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concentration and
enzyme inhibitor on
enzyme activity
Determination of
Alkaline Phosphatase
activity
Introduction:
Alkaline Phosphatase
1- Hydrolytic
R-O- P + HOH
2- Phosphotransferase
R-O- P + R`-OH
3- Pyrophosphatase:
R-O- P -O- P -O- R` + HOH
ROH + H3PO4
R- OH + R`- O- P
R-O- P + R`-O- P
Sources:
Osteoblasts
in the bone
Bile canaliculi in liver
Small intestinal epithelium
Proximal tubules in the kidney.
The placenta
Lactating breasts
In all these sites, it seems to be
involved in the transport of phosphate
across membrane
ALP of normal serum is mainly derived
from liver (the bone isozyme is absent)
Inhibitors of ALP:
Principle
of the test:
PROCEDURE:
Into 10 test tubes, add the following (in
ml):
Blank
Buffer
Standard
Test
Without Inhibitor
Inhibitor
Test
With
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Substrate
0.25
0.50
0.75
1.0
Substrate + Inhibitor
0.25
0.50
0.75
1.0
Standard
1.0
1.1
0.1
0.75
0.50
0.25
0.75
0.50
0.25
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Dist. Water
Serum sample
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
Na HCO3 0.5 N
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
4-amino-antipyrine
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Potassium ferricyanide
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
X 10
= mg of phenol produced/100 ml
serum in 15 minutes
= King Armistrong (KA) units/100 ml
1. Calculation:
+I
-I
Test
tube
O.D. B = 0.0
O.D. S =
O.D. C = 0.04
[S] = 2.54 X volume
Sub.volume
(ml)
[S]
1/[S]
0.25
0.635
1.575
0.50
1.27
0.787
0.75
1.905
0.525
1.00
2.54
0.394
0.25
0.635
1.575
0.50
1.27
0.787
0.75
1.905
0.525
1.00
2.54
0.394
O.D.T
T- C
V = --------- X 10
SB
1/V
2. Type of inhibition:
Using 1/V and 1/[S], draw
the Linweaver-Burk plot to calculate the K m and Vmax of
the reaction in presence
and absence of the inhibitor
1/Vmax(-I) =
- 1/Km (-I) =
Vmax (-I) =
Km (-I) =
1/Vmax(+I) =
Vmax (+I) =
- 1/Km (+I) =
Km (+I)
Vmax is the
same in
presence of
a
competitive
inhibitor
Competitive
Inhibitor
1/vi
1/vi
+ Inhibitor
1/Vmax
Apparent Km is
increased in presence
of a competitive
Inhibitor
No Inhibitor
No
Inhibitor
1/Vmax
- 1/Km
- 1/Km
1/[S]
Competitive Inhibitor
1/vi
+ Inhibitor
No Inhibitor
1/Vmax
- 1/Km
Uncompetitive Inhibitor
1/[S]
V - Vi
% inhibition = ---------------- X 100
V
where V = rate without inhibitor, Vi = rate with inhibitor
[S]
Vi
%
inhibition
0.635
1.27
1.905
2.54