Title: Experiment 3: Kinetic studies with alkaline phosphatase

Objective(s):
1. To prepare the standard curve for p nitrophenol
2. To study the effect of the addition of Mg2+ ions an alkaline phosphate
Introduction:
Phosphatases are enzymes that catalyze the hydrolysis of esters of phosphoric acid. They
occur in the cells and extracellular fluids of a wide range of organisms. This large and complex
group of enzymes falls into four general types based on the chemical nature of the substrate or
the type of hydrolytic reaction that is catalyzed .This large and complex group of enzymes falls
into four general types based on the chemical nature of the substrate or the type of hydrolytic
reaction that is catalyzed.

One group, the phosphomonoesterases, hydrolyzes monoesters of phosphoric acid such as glycerophosphate or glucose 6-phosphate. Some phosphomonoesterases are highly substratespecific. For example, in gluconeogenesis, fructose-1, 6-bisphosphatase specifically converts
fructose

1,6-bisphosphate

to

fructose

6-phosphate

and

inorganic

phosphate.

Other

phosphomonoesterases react with a broad range of substrates, which share common structural
motifs. The phosphomonoesterases that lack substrate specificity are classified as acid or
alkaline phosphatases based on their pH optima. Acid phosphatases function best at around pH
5.0 and are inhibited by fluoride ion but not by divalent cation-chelating agents. The alkaline
phosphatases have pH optima of about 9.0 and are not generally sensitive to fluoride ion but
are inhibited by divalent cation-chelating agents like EDTA (ethylene diamine tetraacetic acid,
disodium salt).
Low level of alkaline phosphatase can cause, Hypophosphatasia. Hypophosphatasia is a
genetic metabolic bone disease that is quite rare in occurrence but fatal to the sufferer. Some of
the identifiable symptoms are skeletal hypomineralization, mild respiratory problems,

progressive osteomalacia, etc. The patients of hypophosphatasia have very low alkaline
phosphatase levels in their blood serum. Such patients often lose their primary teeth much
before the standard age. Other than that it can also cause aolastic anemia or chronic
Myelogenous Leukemia.
In this experiment, the alkaline phosphatases kinetic studies are carried out on the effect of pH,
inhibitors and divalent cation. At the beginning of the experiment the standard curve is drawn
against the amount of p-nitrophenol. The p-nitrophenol amount used in the assay is determined
from the formula of;
(Amount of 0.3mM p-nitrophenol) x 10-3L X (0.3 X 10-3mol/L)
This graph is used as standard to determine the amount of p-nitrophenol have been consumed
in the pH, inhibitor and divalent cations reaction. The alkaline phosphatases have binding sites
for Zn2+ and Mg2+, on which enzyme activity is dependent. In this experiment, the effects of
addition of Mg2+ ions on alkaline phosphatase are carried out.

Next, with variable pH,

beginning from pH 7.0, pH 7.5, pH 8.0, pH 8.5 and pH 9.0. Varying pH levels may have a direct
effect on the alkaline phosphatase due to the presence of ionizable residues in the catalytic site
of the enzyme.

Materials:
Bovine or calf intestinal alkaline phosphatase (Sigma), diluted 1:10,000 with 50 mM Tris-HCl,
pH 8.0 buffer, containing 1 mg/ml bovine serum albumin, water bath, p-nitrophenol phosphate
in buffer (2.70 mM),

p-nitrophenol in buffer (0.3 mM),

phenylalanine, 50 mM Tris-HCl, pH 7.0, 7.5, 8.0, 8.5, 9.

Procedure:

monobasic sodium phosphate, L-

A) Preparation of standard curve for p-nitrophenol

Tube
0.3nM p-nitrophenol ( ml)
Distilled water(ml)

3.0

0.05
2.95

0.1
2.90

0.2
2.80

0.4
2.60

0.6
2.40

1. Following tubes were prepared

2. Absorbance was read at 400 nm using tube 1 as a blank
3. The amount in mol of p-nitrophenol for each tube
4. A graph has been plotted ( absorbance against amount of p-nitrophenol in umol)
B) Effects of divalent cation
Tube 1
2.70 mM substrate( ml)
50mm tris-HCl pH 8.0
15nM MgCl2

