypeptide chains (33,890 Daltons), hemin plus Ca2+ (about 700 Daltons) and carbohydrates (about 9,400 Daltons). There are at least 7 HRP isoenzymes. The isoelectric point of horseradish peroxidase isoenzymes ranges from 3.0 to 9.0. The mechanism of action, enzymatic properties, isoelectric point, etc. of horseradish peroxidase are closely related to its structure.

HRP Biochemical/Physiological Effects

HRP easily combines with hydrogen peroxide (H2O2), and the resulting [HRP-H2O2] complex can oxidize various hydrogen donors to generate free radicals and water. There are many hydrogen donors, such as aromatic compounds, phenols, indoles, amines and sulfonates. In practical applications, the solution of hydrogen donor DH2 is usually a colorless substance, and the colored substance is generated through the reaction. The required value can be obtained by measuring the color 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. Most of the optimal pH is 6.0-6.5, and the enzyme is most stable in the pH range of 5.0-9.0. The reaction temperature is generally around 25°C. HRP can be conjugated to antibodies through several different methods, including glutaraldehyde, periodate oxidation, through disulfide bonds, and through amino- and thiol-directed cross-linkers. It is smaller and more stable than the enzyme labels β-galactosidase and alkaline phosphatase. Additionally, its glycosylation results in lower nonspecific binding. When incubated with substrate, horseradish peroxidase produces colored, fluorescent, or luminescent derivatives of labeled molecules, allowing quantification. Horseradish peroxidase has been shown to slightly reduce inhibition levels in cydAB mutants. Known inhibitors include sodium azide, cyanide, L-cystine, dichromate, ethylene thiourea, hydroxylamine, sulfide, vanadate, p-aminobenzoic acid, and Cd2+, Co2+, Cu2+, Fe3+, Mn2+, Ni2+ and Pb2+ ions.

HRP Application

Because HRP has stable properties, abundant natural sources, and small molecular weight, it is easy to prepare pure enzymes with higher activity, so it is widely used.

  • HRP is currently one of the most widely used labeling enzymes in ELISA, mainly because it is easy to prepare, relatively low-priced, stable in nature, resistant to heat and organic solvents, and has high activity after coupling with antigens or antibodies.
  • HRP can also be used for chemiluminescence detection, immunoblotting and immunohistochemistry experiments, and combined with glucose oxidase to detect glucose content, etc.
  • 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 serious environmental pollution. HRP can use these pollutants as substrates to oxidize them into free radicals, and the free radicals aggregate into precipitation polymers, thus Greatly reduce environmental pollution.
  • HRP has been studied as a food additive in recent years. Since HRP itself is non-toxic and harmless, the reaction conditions are high and the reaction is easy to control, it has been studied in recent years for use in food preservation, detoxification and detection.

Related Services

Catalog Number Product Name Category
SCI020101 Ready-To-Use AEC HRP (Red) For HRP Detection Systems Inquiry
SCI020102 Liquid DAB HRP Kit (Brown) For HRP Detection Systems Inquiry
SCI020103 DAB HRP Buffer (Brown) For HRP Detection Systems Inquiry
SCI020104 DAB HRP Chromogen (Brown) For HRP Detection Systems Inquiry
SCI020105 HRP Kit (Black) For HRP Detection Systems Inquiry
SCI020106 HRP Kit (Green) For HRP Detection Systems Inquiry
SCI020107 HRP Kit (Blue) For HRP Detection Systems Inquiry
SCI020108 HRP Plus Kit (Blue) For HRP Detection Systems Inquiry
SCI020109 HRP Kit (Mulberry) For HRP Detection Systems Inquiry
SCI020110 HRP Kit (Fuchsia) For HRP Detection Systems Inquiry
SCI020111 HRP Kit (Yellow) For HRP Detection Systems Inquiry
  1. Wang X, et al.; Development of a highly sensitive and selective microplate chemiluminescence enzyme immunoassay for the determination of free thyroxine in human serum. Int J Biol Sci. 2007, 3(5):274-80.

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