Alkaline Phosphatase and Phospholipase C Assays

1. Grow bacteria overnight in Pi sufficient and Pi deficient media.
2. Read OD₆₀₀ of overnight culture. One OD₆₀₀ unit = 0.3 mg protein.
3. Pellet 1 ml of culture in microfuge (5 min). Save supernatant.
4. Add 500 µl of resulting supernatant to 500 µl of alkaline phosphatase substrate or phospholipase C substrate.
   

   Alakaline phosphatase substrate:

   • 2 mg/ml of p-nitrophenyl phosphate
   • One Sigma 104 tablet (5mg) per 2.5 ml of 0.1 M Tris-HCl, pH 8.5

   Phospholipase C substrate:

   • 6 mg/ml nitro-phenylphosphorylcholine
   • 0.25 M Tris-HCl pH 7.3
   • 60% (v/v) glycerol
5. Follow the A₄₁₀ “absorbance change” using the Perkin-Elmer spectrophotometer and attached chart recorder. 1-2 min is enough.
6. Let one sample hydrolyse the substrate to completion (i.e. no more change in absorbance) then read the A₄₁₀. This is the A₄₁₀ FINAL which is used to calculate the extinction coefficient.

Calculations:

One OD₆₀₀ culture = 0.3 mg/ml proteinMW Phospholipase C substrate = 304.2; 3 mg/ml final concentration = 9.9 umoles/ml

MW Alkaline phosphate substrate = 263.1;
1 mg/ml final concentration = 3.8 umoles/ml

Extinction coefficient = umoles substrate hydrolyzed/A₄₁₀ FINAL
(e.g. for APase: Ext. coef. = 3.8 umoles/A₄₁₀ FINAL)

Enzyme Activity = A₄₁₀ “absorbance change”/min x Extinction coefficient/mg protein = umoles substrate hydrolyzed/min./mg protein.