Effect of substrate

concentration and
enzyme inhibitor on
enzyme activity
Determination of
Alkaline Phosphatase
activity

Introduction:

Phosphatases are enzymes which

catalyze the splitting off phosphoric
acid from nonphosphoric esters.
Two common types are estimated in
serum:
Alkaline Phosphatase (optimum pH 10)
Acid Phosphatase (optimum pH 5 6)

Alkaline Phosphatase

Purified forms from different sources

undergo 3 types of activity:

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)

ALP requires metal ions :

Mg2+, Zn2+ and to a lesser extent Mn2+

Inhibitors of ALP:

Cu2+, Hg2+, EDTA (chelating Mg2+),

Phosphate & some amino acids e.g. Lphenylalanine

 Principle

of the test:

ALP from human serum will hydrolyze

the artificial substrate, disodium phenyl
phosphate to phenol which will react
with 4-aminoantipyrine in the alkaline,
oxidizing agents giving a red purple
color which can be measured at 520 nm

Objective of the test

In this experiment we will investigate

the effect of changing substrate
concentration on enzyme activity in
presence and in absence of the
inhibitor (inorganic phosphate) to
identify the type of inhibition.

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

Mix and incubate the tubes at 37C for exactly 15 min.

NaOH 0.5 N

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

Mix and read the tubes against blank at 520 nm

Calculate ALP activity (v) for each

substrate
concentration using the following
formula:
O.D test O.D control

ALP activity (v) = O.D Standard O.D blank

X 10

= mg of phenol produced/100 ml
serum in 15 minutes
= King Armistrong (KA) units/100 ml

Calculation of substrate concentration:

Substrate concentration = 0.01 M
Substrate M. Wt.
= 254
Substrate concentration = 0.01 X 254 =
2.54
[S] = substrate conc. X volume
= 2.54 X volume

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]

Non - Competitive Inhibitor

Competitive Inhibitor

1/vi

+ Inhibitor

No Inhibitor
1/Vmax

- 1/Km

Uncompetitive Inhibitor

1/[S]

3. Calculation of the percentage inhibition:

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

4. Does the % inhibition change as [S] increase?

5. Do the results of this calculation confirm your
conclusions as to the type of inhibition?