Peroxidase (HRP), also known as Horseradish Peroxidase, is a glycoprotein composed of enzyme protein and iron porphyrin, with a sugar content of 18%.
The enzyme is composed of multiple isozymes and is a commonly used enzyme in clinical test reagents.
Peroxidase is widely distributed in the plant kingdom, and its content is high in horseradish, so it is often extracted from horseradish.
HRP is composed of multiple isoenzymes with a molecular weight of 40,000 and an isoelectric point of pH 3 to 9. The optimum pH for enzyme catalysis varies slightly due to different hydrogen donors, but it is mostly around pH 5.
The maximum absorption spectra of the prosthetic group and enzyme protein of HRP are 403nm and 275nm, respectively.
Therefore, people usually use the RZ value (ie the ratio of OD403nm/OD275nm) to represent the purity of Peroxidase. The larger the RZ value, the higher the purity.
This product is not only widely used in many biochemical detection projects, but also widely used in immune type (ELISA) kits.
Peroxidase, as a key component of the chromogenic system of multiple kits, has an important impact on the quality of the kits.
Peroxidase is relatively stable to heat and organic solvents, and its activity cannot be changed by treating with toluene and paraffin sections or by fixing with pure ethanol or 10% formaldehyde solution for frozen sections.
Cyanide or sulfide can reversibly inhibit HRP at a concentration of 10–5–10–6 mol/L.
Fluoride, azide or hydroxylamine inhibited HRP only at concentrations higher than 10-3 mol/L.
Peroxidase is also irreversibly inhibited by hydroxymethyl hydroperoxide.
Strong acid is also a strong inhibitor of HRP. Therefore, some of the above-mentioned compounds such as sodium fluoride, sodium azide and strong acid are often used as terminators for enzyme reactions in enzyme immunoassays.
In addition, to prevent enzyme inactivation, the use of sodium azide as a preservative should be avoided when preparing dilution buffers for enzyme immunoassays.
The isoenzymes of Peroxidase can be mainly divided into three types:
The HRP used in the enzyme immunoassay is mainly composed of the so-called “C” isozyme with a PI of 8.7 to 9.0, and the activities of other isozymes are very low.
The covalent structure of the “C” isozyme is composed of two closely adjacent regions, and the heme group is located in the middle to form a sandwich structure, and the sugar chain is bound to the polypeptide at 8 different parts.
The natural enzyme has very little pure charge, no free, -amino groups, only 2 detectable histidines, and 6 lysines that appear to be completely covered by the sugar chain shell.
Therefore, Peroxidase generally has only 1-2 amino groups available for coupling.
According to the catalytic properties of Peroxidase, hydrogen peroxide (H2O2) is generally used as one of the HRP substrates in ELISA. In the presence of a hydrogen donor (ie, a chromogen substrate), the reaction between HRP and H2O2 is rapid and specific.
30% H2O2 is not stable. Since H2O2 is both a substrate and an inhibitor of HRP, in order to obtain satisfactory results by ELISA, H2O2 must be limited to a certain concentration range, and the final concentration is usually 2-6 mmol. /L.
However, in practical research work, generally little attention is paid to this point.
The concentration of H2O2 used by most investigators is often 2 to 4 times greater than that required for the ideal reaction.
HRP adsorbed on the solid phase is more susceptible to inhibition by excess H2O2 than free HRP.
If the concentration of the 30% H2O2 stock solution is confirmed to be 30% by measurement, a dilution of 10 000-12 000 times is often an ideal substrate.
The molar extinction coefficient of H2O2 is 43.6 at a wavelength of 240 nm with an optical diameter of 10 mm. Therefore, the concentration of H2O2 working solution can be detected in this way.
In solid-phase ELISA, when the temperature is higher than 20oC, the activity of Peroxidase is often low. Adding non-ionic detergent polysorbate-20 or TritonX-100 to the substrate solution can delay the inactivation of HRP, and can make the reaction temperature increased, but the protective effect of this enzymatic activity of non-ionic detergents varies with different hydrogen donors.
If 2,2′-diazo-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS) is used as the hydrogen donor, only 20% of the enzyme activity can be protected, while the The protection of enzymatic activity was increased to 90% when amine (ODA) was the hydrogen donor.
The reason why Peroxidase is by far the most widely used labeling enzyme in ELISA is mainly because of its easy extraction and relatively low price.
On the other hand, HRP has stable properties, heat resistance and organic solvents, and its activity is rarely lost after coupling with antigen or antibody.
I’m william, CEO of Look Chemical Co., Ltd. Welcome to visit our company. As China’s top Global Supply Specialist of chemicals, we have always been adhering to the principle of “service first, quality first, mutual benefit”, and we insist on Create value for customers and grow together with customers.
In the past few years , we have provided efficient and satisfactory services to more than 6000 customers and have been widely praised internationally. In the future, we will strive to provide more than 100,000 customers with professional and thoughtful services, and strive to become your first choice for worry-free purchasing chemicals in China , Hope to bring you a good cooperation experience. If you have any questions, please feel free to contact us.