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Assay Procedure for Alkaline
Phosphatase (Ref. No. FGAP014)
Based on that of Bessey et al in which the rate
of formation of the yellow colour of p-nitrophenol (p-NP) produced by hydrolysis of
p-nitrophenylphosphate (p-NPP) in alkaline solution is measured spectrophotometrically at
405nm and 37°C.
|
Alkaline phosphatase |
|
| p-NPP + H2O |
 |
p-NP + Pi |
Unit Definition
That amount of enzyme which catalyses the liberation of 1
micromole p-nitrophenol per minute at 37°C.
Reagents
| A |
0.3M 2-Amino -2 Methylpropane -1,3 Diol/0.002M MgCl2
Buffer pH 10.25 Dissolve 3.16g AMPD[MM 105.14] in 80 ml
distilled H2O, adjust to pH10.25 (with 5M HCl), add 40.6mg MgCl2.6H2O
[MM203.3], dissolve, dilute to 100ml and recheck pH 10.25. Adjust with 1M NaOH or 5M HCl.
Store diluent on ice, and store buffer at 37°C.
|
| B |
Substrate [0.4M p-Nitrophenyl Phosphate] Dissolve 105mg Na2p-NPP [MM263.05] or 148.46 mg Na2p-NPP.6H2O[MM
371.15] / ml distilled H2O. Store on ice.
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| C |
Sample For F/D product,
dissolve 1mg enzyme/ml ice-cold buffer [3A]. Immediately before assay, dilute to yield ±
0.15 u/ml ice-cold buffer. [DA/min 0.09 - 0.10]. |
Procedure
l = 405nm
Temperature = 37°C
Cuvette volume = 3.0ml
Light Path = 10mm
Into a 10mm quartz cell, pipette:
| Buffer [A] |
2.8 ml |
| Substrate [B] |
0.1 ml |
| Equilibrate at 37°C and monitor DA/min. |
|
| Enzyme [C] |
0.1 ml |
|
3.00 ml |
Record rate of increase in absorbance at 405nm for ± 5 minutes.
Caclulation
|
DA 405/min
x 3 x dilution |
| Activity [u/mg] = |
 |
|
18.8 x 0.1 x mg enzyme / ml original solution |
[e = 18.8; 3 =
cuvette volume; 0.1 = enzyme volume]
Unit Conversion
- One unit of activity as described above is the same as one unit
using diethanolamine buffer at pH9.8 at 37°C (DEA Unit).
- One Glycine unit is equivalent to approximately three DEA where
the glycine unit is defined as: That amount of enzyme causing the hydolysis of one
micromole of p-nitrophenol phosphate per minute at pH9.6 and 25°C in glycine
buffer.
Reference
Bessey, O.A., Lowry O.H. and Brock M.J.:(1946) J.Biol. Chem. 164
321 |
Specialists in the Isolation and Purification of Horseradish
Peroxidase and a Select Range of Enzymes