| Species : |
Bovine |
| Source : |
Milk |
| Description : |
Lactoperoxidase (LPO) is a glycoprotein with a hemin prosthetic group which may occur as a mixture of two isozymes. It has a molecular weight of 77,500 daltons. LPO catalyzes the hydrogen peroxide oxidation of iodide according to the following reaction:
2I- + H2O2 + 2H+ → I2 + 2H2O.
Iodide reacts directly with the heme group; upon addition of H2O2 the complex iodinates the substrate. LPO is inhibited by hydrazines. The assay procedure has been updated from that of Morrison to an ABTS®/H2O2 based method with increased sensitivity and reproducibility. |
| Form : |
A lyophilized powder. |
| Bio-activity : |
≥35 units/mg dry weight ABTS |
| Molecular Mass : |
77.5 kDa |
| Purity : |
Chromatographically purified. A412/A280 = 0.93-0.96 |
| Storage : |
Store at -20 centigrade. |
| EC : |
1.11.1.7 |
| CAS : |
9003-99-0 |
| Unit definition : |
One Unit reduces one micromole of hydrogen peroxide per minute at 25 centigrade, pH 6.0. Note: This unit definition is based upon a new assay method. The previous method yielded approximately 2.8 times higher results. |
| Usage : |
Method: The assay procedure is a modification of that described by Putter and Becker. The increase in the absorbance at 405 nm resulting from the oxidation of 2,2-azino-di(3-ethyl-bnzothiazoline-6-sulphonic acid), diammonium salt, ABTS. Two moles of ABTS are oxidized for every mole of peroxide that is reduced.
Reagents:
0.08 M Sodium-Potassium Phosphate Buffer pH 6.0: Prepare by adjusting the pH of 0.08 M sodium phosphate, dibasic, to 6.0 using 0.08 M potassium phosphate, monobasic.
Sodium Potassium Phosphate Buffer, containing 1 mg/mL Bovine Serum Albumin: Dissolve BSA at a concentration of mg/ml in an aliquot of the above buffer. This will be used as enzyme diluent.
0.0017 M Hydrogen peroxide. Prepare by diluting 1 ml of 30% hydrogen peroxide to 100 ml with reagent grade water. Further dilute 1 ml of this solution to 50 ml with reagent grade water. Prepare fresh daily.
0.051 M ABTS: Dissolve ABTS in 0.08 M sodium phosphate buffer, pH 6.0
Enzyme: Dissolve at one mg/ml in sodium-phosphate buffer. Immediately prior to use, dilute further in phosphate buffer with BSA to obtain a rate of 0.008-0.025 DA/minute.
Keep all dilutions on ice.
Procedure: Set spectrophotometer at 25 centigrade and 405 nm. Pipette into each cuvette 2.65 ml sodium- phosphate buffer, 0.15 mL hydrogen peroxide, and 0.10 ml ABTS. Incubate in spectrophotometer at 25 centigrade for 3-5 minutes to achieve temperature equilibration and establish a blank rate, if any. Add 0.10 ml diluted enzyme and record the increase in A405 for 4-6 minutes. Calculate the rate from the maximum initial linear portion of the curve. The reaction is linear for approximately two minutes.
Calculation:
(Units/mg)=(ΔA350/min×3.0×dilution)/(18.6×2×sample volume)
18.6= extinction coefficient of ABTS at 405nm
2 ABTS: 1 H2O2 |
| Extinction Coefficient : |
13.9 |
| Inhibitors : |
LPO is inhibited by hydrazines. Dolman et al. (1968) report on the kinetics of cyanide inhibition. |
