- Horseradish peroxidase (HRP) is a heme-containing enzyme that catalyzes the oxidation of a wide range of organic and inorganic substrates in the presence of hydrogen peroxide (H₂O₂).
- It is extracted primarily from the roots of the horseradish plant (Armoracia rusticana) and has become one of the most widely used enzymes in biochemistry, molecular biology, and clinical diagnostics. Its utility stems from its high catalytic activity, broad substrate specificity, and the ease with which its enzymatic activity can be coupled to colorimetric, chemiluminescent, or fluorescent detection systems.
- The biochemical basis of HRP activity involves its heme prosthetic group, which facilitates electron transfer reactions. When H₂O₂ is present, HRP undergoes a catalytic cycle in which it oxidizes various chromogenic or luminescent substrates. Commonly used substrates include 3,3′-diaminobenzidine (DAB), which produces a brown insoluble precipitate for tissue staining, and tetramethylbenzidine (TMB), which produces a soluble blue color often used in enzyme-linked immunosorbent assays (ELISA). Chemiluminescent substrates, such as luminol, emit light upon oxidation, enabling highly sensitive detection in blotting techniques. This versatility makes HRP a universal detection enzyme across diverse laboratory applications.
- In immunoassays, HRP is most often conjugated to antibodies or streptavidin to enable targeted detection. In immunohistochemistry (IHC), HRP-linked secondary antibodies bind to primary antibodies localized at specific antigens in tissues, producing a visible color when exposed to a chromogenic substrate. In ELISA and Western blotting, HRP serves as a reporter enzyme that amplifies the detection signal, allowing precise quantification of proteins, antigens, or antibodies. Because of its high turnover rate and sensitivity, HRP-based detection systems often surpass those based on alternative enzymes like alkaline phosphatase.
- Beyond immunological techniques, HRP has applications in biotechnology and environmental sciences. It is employed in biosensors for detecting hydrogen peroxide, phenolic compounds, or pollutants, and in bioremediation studies for breaking down aromatic hydrocarbons. The enzyme’s ability to catalyze oxidative polymerization reactions has also been harnessed in synthetic chemistry and nanomaterial development. Additionally, engineered variants of HRP have been developed to improve stability, broaden substrate compatibility, and adapt to different assay conditions.
- Despite its strengths, HRP has certain limitations. It is sensitive to high concentrations of hydrogen peroxide, which can inactivate the enzyme, and it may lose activity under harsh experimental conditions. Moreover, the insoluble precipitates produced by some chromogenic reactions can obscure fine tissue details in microscopy. Nevertheless, careful optimization of substrate choice and reaction conditions allows researchers to overcome these challenges.
