How are HRP-Conjugates used?

Horseradish peroxidase is a key enzyme used in many bio- and immunochemical applications. Historically it has been use as a marker enzyme in ELISA protocols as well as in amperometric sensors. However, many more applications have been developed more recently.

Several structures for horseradish peroxidase have been solved and reported over the years. Three views (A, B, and C) for the structure of recombinant horsradish peroxidase C in complex with ferulic acid are shown below. The heme group and the ferulic acid can be seen in all three models. In model C, for a better view, the heme group is colored in magenta, the ferulic acid in green, and the protein backbone in cyan.

Recombinant Horesradish Peroxidase C Complex With Ferulic Acid [6ATJ].

HRP-conjugates can be used:

  • As a reporter enzyme: In histochemical staining and diagnostic assays. For example, HRP-conjugated secondary antibodies were used to detect HIV-1 envelope peptides expressed in cell culture via a cell-based ELISA. HRP activity can either be detected by the formation of a chromogenic or fluorogenic product, or by an electrochemical signal due to the redox nature of HRP catalysis.
  • As a biosensor: In the majority of HRP-based biosensor systems H2O2 is detected.  Other molecules, such as glucose, ethanol, DNA and RNA, L-phenylalanine, citrinin, pyrogallol and hydroquinone, phenols, the milk allergen β-lactoglobulin, rotavirus titers, and tumor markers are detected via H2O2.
  • Quantification of protein kinase activity: HRP reporter activities also allow the quantification of protein kinase activity, important in cases of kinase-related drug discovery, therapy, and clinical diagnosis.
  • Human pathogen detection: For example, HRP conjugates were used for the detection of DNA from the human pathogen Mycobacterium tuberculosis.
  • High throughput assays: HRP as a major component of high throughput assays in enzyme engineering, detecting H2O2 as a side product of biooxidants or products of coupling reactions after biohydroxylations.
  • Bioremediation: degradation of synthetic dyes, removal of phenolic contaminants from wastewater, or removal of endocrine-disrupting compounds such as synthetic estrogens.
  • Cancer treatment: chitosan nanoparticles encapsulating HRP were shown to induce cell death in a human breast cancer cell line.
  • Hydrogelation for biomedical applications.
  • Nanomaterials, HRP is immobilized on silica nanoparticles.

However, a consistent enzyme quality is needed in medical diagnostics.




Krainer, F. W., & Glieder, A. (2015). An updated view on horseradish peroxidases: recombinant production and biotechnological applications. Applied Microbiology and Biotechnology, 99, 1611–1625. [Pubmed]


Solved Structures