(Lára, n.d.
BIOCHEMISTRY
BIO462
LABORATORY PRACTICAL
EXPERIMENT 3: ENZYMOLOGY
LECTURER: DR. NUR MAISARAH SARIZAN
GROUP: RAS2012B
GROUP MEMBERS:
NO. NAME STUDENT ID
1. ADDINA BINTI AHMAD HAZRIZAL 2023680198
2. NUR AMIRA FATINI BINTI AHMAD FATHILLAH 2023696428
3. NUR HANIS BINTI SHUHAIMI 2023415484
4. NUR SYAHIRA IZWANI BINTI FAUZI 2023239906
5. NURUL NAJWA BINTI NAZRI 2023423622
INTRODUCTION
OBJECTIVES
MATERIALS
LAB ACTIVITY 1: CALIBRATION AND SETTINGS FOR SPECTROPHOTOMETER
Spectrophotometer
Calibration cuvette
Starch in dropper bottle
Distilled water
Parafilm squares
LAB ACTIVITY 2: THE EFFECT OF ENZYME CONCENTRATION ON THE RATE OF
REACTION
Spectrophotometer
4 cuvettes
Starch in dropper bottle
Amylase
Dropper (for enzyme mixture)
Distilled water
Marking pen
Plastic ruler
Parafilm squares
Data collection sheet
LAB ACTIVITY 3: THE EFFECT OF SUBSTRATE CONCENTRATION ON THE OF
REACTION
Spectrophotometer
7 cuvettes
Starch in dropper bottle
Amylase
Dropper
Distilled water
1
Marking pen
Plastic ruler
Parafilm square
Data collection sheet
LAB ACTICITY 4: THE EFFECT OF PH ON ENZYME ACTIVITY
Spectrophotometer
6 cuvettes
pH buffers 3, 5, 7, 9 and 11
Starch in dropper bottle
Amylase
Dropper
Distilled water
Marking pen
Parafilm squares
Spreadsheet
PROCEDURES
LAB ACTIVITY 1: CALIBRATION AND SETTINGS FOR SPECTROPHOTOMETER
1. The spectrophotometer has been turned on and let warmed for 5 minutes.
2. The calibration cuvette has been prepared:
a) The cuvette has been marked at the 2 cm level (from the bottom of the
cuvette it has been measured). The mark has been placed on the side of the
cuvette so it will not interfere with the light beam of the spectrophotometer.
b) The distilled water has been added up to the 2 cm level.
c) 10 drops of starch have been added, the parafilm has been placed over the
opening of the cuvette and it has been held with the finger. The contents have
been mixed with the contents by inverting the tube several times.
3. The spectrophotometer has set to 410 nm.
4. The spectrophotometer has been set to Absorbance (A).
5. The chamber cover has been opened.
6. The calibration cuvette has been inserted into the holder.
7. The chamber cover has been closed.
8. The absorbance has been read and recorded.
9. The calibration cuvette and its contents has been saved.
2
LAB ACTIVITY 2: THE EFFECT OF ENZYME CONCENTRATION ON THE RATE OF
REACTION
1. The cuvettes were numbered as 1, 2, 3 and 4. The numbers was written at the top of
the cuvette. Do not wrote them very large as they may interfere with the light beam of
spectrophotometer.
2. All the 4 cuvette was measured up to 2 cm from the bottom by using the plastic ruler
and the mark was placed with the marking pen.
3. All four cuvette were filled with distilled water to 2 cm mark.
4. The cuvette contents were prepared by aerate the tubes every minute for 5 minutes
by covering the cuvette with parafilm and shaking them.
a) 5 drops of enzyme were added in cuvette 2, 15 drops were added in cuvette 3
and 45 drops of enzyme in cuvette 4.
b) 0 drops of starch were added to each of the 4 tubes.
c) The cuvette was let to set for 5 minutes.
5. Cuvette 1 was placed in the spectrophotometer at the end of 5 minutes and the
absorbance was measured with the previously calibrated spectrophotometer.
6. The step 5 was repeated for cuvette 2, 3 and 4.
7. The results were recorded.
8. The cuvette’s content was empty into waste container provided.
9. The cuvette was rinse out and dried them. They can be used in subsequent lab
activities for this lab.
LAB ACTIVITY 3: THE EFFECT OF SUBSTRATE CONCENTRATION ON THE OF
REACTION
1. Seven cuvettes were numbered as 1, 2, 3, 4, 5, 6 and 7.
2. By using the plastic ruler, 2 cm have been measured from the bottom of the cuvette
and mark have been placed by using the marking pen.
