EXPERIMENT THREE
ASSAY OF ACTIVITY OF
ALKALINE PHOSPHATASE
2012-09
1
Aims
•
•
To master the basic method of the assay
of the AKP (Alkaline phosphatase ). To
strengthen the comprehension of
enzyme activity and its inhibition.
To be familiar in the meaning of the
activity unit, total activity , ratio activity
and how to do the calculation of AKP
activity.
2
Some important concepts
• An enzyme unit is that amount of enzyme which
can catalyze the transformation of 1μmol of
substrate per minute at 37℃ under optimal
conditions for that enzyme.
• International unit (IU): 1µmol/min
• Katal unit:
1mol/sec
1 IU = ? katal
1 IU=16.67×10-9kat
3
△S
• V=
△P
or
△t
△t
4
Some important concepts
• Total activity refers to the total unit of
enzyme units of enzyme in the
sample
• Specific activity is the number of
enzyme units per milligram of
protein(unit/mg).
5
Total enzyme activity and specific activity
For example
Sample
A
B
Units of AKP activity
100
100
Volume (ml)
100
10
Do you think whether the AKP activity of this two
samples are same or not?
How to compare the activity of AKP in A sample
with in B sample?
6
Total enzyme activity and specific activity
For example
Sample
A
B
Units of AKP activity
100
100
Volume (ml)
100
10
1 U/ml
10 U/ml
Specific activity (U/ml)
7
Principle
• Alkaline phosphatase (AKP ) can catalyze
disodium phenyl phosphate decomposition in
basic condition and produce phenol and
disodium hydrogen phosphate.
• Phenol can react with 4-aminoantipyrene and
K3Fe(CN)6 to generate red complex which has
the highest absorbance at 510 nm.
8
Mg2+ is the activator for AKP, EDTA is the inhibitor
9
Principle
• This red compound has the most
absorbance atλ510nm. According to this
we can calculate AKP enzyme unit.
10
Protocol
1. Make the standard curve
Reagents
H2CO3-NaHCO3 buffer
(pH10)
Phenol standard solution
H 2O
ml
blank
1
2
3
4
5
6
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0
0.05
0.1
0.2
0.3
0.4
0.5
1.0
0.95
0.9
0.8
0.7
0.6
0.5
37℃, 5min
0.2mol/L NaOH solution
1.0
1.0
1.0
1.0
1.0
1.0
1.0
4-aminoantipyrene
1.0
1.0
1.0
1.0
1.0
1.0
1.0
K3Fe(CN)6
2.0
2.0
2.0
2.0
2.0
2.0
2.0
Mixing, waitting for ten minutes，read A510
A510
Phenol (μmol)
0
0.05
0.1
0.2
0.3
0.4
0.5
Correspond enzyme unit
0
2
4
8
12
16
20
11
Protocol
Draw the standard curve by using A510 as ycoordinate and correspond enzyme unit as xcoordinate
A510
0
Enzyme unit
12
Operator
A
Date
Apparatus
Correspond enzyme unit
0
Name of the curve
13
【Calculation】
Corresponding enzyme activity (mU/tube)
= corresponding µmol value ×
1
× 1000
25
= corresponding µmol value × 40
25, is the time extent for water bath, or reaction time
× 1000, to change U to be mU
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Protocol
2. Assay of AKP activity
ml
Assay1 Assay2(Mg2+)
Reagent
Blank
Substrate (0.02mol/L) (No
Mg2+ and EDTA)
0
0.3
0.3
0.3
Mg2+(0.012mol/L)
0
0
0.1
0
EDTA(0.012mol/L)
0
0
0
0.1
H2 O
1
0.7
0.6
0.6
Enzyme (0.5mg/ml)
0.1
0.1
0.1
0.1
H2CO3 buffer (pH10)
0.9
0.9
0.9
0.9
Assay3(EDTA)
37℃, 25min
NaOH
1.0
1.0
1.0
1.0
4-aminoantipyrene
1.0
1.0
1.0
1.0
K3Fe(CN)6
2.0
2.0
2.0
2.0
15
Protocol
2. Assay of AKP activity
According to the data of your results, calculate the activity
units of AKP and its specific activity of AKP, filling the
following table, please:
Reagent
Blank
Assay1 Assay2( Mg2+)
Assay3(EDTA)
Mixing, let the test tubes stand on the table for ten minutes，then
measure A510 each tube
A510
0
Total activity(mU/tube)
0
mU/ml
0
mU/mg
0
16
Items
Blank
Assay1 Assay2( Mg2+)
A510
0
0.292
Total activity(mU/tube)
0
6
mU/ml
0
60
mU/mg
0
120
Assay3(EDTA)
A
C
0
2
4
6
8
X-coordinate
10
17
Caution
1. The AKP come from cobra snake venom,
so carefully to handle the enzyme solution
2. To add the basic solution (NaOH) quickly
to stop the enzyme-catalyzed reaction
when the time is due, maintaining the time
extent of reaction identical in each test
tube
18
Discussion
1.Why is it needed to add NaOH solution to
stop the reactions?
2. What are the action of Mg2+ and EDTA in
this AKP enzyme-catalyzed reaction,
respectively?
3.Do you know anything about AKP and the
relationship between AKP and some clinic
diseases?
19
Reagents
1. 0.1mol/L pH10 H2CO3-Na2CO3 buffer ( Carbonate
buffer )
2. 0.02mol/L C6H5Na2PO4 ( Disodium phenyl phosphate )
3. 1μmol/ml C6H5OH ( Phenol standard solution )
4. 0.012mol/L MgCl2 ( Magnesium chloride )
5. 0.012mol/L EDTA ( Disodium ethylene diamine
tetraacetate )
6. 0.2mol/L NaOH (Sodium hydroxide )
7. 0.5mg/ml snake venom
8. 0.3% 4-aminoantipyrene
9. 0.5% K3Fe(CN)6 ( Potassium ferricyanide )
20