1
0.6
2.4
-

2
0.6
2.3
0.1

3
0.6
2.2
0.2

4
0.6
2.0
0.4

5
0.6
1.8
0.6

6
0.6
1.6
0.8

7
0.6
1.4
1.0

1. The following tubes were prepared

2. Each of the above reaction mixture is added to a suitable cuvette and the reaction
was started by adding 20 ul of an enzyme.
3. Was mixed by invert
4. The cuvette was placed in the spectrophotometer
5. Absorbance was read for every 30 s for 3 min at 400 nm
6. The A/min was determined.Agraph was plotted in (umol/min) against the MgCl 2
concentration

C) Effect of the pH
1. The tubes were prepared same as in B table but the buffer was substituted with
buffer with different pH which are 7.0,7.5,8.0,8.5,9.0
2. A graph of activity against pH was plotted.

Results:
(A) Preparation of standard curve for p-nitrophenol

Tube
1
0.3mM p- nitrophenol
(ml)

2
0.05

3
0.1

4
0.2

5
0.4

6
0.6

Distilled
water (ml)

2.95

2.9

2.8

2.6

2.4

[pnitrophenol]
(M)

0.015

0.030

0.060

0.120

0.18

Absorbance 0
at 400nm

0.018

0.041

0.056

0.072

0.136

mM convert to M
Tube 2:
0.3m

mol
L

0.00005 L = 1.5 10-5 1000 mol

= 0.015 mol

Tube 3:
0.3m

mol
L

0.0001 L = 3 10-5 1000 mol

= 0.030 mol

Tube 4:
0.3m

mol
L

0.0002 L = 6 10-5 1000 mol

= 0.060 mol
Tube5:
0.3m

mol
L

0.0004 L = 1.2 10-4 1000 mol

= 0.120 mol

Tube6:
0.3m

mol
L

0.0006 L = 1.8 10-4 1000 mol

= 0.180 mol

Graph of absorbance vs p-nitrophenol concentration

0.16
0.14
0.12 f(x) = 0.74x
R = 0.97
0.1
absorbance

0.08
0.06
0.04
0.02
0
0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

concentration of p-nitrophenol

Figure 1 Graph of absorbance againt amount of p-nitrophenol

0.16

0.18

0.2

(B) Effect of divalent cations

Tub
e
Time
(min)
0
0.5
1.0
1.5
2.0
2.5
3.0

Absorbance at 400nm
4
5

0.033
0.035
0.039
0.041
0.045
0.048
0.051

0.030
0.035
0.038
0.042
0.046
0.049
0.053

0.039
0.048
0.052
0.056
0.061
0.065
0.070

0.050
0.054
0.058
0.063
0.068
0.073
0.078

0.032
0.036
0.041
0.047
0.052
0.059
0.064

Table 1: Absorbance vs Time (min)

Calculation of p-nitrophenol
Absorbance = slope (m) concentration
Y = mx, Y is absorbance, m is slope, x is concentration
Slope from standard curve from part A, so m = 0.7371
In order to find concentration, use the absorbance / slope
Example:
Tube 1 with Time = 0:
Concentration =

0.033
0.7371

= 0.045 mol
Time = 0.5 min
Concentration =

0.035
0.7371

= 0.047 mol
Time = 1 min
Concentration =

0.039
0.7371

= 0.053 mol

0.045
0.050
0.055
0.060
0.066
0.071
0.077

0.055
0.059
0.065
0.071
0.076
0.082
0.088

Time = 1.5 min

Concentration =

0.041
0.7371

= 0.056 mol

e
Time
(min)
0
0.5
1.0
1.5
2.0
2.5
3.0

Tub [p-nitrophenol] (mol)

1
2
3
0.045
0.047
0.053
0.056
0.061
0.065
0.069

0.041
0.047
0.052
0.057
0.062
0.066
0.072

0.053
0.065
0.071
0.076
0.083
0.088
0.095

0.068
0.073
0.079
0.085
0.092
0.099
0.110

0.043
0.049
0.056
0.064
0.071
0.080
0.087

0.061
0.068
0.075
0.081
0.090
0.096
0.104

0.075
0.080
0.088
0.096
0.103
0.111
0.120

Table 2: [p-nitrophenol] (mol) vs Time (min)

