Summary
- Horseradish peroxidase (HRP) exhibits high specific activity, stability, and a small molecular weight. The pure enzyme is easily prepared, making it the most commonly used peroxidase. HRP is widely distributed in the plant kingdom, particularly abundant in horseradish. It is a glycoprotein consisting of a colorless enzyme protein and brown iron porphyrin, with a sugar content of 18%.
Brown freeze-dried powder.
Peroxidase, classified enzymatically as EC 1.11.1.7.
Donor + H2O2 → Oxidized donor + 2 H2O.
HRP comprises multiple isozymes with a molecular weight of 40,000 and an isoelectric point ranging from pH 3 to 9. The optimal pH for enzyme catalysis varies slightly depending on different hydrogen donors but is mostly around pH 5. The enzyme is soluble in water and ammonium sulfate solutions with a saturation of less than 58%.
The maximum absorption spectra for HRP's prosthetic group and enzyme protein are 403 nm and 275 nm, respectively. Enzyme purity is commonly expressed by the OD403 nm/OD275 nm ratio, known as the RZ (Reinheit Zahl) value. Highly pure enzymes typically have an RZ value around 3.0 (up to 3.4). A smaller RZ value indicates a higher presence of non-enzymatic proteins.
HRP is a glycoprotein with a molecular weight of 44,000, composed of a colorless enzyme protein and dark brown iron porphyrin. Neutral sugars and amino sugars account for approximately 18%, including mannose, xylose, arabinose, and hexosamine. Each HRP molecule contains hemin IX as a prosthetic group, with a maximum absorption peak at 403 nm. The enzyme protein without the prosthetic group absorbs maximally at 275 nm. The RZ value only reflects the heme group content in HRP, not its true purity or enzyme activity. Additionally, a high RZ value does not necessarily indicate high enzyme activity.
Pure HRP remains stable when dried and stored at -20°C. Cryopreservation is achieved using a solution containing 1.36 mol/L glycerol, 10 mmol/L sodium phosphate, 30 μmol/L bovine serum albumin, and 20 μmol/L cytochrome C (pH 7.4). The enzyme conjugate remains stable for several years. HRP exhibits relative stability against heat and organic solvents, unaffected by toluene and paraffin sectioning or fixation with pure ethanol or a 10% formaldehyde aqueous solution for frozen sectioning. Reversible inhibition of HRP occurs with cyanide or sulfide at concentrations of 10^-5 to 10^-6 mol/L, while fluoride, azide, or hydroxylamine inhibit HRP only at concentrations higher than 10^-3 mol/L. Irreversible inhibition is caused by hydroxymethyl hydroperoxide. Strong acids also act as potent inhibitors of HRP.
Therefore, some of the aforementioned compounds, such as sodium fluoride, sodium azide, and strong acids, are commonly employed as terminators in enzyme immunoassays to halt enzyme reactions. However, it is advisable to avoid using sodium azide as a preservative in the dilution buffer for enzyme immunoassays to prevent enzyme inactivation.
The HRP isoenzymes can be categorized into three types:
Acidic isoenzymes with high sugar content.
Isoenzyme with relatively low sugar content and an isoelectric point close to neutral (or slightly alkaline).
Alkaline isoenzymes (PI>11) with low sugar content.
In enzyme immunoassays, the HRP used mainly comprises the "C" isoenzyme with a PI ranging from 8.7 to 9.0, and activities of other isoenzymes are minimal. The "C" isoenzyme's covalent structure exhibits two closely adjacent regions, forming a sandwich structure with the heme group situated in between. The sugar chain is bound to the polypeptide at eight different sites. Natural enzymes possess very few pure charges, with only 2 detectable histidines and 6 lysines, all seemingly covered by the sugar chain shell. Consequently, HRP generally has only 1 to 2 amino groups available for coupling.
Based on HRP's catalytic properties, hydrogen peroxide (H2O2) is typically utilized as one of the substrates in ELISA. In the presence of a hydrogen donor (i.e., chromogen substrate), the reaction between HRP and H2O2 is rapid and specific. As illustrated in Figure 4, HRP is divalently oxidized by H2O2 to form complex I, which can be reduced to the original state through two consecutive monovalent interactions with a hydrogen donor. Complex II is an oxidized intermediate product with one electron. Excessive H2O2 inhibits enzyme activity due to the formation of complex III or IV.
To achieve accurate measurements in ELISA, the concentration of H2O2 must be limited to a specific range, typically 2 to 6 mmol/L. However, this aspect is often overlooked in practical research, with researchers frequently using H2O2 concentrations 2 to 4 times greater than required for the ideal reaction. HRP adsorbed on the solid phase is more prone to inhibition by excess H2O2 than free HRP. Diluting a 30% H2O2 stock solution 10,000-12,000 times is often an ideal substrate, considering its molar extinction coefficient of 43.6 at a 10mm light path and 240nm wavelength.
In solid-phase ELISA, when the temperature exceeds 20°C, HRP activity tends to decrease. Incorporating non-ionic detergents such as polysorbate-20 or TritonX-100 into the substrate solution can postpone HRP deactivation and enhance the reaction temperature. However, the protective effect of non-ionic detergents on enzyme activity varies depending on the hydrogen donor. For instance, 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-dianisidine (ODA) as a hydrogen donor increases the protection of enzyme activity to 90%.