Wednesday, October 01, 2003

Description
Alkaline phosphatases (APs) are highly ubiquitous enzymes, present in all species from bacteria to man

Procedure
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 l of resulting supernatant to 500 l of alkaline phosphatase substrate or phospholipase C substrate.
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.


Recipes
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

Supplies


Tips
CALCULATIONS:

One OD600 culture = 0.3 mg/ml protein

MW 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.