Everything You Want to Know About Horseradish Peroxidase

Posted by Iva Colter on June 22nd, 2022

Peroxidase, usually derived from horseradish, is a common enzyme used in clinical testing reagents. It is widely used not only in several biochemical assays but also in immunoassay (ELISA) kits. As a key component of the color development system of several kits, peroxidase has an important impact on the quality of the kits.

Horseradish peroxidase (HRP) is the most commonly used enzyme because of its high activity, stability, small molecular weight and easy preparation of pure enzyme. It is composed of multiple isoenzymes, with a molecular weight of 40,000 and an isoelectric point of PH3 to 9. The optimum pH for enzyme catalysis varies slightly due to different hydrogen donors, but it is mostly around PH5. The enzyme is soluble in water and ammonium sulfate solution below 58% saturation. The maximum absorption spectra of the prosthetic group and enzyme protein of HRP are 403nm and 275nm, respectively, and the purity of the enzyme is generally expressed by the ratio of OD403nm/OD275nm RZ. The RZ value of high purity enzyme should be around 3.0 (up to 3.4), and the smaller the RZ value, the more non-enzymatic proteins are present.

HRP in different activities

1) RZ > 3 activity > 250 u/mg, mainly used in immunology, is a high purity peroxidase. Special chromatographic purification techniques are used to remove isozyme B which can affect immunological reactions.

(2) RZ > 2 activity > 180 u/mg is mainly used in clinical chemistry, but some of the customers have also used this size product in immunological studies. At this point, a standardized analytical method becomes particularly important.

3) RZ > 1 activity > 100 u/mg, mainly used in blood glucose test strips and urine analysis test strips.

(4) RZ > 0.6 activity > 60 u/mg, mainly used in urine analysis test strips.

Horseradish peroxidase is a 44,173.9-dalton glycoprotein with 6 lysine residues that can bind label molecules. The luminescent derivatives of the labeled molecules can be detected and quantified when incubated with appropriate substrates that produce colored fluorescence. HRP is commonly used in conjugates (molecules that have been bound by genetic or chemical means) to determine the presence of molecular targets. For example, antibodies conjugated to HRP proteins can be used to detect small amounts of specific proteins. Here, the antibody provides the specificity to localize the target protein, and the HRP enzyme produces a detectable signal in the presence of a substrate. Horseradish peroxidase is also commonly used in applications such as ELISA and immunohistochemistry.

Horseradish peroxidase is ideal for these applications in many ways because it is smaller, more stable and less expensive than other popular alternatives, such as alkaline phosphatase. It also has a high turnover rate and can generate strong signals in a short period of time. High concentrations of phosphate can severely reduce the stability of horseradish peroxidase. Besides biomedical applications, horseradish peroxidase is also one of the enzymes with important environmental applications. The enzyme is suitable for the removal of hydroxylated aromatic compounds (HACs), which are considered major contaminants in a variety of industrial wastewaters.

Due to the high efficiency and specificity of the enzyme-catalyzed reaction, it has higher selectivity than conventional oxidation reactions, and the reaction conditions are relatively mild. Horseradish peroxidase has been widely used in analytical chemistry, environmental chemistry and clinical chemistry, as well as in biosensors, materials science, etc.