PRACTICAL REPORT

Practical 3 : Factors Affecting

Enzyme Activities

Group Members :

Name Matrics Number

NURUL ALIA SHAFYKA BINTI REDZOAN A188275
NUR AYESHAH AMEERA BINTI NORAZILEE A188766
ARYSSA BINTI AZRI A189578
 SHRRUTI A/P SURESH KUMAR A189624

Date : 3/11/2021
Lecturer : PROF. MADYA DR. GOON JO AAN​

A. CALIBRATION CURVE

Reagents

1. 50 µM of p-nitrophenol
2. 0.2 M of sodium hydroxide (NaOH)

Method

1. Prepare the test tubes as follows:

Test tube (number) Reagent 1 2 3 4
m
blank
@
50 µM of p-nitrophenol (ml) 0 2.0 4.0 6.0 8.0

Distilled water (ml) 8.4 6.4 4.4 2.4 0.4

0.2 M of NaOH (ml) 1.0 1.0 1.0 1.0 1.0

2. Mix the content of the test tubes.

3. Measure and record the absorbance at 410 nm.

Test Amount (ml) of 50 µM Amount of p- OD (410 Actual

Tube p-nitrophenol used nitrophenol (µmol) nm) OD
Reagent 0.00 0.00 0.008 0.000
blank
1 2.00 0.10 0.198 0.190
2 4.00 0.20 0.386 0.378
3 6.00 0.30 0.572 0.564
4 8.00 0.40 0.761 0.753
4. Plot a calibration curve from the obtained data.

​
B. EFFECT OF pH ON THE ACTIVITY OF ALKALINE
PHOSPHATASE Reagents
1. 0.1 M of pH 4, pH 6, pH 9 and pH 13 glycine buffer solution
2. 5 mM p-nitrophenyl phosphate (substrate)
3. Enzyme solution: 0.1 mg/ml alkaline phosphatase
4. 0.025 M of sodium hydroxide (NaOH)

Method

1. Prepare test tubes with the following solutions:

Test tube (number) Reagent 1 2 3 4
blank (pH 9) (pH (pH (pH
4) 6) (pH 9) 13)
Distilled water (ml) 0.2 - - - -

Glycine buffer solution 1.0 1.0 1.0 1.0 1.0

(ml)
5 mM of p-nitrophenyl 0.2 0.2 0.2 0.2 0.2
phosphate (ml)
(substrate)

2. Incubate each test tube in water bath (37°C) for 5 minutes.

3. EXCEPT for the REAGENT BLANK tube, add 0.2 ml alkaline phosphatase (0.1
mg/ml) and mix the solution in each test tube. Incubate all test tubes including the
reagent blank in 37°C waterbath for 15 minutes.

4. After 15 minutes, add 8 ml of 0.025 M NaOH to all test tubes including the
reagent blank.

5. Measure and record the absorbance of each test tube at 410 nm.
Test OD Actual Amount of p- Enzyme activity (m
tube (410nm) OD Nitrophenol (m mol) mol/min)
Control 0.030 0.000 0.000 0.0000
1 (pH4) 0.039 0.009 0.005 0.0003
2 (pH6) 0.046 0.016 0.006 0.0004
3 (pH9) 0.283 0.253 0.132 0.0009
4 0.034 0.004 0.002 0.0001
(pH13)

6. By referring to the calibration curve prepared in Part A, determine the amount

of p-nitrophenol (µmol) and calculate the enzyme activity in µmol/min.
7. Plot a graph of alkaline phosphatase activity versus pH.

C. INFLUENCE OF TEMPERATURE ON THE ACTIVITY OF ALKALINE

PHOSPHATASE

Reagents

1. 0.1 M of pH 9 glycine buffer solution

2. 0
5 mM p-nitrophenyl phosphate (substrate)
3. Enzyme solution: 0.1 mg/ml alkaline phosphatase
4. 0.025 M sodium hydroxide (NaOH)
Method

1. Prepare the test tubes as shown in the table below.

Test tube Reagent 1 2 3 4
blank (5°C) (Room (37°C) (70°C)
(37°C) temperature
29°C)
Distilled water (ml) 0.2 - - - -

5 mM of p-nitrophenyl 0.2 0.2 0.2 0.2 0.2

phosphate (substrate)
(ml)
Glycine buffer 0.5 0.5 0.5 0.5 0.5
solution (ml)

2. Shake each test tube and incubate for 5 minutes at the designated temperature.

3. EXCEPT for the REAGENT BLANK tube, add 0.2 ml alkaline phosphatase (0.1
mg/ml) and mix the solution in each test tube. Incubate all test tubes including the
reagent blank for 15 minutes at the designated temperature.

