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Chinese Name 辣根过氧化物酶 HRP ALIAS Donor: Hydrogen-peroxide oxidoductase; Horseradish Peroxidase; HRP Pod Appearance Brown crystalline frozen dried powder Source From Horseradish Solubility 10 mg/mL in water Note: POD is a hemoglobin with oxidizing enzyme nature. For biochemical research, it is a major enzyme used in the enzyme standard method. It is also used in the measurement of glucose and lactose in biological liquids
HRP is a molecular amount of 44,000 glycoprotein, which is combined with colorless enzyme protein and dark brown iron phenein. Formed, about 18 % of neutral sugar and amino sugar, mainly as manngose, xylose, Arabian sugar, and hexide. Each HRP molecule contains a chloride hemantan IX as auxiliary group. The auxiliary group has a maximum absorption peak at the 403nm wavelength, and the enzyme protein that remove the auxiliary group has a maximum absorption at the 275nm wavelength. The ratio of HRP's OD value at 403nm to 275nm is the so -called RZ (Reinheits Zahl) value. The RZ value only indicates the content of the hemoglobin group in HRP, does not represent the true purity of the HRP preparation, and the HRP preparation with a high RZ value does not mean that the enzyme activity is also high. However, a solid enzyme solution can calculate the enzyme concentration at the inhalation of the 10mm light diameter 403nm wavelength. Pure HRP is stored in -20oc can be kept stable, using 1.36 mol/L glycerin, 10 mmol/L sodium phosphate, 30umol/L beef serum albumin, and 20umol/L cytochrome C (pH 7.4) solution as a matrix frozen preservation, which can make it can make it frozen, which can make it can make it frozen, which can make it can cause freezing. Enzyme binding is stable for several years. The effect of HRP on thermal and organic solvents is relatively stable. Treatment of tither and paraffin is used or using pure ethanol or L0 % formaldehyde solution for freezing slices, which cannot change its activity. Cyanide or sulfides can inhibit HRP in a reversible manner at a concentration of 10-15 ～ 10–6 mol/L. Can be suppressed by hydrogen peroxide perfuity. Strong acid is also a strong inhibitor of HRP.
Therefore, some compounds such as sodium fluoride, sodium nitrogen, and strong acids are often used in enzyme immunization measurement. In addition, in order to prevent enzymes from losing activity when preparation of enzyme -proof measurement, sodium nitrogen should be avoided as preservatives. HRP's homogeneous enzymes can be divided into three types: ① acidocyte enzymes with high sugar content. ② Equal electricity points are close to the relatively low sugar content of neutral (or microba). ③ Alkaline (Pi\u003e 11) wasozymes with low sugar content. The HRP used in enzyme immunogenesis is the main component of the so -called \"C\" mobin enzymes with PI as 8.7 to 9.0, and the activity of other companion enzymes is very low. The covalent structure of the \"C\" is composed of 2 closely close areas, and the hemoglobin group is located in the middle of the sandwich structure. The sugar chain is combined with 8 different parts to the polypeptide. Natural enzymes are very small; without free. - Amino -based, there are only 2 measured histidine, 6 lysine seems to be sugarCovering the chain shell. Therefore, HRP generally has only 1 or 2 amino groups that can be used for coupling.
According to the catalytic characteristics of HRP, the use of hydrogen peroxide (H2O2) as one of the HRP substrates in ELISA is generally used as one of the HRP substrates. When the hydrogen supply (that is, the original substrate) exists, the response of HRP and H2O2 response is rapid and dedicated. Essence It can be seen from Figure 4 that HRP is oxidized by H2O2 binary land to form a complex i, and the complex I can be restored to the starting state with the two -step unit price of the hydrogen supply. The complex Ⅱ is an intermediate product with an oxidized electron. When the H2O2 is excessive, due to the formation of the complex Ⅲ or ⅳ, the enzyme activity is suppressed (later supplemented). 30 % H2O2 is not stable. Because H2O2 is both a substrate for HRP and a inhibitor, if ELISA wants to get a satisfactory measurement result, H2O2 must be limited to a certain concentration range, and the final concentration is usually 2 to 6 mmol/L. However, in actual research work, we rarely pay attention to this. The concentration of H2O2 used by most researchers is often 2 to 4 times larger than the amount required for ideal reactions. HRP adsorbed by solid -phase is more likely to be suppressed by excessive H2O2 than excessive H2O2. If the concentration of 30 % H2O2 storage liquid is determined to be 30 % after being measured, the diluted 10,000-12 000 times is often an ideal substrate. H2O2's Moore's consolidation coefficient is 10mm at 43.6 at a wavelength of 20mm. Therefore, the concentration of H2O2 working solution can be detected in this way.
In the solid phase ELISA, when the temperature is higher than 20OC, the HRP activity is often low. Add non-ion-deodbeat agent in the substrate solution to The response temperature is increased, but the enzyme activity protection effect of non -ions deodorant is different according to the different supply of hydrogen. For example, 2,2'-linked nitrogen-double- [3-ethylene pyramodoline] -6-sulfonic acid (ABTS) is a hydrogen supply body, which can only protect the enzyme activity of 20 %, and the neighboring fennel When the amine (ODA) is a hydrogen supply, the protection of enzyme activity is increased to 90 %.
The reason why HRP is by far has the most widely used labeling enzyme in ELISA, mainly because it is easy to extract on the one hand and the price is relatively low; on the other hand, the role of heat resistance and organic solvent After the antibody occasionally couples, the activity is rarely lost.
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