3. Each of the cuvette have been filled to the 2 cm mark with the distilled water.
4. Preparation of cuvette contents: NOTE: the cuvettes has been aerated every minute
over 10 minutes incubation by covering the cuvettes with parafilm and the cuvettes
was shaken.
a) 1 drop of starch was added to cuvette 1, 3 drops of starch were added to
cuvette 2, 5 drops of starch were added to cuvette 3, 10 drops of starch were
added to cuvette 4, 20 drops of starch were added to cuvette 5, 40 drops of
starch were added to cuvette 6 and 60 drops of starch were added to cuvette
3
7. NOTE: cuvettes 6 and 7 were poured into a test tube for the incubation
3
time and then poured back into a cuvette by filling it to approximately full.
4
b) 10 drops of enzymes were added to each 7 cuvettes.
c) The cuvettes have been let set for 10 minutes.
5. At the end of 10 minutes, cuvette 1 was placed in the spectrophotometer and the
absorbance were measured.
6. Step 5 were repeated with cuvette 2, 3, 4, 5, 6 and 7.
7. The result was recorded.
8. The cuvette contents were emptied into waste container provided. The cuvettes were
then rinsed and dried.
LAB ACTICITY 4: THE EFFECT OF PH ON ENZYME ACTIVITY
1. Six cuvettes have been numbered as 1,2,3,4,5, and 6.
2. Using the small plastic, 2 cm from the bottom of the cuvette has been measured up
and a mark has been placed with the marking pen.
3. Preparation of cuvette contents: NOTE: the cuvettes has been aerated every minutes
over 5 minutes by covering the cuvettes with parafilm and was shaken them.
a) Cuvette 1 has been filled with up to 2 cm mark with pH3 buffer solution and
10 drops of enzyme.
b) Cuvette 2 has been filled with up to 2 cm mark with pH5 buffer solution and
10 drops of enzyme.
c) Cuvette 3 has been filled with up to 2 cm mark with pH7 buffer solution and
10 drops of enzyme.
d) Cuvette 4 has been filled with up to 2 cm mark with pH9 buffer solution and
10 drops of enzyme.
e) Cuvette 5 has been filled with up to 2 cm mark with pH11 buffer solution and
10 drops of enzyme.
f) Cuvette 6 has been filled with up to 2 cm mark with distilled water and 10
drops of enzyme.
g) 10 drops of starch have been added to cuvettes 1, 2, 3, 4, 5 and 6
respectively.
h) The cuvettes have been let stood for 5 minutes.
4. At the end of 5 minutes the cuvette 1 has been placed in the spectrophotometer and
its absorbance has been measured with the previously calibrated spectrophotometer.
5. Step 4 has been repeated with cuvettes 2, 3, 4, 5 and 6.
6. The results have been recorded on the data collection sheet and spreadsheet.
4
7. The cuvette contents have been emptied into the waste container provided.
8. The cuvettes have been rinsed out and dried. They can be used in subsequent lab
activities for this lab.
RESULTS
a. Enzyme concentration
OD OD OD
Cuvette Mean ± S.D
(replicate 1) (replicate 2) (replicate 3)
1 0.070 0.071 0.073 0.071 ± 0.0153
2 0.222 0.222 0.222 0.222 ± 0
3 0.521 0.525 0.524 0.523 ± 0.0021
4 1.024 1.025 1.025 1.025 ± 0.0005
b. Substrate concentration
Cuvette OD OD OD Mean ± S.D
(replicate 1) (replicate 1) (replicate 1)
1 0.355 0.355 0.354 0.355 ± 0.001
2 0.372 0.370 0.370 0.371 ± 0.001
3 0.436 0.435 0.435 0.435 ± 0.001
4 0.370 0.368 0.366 0.368 ± 0.002
5 0.335 0.336 0.336 0.336 ± 0.001
6 0.280 0.286 0.287 0.284 ± 0.004
7 0.259 0.258 0.260 0.259 ± 0.001
c. pH
OD OD OD
Cuvette Mean ± S.D
(replicate 1) (replicate 2) (replicate 3)
1 0.444 0.447 0.446 0.446
2 0.368 0.365 0.365 0.366
5
3 0.470 0.467 0.467 0.468
4 0.408 0.403 0.404 0.405
5 0.389 0.390 0.389 0.389
6 0.374 0.375 0.376 0.375
GRAPH
a. Enzyme concentration
The Effect of Enzyme Concentration
on the Rate of Reaction
1.2
1 f(x) = 0.024210989010989 x
R² = 0.967400500642574
Absorbance
0.8
0.6
0.4
0.2
0
0 5 10 15 20 25 30 35 40 45 50
Eznyme Concentration (drops)
b. Substrate concentration
6
The Effect of Substrate Concentration on the
Rate of Reaction
0.500
0.450
0.400 f(x) = 0.00714036617262424 x
Absorbance (410 nm)
0.350 R² = 0.344516487237925
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0 10 20 30 40 50 60 70
Substrate Concentration (Drops)
c. pH
DISCUSSION
Spectrophotometers are important instruments for researchers to analyse a wide
variety of substances and components. The detector at the bottom of the device calculates
the absorbance and transmittance values of the sample. The detector will generate the
absorbance and plot the calibration curve. In this experiment, the analyser is calibrated to
zero, then adjusted to 410 nm. The analyser is used to determine the reaction rate of the
enzyme and the substrate concentration and to determine how pH affects enzyme activity.
