DETERMINATION OF HUMAN SPUTUM LYSOZYME ACTIVITY (45)
1. Buffer; 0.1 M Tris pH 7.5 prepared at 25°C. Dissolve 12.70 g Tris-HCl and 2.36 g Tris-base in 900 ml H2O. Determine the pH and titrate if necessary with 0.1 M HCl or 0.1 M NaHO to pH 7.5 at 25°C. Bring to 1000 ml with H2O.
2. Substrate suspension; 0.04% w/v. Weigh 40 mg of lyophilized Micrococcus Lyso-deikticus into a 200 ml conical flask. Add 100 ml of Tris (prepared in step 1.) and stir until the suspension is homogenous. Prepare fresh daily.
3. Enzyme solution. Dissolve 1.0 mg per ml in the Tris buffer. Prepare a secondary dilution of 0.05 mg per ml in the buffer. Keep the enzyme solutions cold in an ice bath.
1. Equilibrate the spectrophotometer to 450 nm and the cell temperature to 25°C.
2. Dispense 3.0 ml of substrate suspension in the cell and equilibrate to 25°C.
3. Add 0.005 ml of the 0.05 mg per ml lysozyme solution, mix and determine the rate of decrease in absorbency at 1 minute intervals. The rate of decrease should be linear for 3 to 6 minutes.
Calculation of Specific Activity
Vol = 3.005 A = 450 nm T = 25°C Light path = 1 cm
Î, 1%, 1 cm = 17.60 mg/ml = A280 x 0.568
Units = ∆A / min x 1000 ml
mg 8.8 x 0.0025 mg