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.

Reagents

• 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.

Enzyme

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.

Procedure

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.

Calculation