Revealed: Tips for using horseradish peroxidase (HRP)!
We know that HRP is a glycoprotein with a molecular weight of 44,000. It is composed of colorless enzyme protein and dark brown iron porphyrin. Neutral sugars and amino sugars account for about 18%, mainly including mannose, xylose, Arabinose and hexosamine, etc. Each HRP molecule contains a hemin IX as a prosthetic group, which has a maximum absorption peak at a wavelength of 403 nm, while the enzyme protein that removes the prosthetic group has a maximum absorption at a wavelength of 275 nm.
The ratio of the OD value of HRP at 403nm to the OD value at 275nm is the so-called RZ value. The RZ value only indicates the content of heme groups in HRP, but does not indicate the true purity of the HRP preparation. Moreover, an HRP preparation with a high RZ value does not mean that the enzyme activity is high.
However, the enzyme concentration can be calculated by the absorbance of a pure enzyme solution at a wavelength of 10mm light path and 403nm [the absorbance of 1% (W/V) enzyme solution is 22.5 and the concentration is 227umol/L]. Pure HRP can remain stable when dried and stored at –20°C. Use 1.36 mol/L glycerol, 10mmol/L sodium phosphate, 30umol/L bovine serum albumin and 20umol/L cytochrome C (pH 7.4) solution as the matrix for cryopreservation. Stable enzyme conjugates for several years.
HRP is relatively stable against heat and organic solvents. Its activity cannot be changed by processing it with toluene and paraffin sections or fixing it with pure ethanol or 10% formaldehyde aqueous 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 only inhibit HRP at a concentration higher than 10-3 mol/L; HRP also inhibits HRP. Irreversibly inhibited by hydroxymethyl hydroperoxide.
Strong acids are also strong inhibitors of HRP. Therefore, some of the above compounds, such as sodium fluoride, sodium azide and strong acids, are often used as terminators for enzyme reactions in enzyme immunoassays. In addition, when preparing the dilution buffer for enzyme immunoassay, the use of sodium azide as a preservative should be avoided to prevent enzyme inactivation.
HRP isozymes can be divided into three main types:
①Acidic isoenzyme with high sugar content.
②The isoenzyme with relatively low sugar content has an isoelectric point close to neutral (or slightly alkaline).
③Alkaline (PI>11) isoenzyme with low sugar content. The HRP used in the enzyme immunoassay is mainly composed of the so-called "C" isoenzyme with a PI of 8.7 to 9.0, and the activities of other isoenzymes are very low. The covalent structure of the "C" isoenzyme consists of two closely adjacent regions, with the heme group located in between to form a sandwich structure. The sugar chain is bound to the polypeptide at eight different sites. Natural enzymes carry very few pure charges; none are free. -Amino group, with only 2 detectable histidines and 6 lysines, all seem to be covered by the sugar chain shell. Therefore, HRP generally only has 1 to 2 amino groups available for coupling.
According to the catalytic properties of HRP, hydrogen peroxide (H2O2) is generally used as one of the HRP substrates in ELISA. In the presence of a hydrogen donor (i.e., chromogen substrate), the reaction between HRP and H2O2 is rapid and specific. HRP is divalently oxidized by H2O2 to form complex I, and complex I can be reduced to its original state through two consecutive monovalent interactions with a hydrogen donor. Complex II is an oxidized intermediate product with one electron. When H2O2 is excessive, the enzyme activity is inhibited due to the formation of complex III or IV (to be added later).
30% H2O2 is not stable. Since H2O2 is both a substrate and an inhibitor of HRP, in order to obtain satisfactory measurement results by ELISA, 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, this point is generally rarely paid attention to. The concentration of H2O2 used by most researchers is often 2 to 4 times greater than the amount 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, then diluting it 10,000-12,000 times is often an ideal substrate. The molar extinction coefficient of H2O2 is 43.6 at 10mm light path and 240nm wavelength. Therefore, the concentration of H2O2 working solution can be detected in this way.
In solid-phase ELISA, when the temperature is higher than 20°C, HRP activity is often low. Adding non-ionic detergent polysorbate-20 or TritonX-100 to the substrate solution can delay the inactivation of HRP and make the reaction The temperature increases, but the protective effect of non-ionic detergents on enzyme activity varies depending on the hydrogen donor. For example, if 2,2'-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS) is used as the hydrogen donor, only 20% of the enzyme activity can be protected, while o-dianisine When amine (ODA) is used as a hydrogen donor, the protection of enzyme activity is increased to 90%.
The reason why HRP is the most widely used labeling enzyme in ELISA so far is mainly because it is easy to extract and relatively cheap; on the other hand, it is stable, resistant to heat and organic solvents, and can be coupled to antigens or antibodies. Afterwards, there is little loss of activity.