Enzymes play a critical role in life, catalysing breakdowns, and metabolic processes. This
experiment utilizes a starch solution as the substrate and amylase as the enzyme. Amylase,
an enzyme present in human saliva, breaks down starch into simple sugars.
Firstly, the effect of enzyme concentration on the rate of reaction have resulted in a
continuously increase straight linear graph that’s shows as the enzyme concentration
increases, the rate of reaction increases as well. The graph is linear, demonstrating that
enzyme-substrate complexes form when substrate concentrations rise. According to (Lára,
n.d.), as long as there is enough substrate available, the initial rate of reaction increases
linearly with enzyme concentration. If the amount of substrate is limited, any further increase
in enzyme concentration will not enhance the reaction rate, because the amount of substrate
becomes a limiting factor (Lára, n.d.).
7
Secondly, for the effect of substrate concentration on the rate of reaction experiment
presented in a plotted graph have resulted to a linear graph, however it was supposedly be a
graph with hyperbolic curve which indicates that the reading of absorbance increases as
substrate concentration was added before it reaches a limiting rate.
According to study of (The effect of substrate concentration on enzyme activity, n.d.),
an enzyme-catalysed reaction typically has a hyperbolic relationship between the rate of
reaction and the concentration of substrate. When referring to linear graph we have
identified, at low substrate concentration, the rate of reaction increases exponentially as
substrate concentration increases. For most of the time, the enzyme’s catalytic site is
completely empty, waiting for the substrate to bind. This means that the rate of product
formation is limited by the available substrate concentration. Furthermore, as substrate
concentration increases, the enzyme saturates the catalytic site. As soon as more substrate
is available, it readily binds to the substrate and undergoes reaction. However, this graph
continuously increases indicates rate of reaction keep increasing which is contradictory to
hyperbolic curve graph because when more substrates was added, it will not change the rate
of reaction to any appreciable degree.
For the effect of pH on enzyme activity experiment, its graph shows an enzyme
activity is highest at the optimal pH which is at pH 7 and decreases when the pH exceeds or
falls below the optimal level. This is because at an extreme pH level, the enzyme's structure
changes, and it no longer complements to its substrate. Denaturation is a term used to
signify an impact that is permanent and irreversible (Proteins: Effect of pH on enzyme
activity, n.d.). According to (Effect of pH on Enzymatic Reaction, n.d.), the enzyme's
structure greatly influences its activity. In other words, changes in the structure of the
enzyme influence the pace of chemical reactions. When the pH of the reaction
medium changes, the enzyme's shape, and structure change.
There are some errors that we have identified from this experiment that may have
affected the accuracy of our results especially when handling materials. Moreover, this
experiment utilized drops to quantify concentration, resulting in imprecise measurements.
Using a volume unit, such as millimetres, would have provided more precise findings. In
addition, using an unclean cuvette has introduce random errors and interfere with the
concentration of enzymes or substrates supplied. Systematic errors can also happen when
there is incorrect calibration was performed on the spectrophotometer. To improve, we
suggest repeating the results and graphs to ensure proper depiction.
8
CONCLUSION
References
Lára. (n.d.). Rate: Enzyme Concentration (CIE A Level Biology). Retrieved 2 MAY,
2024, from SAVE MY EXAMS:
https://www.savemyexams.com/a-level/biology/cie/22/revision-notes/3-
enzymes/3-2-factors-that-affect-enzyme-action/3-2-3-rate-enzyme-
concentration/
Proteins: Effect of pH on enzyme activity. (n.d.). Retrieved 2 MAY, 2024, from
BITESIZE: https://www.bbc.co.uk/bitesize/guides/zcr74qt/revision/5
The effect of substrate concentration on enzyme activity. (n.d.). Retrieved 2 MAY,
2024, from UCL-London's Global University:
https://www.ucl.ac.uk/~ucbcdab/enzass/substrate.htm
