An Introduction to Horseradish Peroxidase (HRP) and Its Applications

The Function of Horseradish Peroxidase (HRP)

Horseradish peroxidase is an enzyme isolated from the roots of the horseradish plant¹ and is crucial for catalyzing a variety of reactions in biological applications. Horseradish peroxidase can catalyze the oxidation of various substrates using hydrogen peroxide as an electron acceptor, leading to the formation of reactive intermediates. HRPs versatility in catalyzing diverse chemistry, including chromogenic or fluorescent reactions makes the enzyme indispensable in many applications.

Detection of target molecules

HRP is commonly used as a detection enzyme in various biochemical assays, including ELISA and Western blotting. In these assays, HRP is conjugated to a secondary antibody or protein and used to detect the presence of a target molecule by catalyzing a colorimetric or chemiluminescent reaction that produces a signal.

Degradation of toxic compounds

HRP can catalyze the degradation of a variety of toxic compounds, including phenols, polycyclic aromatic hydrocarbons, and organophosphorus compounds, making it useful for bioremediation applications.²

Cross-linking of proteins

HRP can catalyze the formation of covalent bonds between proteins by oxidizing tyrosine residues to form tyrosyl radicals, which then react with other tyrosine residues in neighboring proteins. This process is often used in protein-protein interaction studies and can help to stabilize protein complexes.

Synthesis of compounds

HRP can be used in the synthesis of a variety of compounds, including polymers, dyes, and pharmaceuticals. HRP can catalyze the formation of covalent bonds between different molecules, leading to the production of new compounds.

Horseradish Peroxidase Biological Research Applications

Horseradish peroxidase is commonly used in various diagnostic tests and assays in the fields of immunology and molecular biology.³

• Enzyme-linked immunosorbent assay (ELISA): HRP is used as a detection enzyme in ELISA, a test that detects the presence of antibodies or antigens in a sample. HRP catalyzes the conversion of a colorless substrate into a colored product, which is detected by a spectrophotometer.
• Western blot (WB): HRP-conjugated secondary antibodies are used in Western blotting, a technique that detects specific proteins in a sample that has been separated by electrophoresis and transferred onto a membrane.
• Immunohistochemistry (IHC): HRP is used as a detection enzyme in IHC, a technique that localizes antigens or proteins in tissue samples. HRP catalyzes the conversion of a chromogenic substrate into a colored product, which is visualized under a microscope.
• In situ hybridization (ISH): HRP-conjugated probes are used in in situ hybridization to detect specific nucleic acid sequences in cells or tissues. HRP catalyzes the conversion of a chromogenic substrate into a colored product that is visualized under a microscope.
• Polymerase chain reaction (PCR): HRP is used as a reporter enzyme in PCR, a technique that amplifies and detects specific DNA sequences. HRP catalyzes the conversion of a fluorescent substrate into a fluorescent product, which is detected by a fluorescence reader.

Horseradish Peroxidase (HRP) in Pharmaceutical Applications

HRP is used in various pharmaceutical applications primarily for its enzymatic properties, which allow it to catalyze reactions that are critical in diagnostics, drug development, and therapeutic processes.

Diagnostic tests

HRP is commonly used in diagnostic tests to detect the presence of specific molecules in a sample. This is achieved by conjugating HRP to antibodies or other molecules that specifically bind to the target molecule. When the HRP-conjugated molecule binds to the target, the HRP enzyme catalyzes a reaction that produces a detectable signal, such as a color change or fluorescence. This type of assay is widely used in clinical laboratories for the diagnosis of diseases, such as cancer and infectious diseases.

Drug Development

In pharmaceutical research, HRP is used in enzyme assays to screen for potential drug candidates by monitoring how they interact with specific targets. The high sensitivity of HRP-based assays allows for the detection of even low concentrations of drug compounds.

