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Lactoperoxidase (LPO)

(Iodide:hydrogen peroxide oxidoreductase; EC
Lactoperoxidase (LPO) is a hemin containing enzyme. It catalyzes the hydrogen peroxide oxidation of iodide as shown below:
2I- + H2O2 + 2H+ ------> I2 + 2H2O 
The enzyme also catalyzes the oxidation of phenols and aromatics in the presence of hydrogen peroxide. Bovine milk is usually used as a source for isolation and purification of LPO. However, the enzyme is also present in the milk of other species and in the secretions of other mammalian glands such as the salivary gland. LPO from bovine milk has a molecular weight of 77,500. It is a glycoprotein and may exist in two isoenzyme forms (Rombauts, W.A., Schroeder, W.A. and Morrison, M.; Biochemistry, 6, 2965, 1967).

LPO can be used for radioiodination of proteins by coupling the enzyme to cyanogen bromide-activated Sepharose-4B. This coupled form of LPO was capable of iodinating all proteins studied and it exhibited activity over a wide range of experimental conditions (David, G.S., Biochem. Biophys. Res. Comm., 48, 464, 1972).

1.Source: Bovine Raw Skim Milk
   Form: Freeze-dried 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: -20 C; -4 F 
   Activity (approx.): 200 U/mg protein 
   Protein (approx.): 200 U/mg protein 
   A415/A280 (approx.): > 0.9 
   Catalog No.: 095A0200 

That amount of enzyme which will catalyze the formation of one micromole of triiodide per minute at 25C, pH 7.0 in 0.033 M sodium phosphate buffer.

The reaction velocity of the LPO-catalyzed reaction is directly proportional to the increase in absorbance at 350 nm resulting from the formation of triiodide [Morrison, M. in Methods in Enzymology, (Tabor, H. and Tabor C.W. eds); Vol. XVII A, pg. 653, 1970].

  1. 0.033 M Sodium phosphate buffer, pH 7.
  2. 0.005 M Potassium iodide in phosphate buffer.
  3. 0.09 M Hydrogen peroxide. Dilute 0.1 ml hydrogen peroxide (30% H2O2 reagent grade) to 10 ml with distilled water.
  4. Lactoperoxidase solution. Prepare stock enzyme solution (1 mg/ml) in phosphate buffer. Immediately prior to use, prepare a dilution in the range of 0.5-1.0 U/ml in phosphate buffer.

  1. Set spectrophotometer at 350 nm and 25C.
  2. Dilute 0.15 ml of 0.09 M hydrogen peroxide to 30 ml with 0.005 M potassium iodide. This is the reaction mixture and it must be used within 30 minutes after preparation.
  3. Into a cuvette, pipette 3.0 ml reaction mixture and incubate in the spectrophotometer at 25C for 5 minutes to achieve temperature equilibration and establish blank rate, if any.
  4. Add 0.01 ml of diluted enzyme (LPO) solution (0.5-1.0 U/ml) to the cuvette. Mix and record the increase in absorbance at 350 nm for 5 minutes.
  5. Calculate (delta)E350 nm/min from linear portion of the curve.

Activity (U/mg) = (ΔE350nm/min)(Total Vol.)(Enz. Diln.)
(26)(Enz. Vol.)(mg Enz./ml)

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