Lactoperoxidase Assay

Method: The assay procedure is a slight modification of that described by Morrison (1970). The reaction velocity is determined by measuring the increase in A350 resulting from the production of triiodide. One unit results in the formation of one micromole of triiodide per minute at 25°C and pH 7.0 under the specified conditions.


  • 0.033 M Sodium phosphate buffer pH 7.0
  • 0.005 M Potassium iodide in phosphate buffer
  • 0.090 M Hydrogen peroxide. Prepare by diluting 0.1 ml hydrogen peroxide (Merck Superoxol or equivalent) to 10 ml with reagent grade water.


Dissolve at one mg/ml in phosphate buffer. Immediately prior to use, dilute further in phosphate buffer to obtain a rate of 0.02-0.04 ΔA/minute.


Set spectrophotometer at 25°C and 350 nm. Prepare reaction mixture by diluting 0.15 ml of 0.090 M hydrogen peroxide to 30 ml with 0.005 M potassium iodide. Store no longer than 30 minutes at room temperature. Pipette 3.0 ml reaction mixture into cuvette and incubate at 25°C for 3-4 minutes to achieve temperature equilibrium and establish a blank rate, if any. Add 0.01 ml diluted enzyme and record increase in A350 for 3-4 minutes. Calculate ΔA350/minute from initial linear portion of curve. Reaction remains linear for no longer than 1-2 minutes.



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