Tube 1: [p-nitrophenol] vs Time

0.08
0.07
0.07

f(x) = 0.01x + 0.04

R = 0.99

concentration of p-nitrophenol 0.06

0.06
0.05
0.05
0 0.5 1 1.5 2 2.5 3 3.5
time (min)

Graph 1: Tube 1 [p-nitrophenol] (mol) vs Time (min)

Tube 2 : [p-nitrophenol] vs Time

0.08
0.07

f(x) = 0.01x + 0.04

0.07 R = 1
0.06
concentration of p-nitrophenol

0.06
0.05
0.05
0.04
0 0.5 1 1.5 2 2.5 3 3.5
Time (min)

Graph 2: Tube 2 [p-nitrophenol] (mol) vs Time (min)

Tube 3 : [p-nitrophenol] vs Time

0.1
0.09 f(x) = 0.01x + 0.06
R = 0.98
0.08
concentration of p-nitrophenol

0.07
0.06
0.05
0 0.5 1 1.5 2 2.5 3 3.5
Time (min)

Graph 3: Tube 3[p-nitrophenol] (mol) vs Time (min)

Tube 4 : [p-nitrophenol] vs Time

0.12
0.11
0.1
concentration of p-nitrophenol

f(x) = 0.01x + 0.07

R = 0.99

0.09
0.08
0.07
0 0.5 1 1.5 2 2.5 3 3.5
Time (min)

Graph 4: Tube 4[p-nitrophenol] (mol) vs Time (min)

Tube 5 : [p-nitrophenol] vs Time

0.09
0.08
0.07
concentration of p-nitrophenol

f(x) = 0.01x + 0.04

R = 1

0.06
0.05
0.04
0 0.5 1 1.5 2 2.5 3 3.5
Time (min)

Graph 5: Tube 5[p-nitrophenol] (mol) vs Time (min)

Tube 6 : [p-nitrophenol] vs Time

0.11
0.1

f(x) = 0.01x + 0.06

R = 1

0.09
concentration of p-nitrophenol

0.08
0.07
0.06
0 0.5 1 1.5 2 2.5 3 3.5
Time (min)

Graph 6: Tube 6 [p-nitrophenol] (mol) vs Time (min)

Tube 7 : [p-nitrophenol] vs Time

0.13
0.12

f(x) = 0.02x + 0.07

R = 1

0.11
concentration of p-nitrophenol

0.1
0.09
0.08
0 0.5 1 1.5 2 2.5 3 3.5
Time (min)

Graph 7: Tube 7 [p-nitrophenol] (mol) vs Time (min)

Calculation of MgCl2 concentration (M) for the graph of activity (mol/min) vs MgCl2
concentration (M)

Tube
1
15 mM MgCl2 (mL)
MgCl2
concentration
(M)

2
0.1

3
0.2

4
0.4

5
0.6

6
0.8

7
1.0

1.5

3.0

6.0

9.0

12.0

15.0

Table 3. Conversion of MgCl2 concentration from mL to M

Conversion of the concentration from mM was converted to M by:
Tube 2
15 m
Tube 3
15 m
Tube 4
15 m
Tube 5
15 m
Tube 6
15 m
Tube 7
15 m

MgCl2
(M)
1.5
3
6
9
12
15

mol
L

x 1x10-4 L x 1000 mol = 1.5 M

mol
L

x 2x10-4 L x 1000 mol = 3.0 M

mol
L

x 4x10-4 L x 1000 mol = 6.0 M

mol
L

x 6x10-4 L x 1000 mol = 9.0 M

mol
L

x 8x10-4 L x 1000 mol = 12.0 M

mol
L

x 1x10-3 L x 1000 mol = 15.0 M

concentration Enzymatic
(mol/min)
0.0101
0.0131
0.0136
0.0149
0.0143
0.0151

activity

Table 4.MgCl2 concentration (M) vs Enzymatic Activity (ol/min).

