ALKALINE PHOSPHATASE from Microorganism
Orthophosphoric-monoester phosphohydrolase (alkaline optimum) (EC 220.127.116.11)
|Activity:||Grade II 30,000 U/ml or more|
|Contaminants:||Adenosine deaminase||≤1.0×10⁻⁴ %|
|Stability:||Store at 4ºC|
|Molecular weight:||approx. 104,000|
|Optimum pH :||9.5(Fig.1)|
|Optimum temperature:||≧ 60 ℃(Fig.2)|
|pH Stability:||pH 5.5-10.4 (25°C, 16hr)(Fig.3)|
|Thermal stability:||below 65℃ (pH 7.0, 60min) (Fig.4)|
This enzyme is useful for molecular biology.
The appearance of p-Nitrophenol is measured at 405nm by spectrophotometry.
One unit causes the formation of one micromole of p-Nitrophenol per minute under the conditions described below.
|A.Diethanolamine buffer:||1 M: Dilute 9.66 mL of diethanolamine (MW ＝ 105.14) in 60 mL of H2O, add 5 mL of 0.1 M MgCl2, and, after adjusting the pH to 9.8 with 2 N HCl, make up to 100 mL with H2O (should be freshly prepared).|
|B. pNPP solution:||0.674 M: 2.5g of p-nitrophenylphosphate disodium salt (MW ＝ 371.16) in 10 mL of diethanolamine buffer (A). Should be freshly prepared.|
|C. Enzyme diluent:||30 mM Triethanolamine, 1 mM MgCl2, 0.1 mM ZnCl2, 0.5 % sodium cholate, pH 7.6|
|Concentration in assay mixture|
|Diethanolamine buffer||0.97 M|
1. Prepare the following reaction mixture in a cuvette (d=1.0cm)
and equilibrate at 37℃ for about 5 minutes.
30 mL Diethanolamine buffer (A)
0.5 mL pNPP solution (B)
2. Pipette 3.0 mL of working solution into a cuvette (d = 1.0 cm) and equilibrate at 37 ℃ for approximately 5 minutes.
3. Add 0.1 mL of the enzyme solution* and mix by gentle inversion.
4. Record the increase in optical density at 405 nm against water for 3 to 5 minutes with a spectrophotometer thermostated at 37 ℃, and calculate the ∆OD per minute from the initial linear portion of the curve (∆OD test).
At the same time, measure the blank rate (∆OD blank) using the same method as the test except that the enzyme diluent (C) is added instead of the enzyme solution.
* Dilute the enzyme preparation to 0.1-0.3U/ml with ice-cold enzyme diluent (C), immediately before assay.
Activity can be calculated by using the following formula :
ΔOD/min (ΔOD test−ΔOD blank) × Vt × df
Volume activity (U/ml) =
18.5 × 1.0 × Vs
- Weight activity (U/mg) = (U/ml)×1/C
- : Total volume (3.1ml)
- : Sample volume (0.1ml)
- : Millimolar extinction coefficient of p-Nitrophenol under the assay condition (cm²/micromole)
- : Light path length (cm)
- : Dilution factor