Lab Report Enzyme Kinetics ( Practical 1 Biochemistry)

FAKULTI FARMASI,
UNIVERSITI SULTAN ZAINAL ABIDIN,

BESUT, TERENGGANU

LABORATORY REPORT
PHM10202 : BIOCHEMISTRY

Name & Matric No. of : Kong Yi Meng (BHFL22073615)
Group Members Ching Shan Yi (BHFL22072187)
: Dr. Che Ku Dahlan Bin Che Ku Daud
Name of Lecturers
: 7/11/2022
Date of Practical : Enzyme Kinetics
Title of Experiment

INTRODUCTION

Enzymes are protein molecules that act as biological catalysts by increasing the
rate of reactions without changing the overall process. With the exception of some
RNA molecules, all enzymes are globular proteins with tertiary structure. Thereby,
there is a region of an enzyme where substrate molecules bind and undergo a
chemical reaction. The active site consists of amino acid residues that form
temporary bonds with the substrate (binding site) and residues that catalyze a
reaction of that substrate (catalytic site). Enzymes are very specific in their action
as particular enzymes only act on particular substrates. Basically, enzymatic
reaction starts when a substrate enters the active site of an enzyme forming a
temporary molecule, known as enzyme-substrate complex, the reaction then
occurs, converting the substrate into products. The products then leave the active
site of the enzyme and the enzyme activity will be reversed. The mechanisms of
enzyme action are based on Lock and Key model and Induced-Fit model.

The central approach for studying the mechanism of an enzyme-catalyzed reaction
is to determine the rate of the reaction and its changes in response with the changes
in parameters such as pH and temperature. This is known as enzyme kinetics. In
this experiment, a glucose standard graph is prepared to study the relationship
between glucose concentration and absorbance (optical density) while the effects
of pH and temperature on enzyme activity are investigated by finding out the
optimum pH and temperature. Based on the theories, the changes in pH and
temperature (extreme values) will lead to denaturation of the enzyme. For instance,
if the enzyme is exposed to severe changes in temperature and pH, the tertiary
structure of enzyme will change and the enzyme will no longer be compatible with
the substrate. Hence, the enzymatic reaction will be decreased.

The enzyme selected in this experiment to show the principles of enzyme kinetics
is sucrase, known as invertase (β-D-fructofuranosidase, fructohydrolase, E.C.
3.2.1.26). This enzyme catalyzes the hydrolysis of sucrose to glucose and fructose.
In this case, glucose and fructose are reducing sugars, while sucrose is not. In this
experiment, enzyme activity can be measured by estimating the total glucose and
fructose produced using Somogyi-Nelson method.

OBJECTIVES
To determine the effect of pH and temperature on enzyme activity.

APPARATUS
Test tube
Beaker
Hot plate
Pipettes
Test tube rack
Label paper
Marker pen
Cuvettes
Spectrophotometer
Pipette filler
Micropipette
Water bath
Thermometer
Ice

REAGENTS
Sucrose solution
Buffer
Enzyme Invertase (Sucrase)
Distilled water

Reagent A
24g/L Na2CO3
12g/L K Na tartarat
16g/L NaHCO3
144g/L Na2SO4

Reagent B
4g/L CuSO4.5H2O
36g/L Na2SO4

*On the day of the experiment, 4 parts of Reagent A are mixed with 1 part of
Reagent B. This reagent is labeled as A+B reagent.

Arsenomolybdate (Ammonium molybdate, Conc.H2SO4, Na2A5SO4.7H2O)
*25g of ammonium molybdate is dissolved in 450 ml of distilled water and 21 ml
of concentrated H2SO4.. 3g of Na2A5SO4.7H2O is dissolved in 25 ml of water. The
two solutions are mixed together.

PROCEDURE

A) Preparing A Glucose Standard Graph
1. A standard glucose solution (200µg/ml) is prepared and a series of test tubes
are prepared according to the following table.
2. The absorbance is read at 510 nm using test tube 1 as blank.
3. The standard curve from the information obtained (absorbance versus
amount of glucose) is drawn using Microsoft Excel.

Test Glucose Water Reagent Arsenomolybdate Water
Tube (ml) (ml) (A+B) (ml) (ml)

1 0 2.0 1.0 1.0 6.0
2 0.2 1.8
3 0.4 1.6 1.0 Boiled 1.0 6.0
4 0.6 1.4 1.0 for 20 1.0 6.0
5 0.8 1.2 1.0 6.0
6 1.0 1.0 minutes. 1.0 6.0
7 1.5 0.5 1.0 Cool.