4. After 15 minutes, add 8 ml of 0.025 M NaOH to all test tubes including the
reagent blank.

5. Measure and record the absorbance of each test tube at 410 nm.

6. By referring to the calibration curve prepared in Part A, determine the amount

of p nitrophenol (µmol) and calculate the enzyme activity in µmol/min.

Test tube OD Actual OD Amount of p- Enzyme activity

(410nm) Nitrophenol (m mol) (m mol/min)
Control 0.036 0.000 0.000 0.0000
1 (5) 0.149 0.113 0.057 0.0038
2 (29) 0.202 0.166 0.085 0.0057
3 (37) 0.250 0.214 0.112 0.0075
4 (70) 0.0.092 0.056 0.030 0.0020
7. Plot a graph of alkaline phosphatase activity versus temperature.

Questions:
1. Explain how changes in pH affects the activity of enzymes by describing the
structural modifications that may occur.
If the pH decreases in the blood, the concentration of hydrogen ions (increases
making the environment more acidic. The structure of enzymes are held by ionic
bonds and hydrogen bonds where the bonds are attracted to the oppositely charged
groups on the amino acid side chains. When there are excessive amounts of , it will
cause the negatively charged amino acids to be protonated; accepting the proton.
The protonation causes disruption to the ionic bonds and hydrogen bonds formation
that holds the tertiary structure of the enzyme in shape. This will cause the enzyme
to denature especially it’s binding side making it hard for it to bind with substrates
and subsequently lower the reaction velocity.
¥
If the pH increases in the blood, the concentration of decreases but the
concentration of hydroxide ions () increases. As mentioned on the influence of ionic
bonds and hydrogen bonds on an enzyme’s shape, the excessive amounts of in the
environment will cause the positively charged amino acids to be deprotonated;
removal of a proton. This disrupts the formation of the ionic bonds and hydrogen
bonds holding the enzyme together. This change in ionization charge alters the
enzyme’s normal conformation therefore the active site loses its ability to bind with
substrates thus lowering enzyme activity.

2. Explain optimum pH using the enzymes given in the graph and reasons for the
differences seen.
Optimum pH is the pH at which the rate of enzymatic reaction is the highest.
Different enzymes have a different optimum pH because each enzyme is functional
at different locations in the human body.

For example, acid phosphatase is most effective in an acidic environment with pH

4.5. The action of acid phosphatase will stop when in high pH as it may be
 still do in
exp as -
ve control
/
denatured. Alkaline phosphatase is most effective in an alkaline environment with pH
0
9.0 and will start to denature when in low pH. Salivary amylase lives in a neutral pH
environment which is 6.5, thus it does not need to adapt to a lower or higher pH
level. The action of salivary amylase will stop when the food passes into the stomach
because of the low pH of gastric juice.
3. Explain how changes in temperature affects the activity of enzymes by describing
the structural modifications that may occur.
At low temperature, an enzyme-catalysed reaction rate is slow because the
substrate molecules are moving at a slow rate. An increase in temperature will
increase the enzymatic activity due to the increase in kinetic energy and velocity.
Increasing velocity will lessen the time between collisions which will increase the ↑
amount of molecules reaching the activation energy thus increasing the rate of temp
reaction. kinetic energy which in turn increases collision rate between molecules. ↓
This will also increase the reaction velocity. ↑ collision (↑⑤t8⑥
rate

At high temperatures that exceed the optimum temperature, the rate will decrease ↓ time
because the enzymes will start to break off and become denatured, thus losing their between
shape and function. The 3D structure of enzymes will be altered and the substrates collision
can no longer fit into the active site of the enzymes ↓
↑ amtofmol
4. What is the optimum temperature of enzymes in the human body? reach actuation
energy
.

37 °C ↓
↑ ROR

5. What happens to enzymes in the body during a high fever?

During a high fever, body temperature is above 37°C which exceeds the optimum
temperature of enzymes in the body. So, the rate of enzyme activity will decrease
and denaturation of enzymes occurs . This is due to the enzyme starting to break off
because of the disruption of hydrogen bonds which hold the enzymes . Because of
this, there is conformational change in the active site and substrates can no longer fit
into the active site and bind to it. These are the reasons for the decrease in enzyme
activity during a high fever.