Drug delivery

HRP is also used in the development of drug delivery systems, particularly for targeted drug delivery. In this approach, HRP is conjugated to a prodrug or a carrier molecule that specifically targets a particular tissue or cell type. When the HRP-conjugated molecule reaches its target, HRP catalyzes a reaction that releases the drug or carrier molecule. This approach can increase the efficacy of drugs while minimizing their side effects.⁴

Immunotherapy

HRP is being investigated as a potential therapeutic agent in cancer immunotherapy. In this approach, HRP is conjugated to tumor-specific antigens that can activate an immune response against the tumor. HRP can also be used to generate reactive-oxygen species, which can damage cancer cells and enhance the immune response against them.

Bioavailability Studies

HRP is used in bioavailability and pharmacokinetics studies to understand how drugs are absorbed, distributed, metabolized, and excreted in the body. Its use in these studies provides crucial data for the development of effective pharmaceuticals.⁵

HRP in Lateral Flow Assays

Horseradish Peroxidase (HRP) is commonly used in lateral flow assays (LFAs) as a reporter enzyme due to its ability to catalyze chromogenic reactions, which produce a visible color change indicating the presence of a target analyte.⁶

In lateral flow assays, HRP is conjugated to a detection antibody or a protein that specifically binds to the target analyte. When the sample is added to the lateral flow strip, the target analyte binds to the detection antibody/protein conjugated with HRP, forming a complex. As the sample flows along the strip, the complex moves towards a capture antibody or protein immobilized on a test line.

When the complex reaches the test line, it binds to the capture antibody/protein, which is specific to the target analyte, resulting in the accumulation of HRP-conjugated complexes on the test line. The excess HRP-conjugated complexes continue to flow along the strip and are captured by a control line containing a different antibody/protein that is not specific to the target analyte. Finally, a substrate containing a chromogenic or chemiluminescent agent is added to the strip, which reacts with HRP to produce a detectable signal. A signal at the test line indicates the presence of the target analyte in the sample.

Advanced lateral flow assays using palladium nanopartical (PdNP)-HRP lateral flow test assays have been shown to be 5-10 fold more sensitive than conventional gold nanoparticle lateral flow assays.⁷

Substrates & specific activity of Horseradish Peroxidase

Horseradish peroxidase chromogenic substrates

• 3,3',5,5'-tetramethylbenzidine (TMB): TMB is a chromogenic substrate that turns blue in the presence of HRP. This substrate is commonly used in ELISA and Western blotting assays.
• 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS): ABTS is a soluble chromogenic substrate that turns green in the presence of HRP. This substrate is used in ELISA and in enzymatic assays for measuring peroxidase activity.
• o-Phenylenediamine dihydrochloride (OPD): OPD is a colorless substrate that turns yellow in the presence of HRP. This substrate is used in ELISA and Western blotting assays.
• Diaminobenzidine (DAB): DAB is a chromogenic substrate that turns brown in the presence of HRP. This substrate is used in immunohistochemistry assays.

Horseradish peroxidase chemiluminescent and fluorescent substrates

• Luminol emits blue light upon oxidation by HRP. This substrate is used in Western blotting, ELISA, and in situ hybridization applications.
• Acridan emits blue light upon oxidation by HRP. This substrate is used in Western blotting, ELISA, and in situ hybridization applications.
• CDP-Star^® substrate emits blue light upon oxidation by HRP. This substrate is used in Western blotting and ELISA.
• Fluorescein diacetate: Fluorescein diacetate is a fluorescent substrate that is cleaved by HRP to release fluorescein, which emits green fluorescence upon excitation by a light source. This substrate is commonly used in fluorescence microscopy and flow cytometry assays.

Specific activity of horseradish peroxidase

The specific activity of HRP is a measure of its enzymatic activity, which is the rate at which it catalyzes the oxidation of a substrate in the presence of hydrogen peroxide.

The specific activity of HRP is typically expressed in units of enzyme activity per milligram of protein (U/mg). One unit of HRP activity is defined as the amount of enzyme that catalyzes the oxidation of 1 micromole of hydrogen peroxide per minute at 25°C and pH 7.0, using guaiacol as a substrate. The specific activity can vary depending on the source of the enzyme and the method used to purify it. However, a typical specific activity for commercially available HRP is 150-250 U/mg.