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Description: POD is a heme protein with oxidoreductase properties. Used in biochemical research, it is a major enzyme used in enzyme labeling. Also used in the determination of glucose and galactose in biological fluids.
Alias: Donor: hydrogen-peroxide oxidoreductase; Horserapish peroxidase; HRP
CAS#:9003-99-0 Appearance: Tan crystal or lyophilized powder Type: Type VI-A
Enzyme activity: 250-330 U/mg solid Solubility: soluble in 0.1 M phosphate buffer, pH 6.0, reference concentration 10 mg/ml
HRP is a glycoprotein with a molecular weight of 44 000. It is composed of a colorless enzyme protein and a dark brown iron porphyrin. The neutral sugar and amino sugar account for about 18%, mainly mannose, xylose and arabinose. And hexosamine and the like. Each HRP molecule contains a hemin IX as a prosthetic group, which has a maximum absorption peak at a wavelength of 403 nm, and the deprotected enzyme protein has a maximum absorption at a wavelength of 275 nm. The ratio of the OD value of HRP at 403 nm to the OD value at 275 nm is also the so-called RZ (Reinheits Zahl) value. The RZ value only indicates the content of the heme group in HRP, and does not indicate the true purity of the HRP preparation, and the HRP preparation having a high RZ value does not mean that the enzyme activity is also high. However, the enzyme concentration can be calculated from the absorbance of a pure enzyme solution at a wavelength of 10 mm and a wavelength of 403 nm [1% (W/V) enzyme solution absorbance of 22.5, concentration of 227 umol / L]. Pure HRP dry storage can be kept stable at –20oC, using 1.36 mol/L glycerol, 10mmol/L sodium phosphate, 30umol/L bovine serum albumin and 20umol/L cytochrome C (pH 7.4) solution as a substrate for cryopreservation. The enzyme conjugate is stable for several years. HRP has a relatively stable effect on heat and organic solvents. It can not be changed by toluene and paraffin section treatment or fixed section with pure ethanol or 10% formaldehyde solution. Cyanide or sulfide reversibly inhibits HRP at a concentration of 10–5–10–6 mol/L; fluoride, azide or hydroxylamine inhibits HRP only at concentrations above 10-3 mol/L; HRP also It can be irreversibly inhibited by methylol hydroperoxide. Strong acids are also strong inhibitors of HRP.
Therefore, in the enzyme immunoassay, some of the above compounds such as sodium fluoride, sodium azide and strong acid are often used as terminators for the enzyme reaction. In addition, in order to prevent enzyme inactivation when formulating the dilution buffer for enzyme immunoassay, the use of sodium azide as a preservative should be avoided. HRP isoenzymes can be mainly divided into three types: 1 acidic isozyme with high sugar content. The 2 isoelectric point is close to the neutral (or slightly alkaline) isoenzyme with a relatively low sugar content. 3 alkaline (PI>11) isoenzyme with low sugar content. The HRP used in the enzyme immunoassay has a so-called "C" isoenzyme with a PI of 8.7 to 9.0 as a main component, and the activity of other isozymes is very low. The covalent structure of the "C" isozyme consists of two closely related regions, the heme group is in the sandwich structure, and the sugar chain is bound to the polypeptide at eight different sites. Natural enzymes carry very little pure charge; no free. - Amino group, only 2 measurable histidines, 6 lysines appear to be completely covered by the sugar chain shell. Therefore, HRP typically has only one to two amino groups that can be used for coupling.
According to the catalytic properties of HRP, hydrogen peroxide (H2O2) is generally used as one of the HRP substrates in the ELISA. In the presence of a hydrogen donor (ie, a chromogen substrate), the reaction of HRP with H2O2 is rapid and specific. As can be seen from Fig. 4, HRP is bivalently oxidized by H2O2 to form complex I, and complex I can be reduced to the initial state by a two-step continuous monovalent interaction with the hydrogen donor. Complex II is an oxidized intermediate with an electron. When H2O2 is excessive, the enzyme activity is inhibited (subsequently added) due to the formation of complex III or IV. 30% of H2O2 is not stable. Since H2O2 is both a substrate for HRP and an inhibitor of HRP, H2O2 must be limited to a certain concentration range, and the final concentration is usually 2-6 mmol/L. However, in actual research work, this is rarely paid attention to. The concentration of H2O2 used by most researchers is often 2 to 4 times greater than that required for an ideal reaction. HRP adsorbed to the solid phase is more susceptible to inhibition by excess H2O2 than free HRP. If the concentration of 30% H2O2 stock solution is confirmed to be 30%, dilution of 10,000-12 000 times is often an ideal substrate. The molar extinction coefficient of H2O2 is 10 mm and the optical diameter is 43.6 at a wavelength of 240 nm. Therefore, the concentration of the H2O2 working solution can be detected in this manner.
In solid phase ELISA, when the temperature is higher than 20oC, the HRP activity is often low. Adding the nonionic 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 enzyme activity of nonionic detergents varies depending on the hydrogen donor. For example, when 2,2'-diazide-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS) is used as a hydrogen donor, it can only protect 20% of the enzyme activity, and When the amine (ODA) is a hydrogen donor, the protection of the enzyme activity is increased to 90%.
HRP is by far the most widely used enzyme for labeling in ELISA, mainly because it is easy to extract on the one hand and relatively inexpensive; on the other hand, it is stable in nature, heat and organic solvent, coupled with antigen or antibody. After that, the activity is rarely lost.
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