Lysozyme Assay

Method: The rate of lysis of Micrococcus lysodeikticus is determined as suggested by Shugar (1952). One unit is equal to a decrease in turbidity of 0.001 per minute at 450 nm at pH 7.0 and 25°C under the specified conditions. A wide range of activities are reported for pure lysozyme preparations under these conditions. The Worthington specific activity of 8,000 u/mg dw is equivalent to 50,000 u/mg dw claimed by other suppliers.

Reagents

• 0.1 M Potassium phosphate, pH 7.0
• Micrococcus lysodeikticus cells: Prepare by suspending 9 mg of dried Micrococcus lysodeikticus (Worthington code: ML) cells in 25 ml of 0.1 M potassium phosphate buffer, pH 7.0. Dilute to a final volume of 30 ml with the same buffer.

Enzyme

Dissolve the enzyme at a concentration of one mg/ml in cold reagent grade water. Keep cool until assay. Immediately prior to assay, dilute to a concentration of 150-500 units/ml with reagent grade water. (The rate should fall between 0.015-0.040 ΔA₄₅₀/minute).

Procedure

Adjust spectrophotometer to 450 nm and 25°C.

Pipette 2.9 ml of Micrococcus lysodeikticus cell suspension into a cuvette and incubate for 4-5 minutes in order to achieve temperature equilibration and to establish blank rate, if any. Add 0.1 ml of appropriately diluted enzyme to cuvette and record the change in A₄₅₀ per minute from the initial linear portion of the curve.

Calculation