Graph of Enzymatic Activity (mol/min) vs of Magnesium Chloride concentration (M)

0.02
0.01
0.01

f(x) = 0x + 0.01
R = 0.68

0.01

Enzymatic Activity (mol/min) 0.01

0.01
0
0
0
0 2 4 6 8 10 12 14 16

Magnesium chloride concentration (M)

Graph 8. Enzymatic Activity (mol/min) vsof Magnesium Chloride concentration (M)

C) Effect of pH on the reaction

Tubes with Absorbance Value

Time (minute)
0
0.5
1.0
1.5
2.0
2.5
3.0

1
0.749
0.755
0.760
0.765
0.773
0.779
0.785

2
0.512
0.513
0.515
0.516
0.519
0.522
0.526

3
0.693
1.013
1.024
1.027
1.030
1.036
1.042

4
0.898
0.798
0.792
0.795
0.801
0.809
0.813

5
0.900
0.899
0.910
0.922
0.936
0.950
0.964

Table 1. Absorbance recorded in 30 seconds time interval with different pH of Tris-HCl

Calculation of Tris-HCl concentration
Concentration =

Absorbance
slope

Slope, m = 0.7371
Example:
Tube 1, time = 0 minute
Concentration of Tris-HCl =

0.749
0.7371

= 1.016 mol

Tube
Time (min)
0
0.5
1.0
1.5

Concentration of Tris-HCl at different pH

(mol)
1 (pH
7)
1.016
1.024
1.031
1.038

2
(pH
7.5)
0.695
0.696
0.699
0.700

3 (pH 8)

4 (pH 8.5)

5 (pH 9.0)

0.940
1.374
1.389
1.393

1.218
1.083
1.074
1.079

1.221
1.220
1.235
1.251

2.0
2.5
3.0

1.049
1.057
1.065

0.704
0.708
0.714

1.397
1.406
1.414

1.087
1.098
1.103

1.270
1.289
1.308

Table 2. Concentration of Tris-HCl at 30 seconds time interval with different pH

[Tris-HCl] vs Time (Minutes) at pH 7.0

1.07
1.06 f(x) = 0.02x + 1.02
1.05 R = 1
1.04
[Tris-HCl] (mol)

1.03
1.02
1.01
1
0.99
0

0.5

1.5

Timr (Minutes)

2.5

3.5

[Tris-HCl] vs Time (Minutes) at pH 7.5

0.72
0.72
0.71 f(x) = 0.01x + 0.69
R = 0.94
0.71

Concentration of Tris-HCl (mol)

0.7
0.7
0.69
0.69
0

0.5

1.5

2.5

3.5

Time (minute)

[Tris-HCl] vs Time (Minutes) at pH 8.0

1.6
1.4 f(x) = 0.11x + 1.17
R = 0.45
1.2
1
Concentration of Tris-HCl (mol)

0.8
0.6
0.4
0.2
0
0

0.5

1.5

Time (Minute)

2.5

3.5

[Tris-HCl] vs Time (Minutes) at pH 8.5

1.25
1.2
1.15

Concentration of Tris-HCl (mol)

f(x) = - 0.02x + 1.14

1.1 R = 0.21
1.05
1
0

0.5

1.5

2.5

3.5

Time (Minutes)

[Tris-HCl] vs Time (Minutes) at pH 9.0

1.32
1.3

f(x) = 0.03x + 1.21

1.28 R = 0.96
1.26

Concentration of Tris-HCl (mol) 1.24

1.22
1.2
1.18
1.16
0

0.5

1.5

Time (minutes)

Tris-HCl pH
7.0
7.5
8.0
8.5

Enzyme activity (mol/min)

0.0165
0.0061
0.1067
-0.0216

2.5

3.5

9.0

0.0310

Table of enzyme activity vs pH of the buffer

Enzyme activity (mol/min)

0.12
0.1
0.08
0.06

Enzyme activity (mol/min)

0.04
0.02
0
6.50
-0.02

7.00

7.50

8.00

8.50

9.00

9.50

-0.04

pH

Discussion
A standard curve is just a plot of two different parameters and the curve reveals the relationship
between the two parameters. Under alkaline conditions, the p-nitrophenolate anion absorbs light
at 400-450 nm. The amount of enzyme present is therefore determined by measuring the
amount of p-nitrophenolate anion produced in the reaction. However to make this estimation a
standard curve must be prepared so that the amount of yellow-orange colour can be translated
into the amount of p-nitrophenol produced.
The equation for this reaction is
p-nitrophenyl phosphate + H2O p-nitrophenol

+ H3PO4
As shown by the graph in result section A , it indicates that the rate of absorbances is linearly
proportional to against amount of p-nitrophenol in mol. And the equation for this graph is Y =
0.7321X.

Enzymes are proteins that act as biological catalysts that are either essential for a reaction to
occur or may increase the speed of the reaction. Some enzyme require the presence of either a
cofactor or a coenzyme to catalyse the reaction. (Brennan, n.d.)
The specific function of the cofactor may vary according to the enzyme. Each enzyme has its
respective sequence of reaction steps which the cofactor play a role in (Brennan, n.d.).
Cofactors are required by the enzymes to facilitate the electrons transfer needed in the
formation and breaking of bonds in the reaction mechanism. (JAKUBOWSKI, 2014)
Divalent cations are cations that have a charge of +2. Magnesium ion, Mg2+ is a divalent cation.
It is a cofactor to alkaline phosphathase. A cofactor is a non-protein molecule or ion required by
the enzyme when underdoing its enzyme activity.
Throughout the experiment the concentration of enzyme, which is alkaline phosphotase, and of
the concentration of the cofactor in the form of magnesium chloride, MgCl 2. Tube 1 acted as a
control to observe the enzymatic activity of phosphotase without the cofactor. Based on Graph
2, the lowest among all the tubes. This indicates that the phosphotase is capable of hydrolyzing
p-nitrophenyl phosphate to p-nitrophenol without the presence of Mg2+. However, the presence
of the cofactor does yield a much higher enzyme activity.

The concentration of MgCl2 increases from Tube 2 7. This will help in identifying the optimal
concentration from maximum product formation and also the effect of the concentration on
enzyme activity when too high, if any. Based on the Enzymatic activity (mol/min) vs
Magnesium Chloride concentration (M) graph it can be clearly seen that when the
concentration of MgCl2 increases, the enzyme activity increases. It can also be deduced that the
optimum concentration of MgCl2 for maximum enzyme activity is higher than 15 mol, which is
the highest concentration tested with ( Tube 7 ).

Binding of the substrate to the active site of an enzyme involves interaction with reactive groups
provided by the side-chains of amino acids at the binding site. The pH of the incubation medium
may affect the ionisation of both the substrate and the amino acid side-chains and will therefore
this will affect binding. It may also affect the ionisation of reactive groups that catalyse the
reaction, although in the micro-environment of the catalytic site, when it is occupied by the
substrate, this is less likely.
Extreme values of pH may also disrupt the tertiary structure of the enzyme, and so distort the
active site, or even denature the enzyme protein. As its name indicates, alkaline phosphatase is
highly pH-sensitive. In the effective buffering range for Tris-HCl, the initial velocity of the
hydrolysis of substrate by alkaline phosphatases hould increases more than 6-fold from pH 7.0
to pH 9.0.
In table 2, it shows that the concentration of Tris-HCl increases with the pH value. However
there is drop in concentration value at 0.5min and 1.0 min on pH of 8.5 and at 0.5min on pH of
9. Even the graphs show concentration of Tris-HCl increases linearly except for graph 5 (pH of
8.5) shows inverse linearly proportional graph.The values of the Km and V max to be affected by
the difference in pH value.

References
1. JAKUBOWSKI, (2014). Chapter 7C - Cofactors and Electron Pushing. [online]
Employees.csbsju.edu.

Available

http://employees.csbsju.edu/hjakubowski/classes/ch331/catalysis/olelectronpush.htm
[Accessed 22 Jun. 2014].

at:

2. Brennan, J. (n.d.). How Would the Lack of a Cofactor for an Enzyme Affect the
Enzyme's Function? | The Classroom | Synonym. [online] Synonym. Available at:
http://classroom.synonym.com/would-lack-cofactor-enzyme-affect-enzymes-function7502.html [Accessed 22 Jun. 2014]