1.0

1.0 1.0 6.0

1.0 1.0 6.0

Information Obtained:

Sample Reading 1 (ml) Reading 2 (ml) Reading 3 (ml) Average (ml)

10 0 00

2 0.0760 0.0780 0.0770 0.0770

3 0.2020 0.2030 0.2040 0.2030

4 0.3290 0.3260 0.3270 0.3273

5 0.3900 0.3900 0.3880 0.3893

6 0.4020 0.3960 0.3970 0.3983

7 0.4300 0.4290 0.4290 0.4293

Figure:

Graph:

Discussion:
The graph of absorbance versus amount of glucose obtained above shows a

relationship that when the amount of glucose (ml) increases, the value of
absorbance also increases. Glucose is a reducing sugar, when reducing sugar
(glucose) is heated with alkaline copper tartrate, the copper from the cupric state is
reduced into cuprous state, forming cuprous oxide. When the cuprous oxide is
treated with arsenomolybdic acid, molybdic acid is reduced into molybdenum blue,
giving the solution a blue colour. Hence, reducing sugars support molybdate
reduction. When more molybdenum blue is present in the solution, the blue colour
of the solution is darker. From the figure of the experiment obtained above, it can
be observed that the blue colour of the solution increases from test tube 1(lowest
amount of glucose) to test tube 5 (highest amount of glucose).

Absorbance value is measured using a spectrophotometer. It is a scientific
instrument that contains an adjustable light source which can produce light of
chosen wavelength (510 nm in this experiment) and it has a detector which can
pick up what proportion of the light that passed through the solutions in the test

tube. In this experiment, absorbance value is measured and is used as an instrument
that indicates the amount of glucose present in the solution. The higher the amount
of glucose present in the solution, the higher the amount of glucose that will absorb
the light of the selected wavelength (510 nm), the higher the value of absorbance
will be. Thus, it can be indicated and concluded that the higher the amount of
reducing sugar present in a given sample, the value of absorbance is higher. The
relationship between absorbance value and amount of glucose is determined.

B) The Effect of pH
The experiment is carried out to determine the rate of the reaction catalyzed by
sucrase measured at different pHs.

Procedure:
1. A series of test tubes as shown in Table 5 are prepared.
2. 4.0ml of sucrose solution is placed with a 2.0 ml buffer (different pHs). It is
shook slowly.
3. The test tubes are incubated in a water bath at 300C for 10 minutes.
4. After 10 minutes, 1.0 ml of enzyme is added to the test tubes that were
placed in the water bath.
5. The test tubes are then incubated in a water bath at 300C for another 10
minutes.
6. After 10 minutes, 2.0 ml of the reaction mixture from each test tube is
pipetted out and is placed inside new test tubes.
7. The concentration of reducing sugar is estimated using the Somogyi-Nelson
method in table 3.
7.1 1 ml of Reagent A+B is added into each test tube containing 2 ml of
sample.
7.2 The test tubes are boiled in a water bath for 20 minutes and left to cool.
7.3 1 ml of arsenomolybdate is added into each test tube followed by 6 ml of
distilled water. The test tubes are shook well.
7.4 The value of absorbance is read at 510 nm for each test tube. Test tube 1
is blank (used as a control value)
8. A graph of reducing sugar against pH is drawn. The optimum pH of this
enzyme is determined.

Table 5

Test tube 1(control/ 2 3 4 5
pH8
blank)
8.0
Sucrose 0ml 4 ml for each test tube

pH of buffer pH 5.0 pH5 pH6 pH7

(2.0ml)

Water 5ml 1.0 ml for each test tube

Incubate in a water bath at 300C for 10 minutes

Enzyme 1.0 ml for each test tube

Total (ml) 8.0 8.0 8.0 8.0

Table 3: Somogyi-Nelson Method

Test tube Sample A+B Arsenomol Water (ml)
Reagent -ybdate
(ml)
(ml)
1( control ) 2.0 ml Boiled for 1 6
2 2.0 ml 1 20 minutes. 1 6
3 2.0 ml 1 6
4 2.0 ml 1 Cool.
5 2.0 ml
1 16

1 16

1

Results: 1 2 3 4 5
5 5 6 7 8
Test Tube 0.0000 1.5974 1.7920 1.5860 1.6620
pH

Absorbance
Value

Figure:

Graph:

Discussion:
This experiment is carried out to study the effect of different pHs on the rate of

sucrase enzyme reaction, then the optimum pH of sucrase enzyme is determined. In
this experiment, 4 different pH values are selected to test the rate of sucrase
enzyme activity, namely pH 5, pH 6, pH 7 and pH 8. 4 ml of sucrose solution is
placed in each test tube, then 2 ml of buffer solution with different pHs are added
into each test tube respectively. Then, each test tube is incubated in the water bath
at 300C for 10 minutes before adding 1 ml of sucrase enzyme (invertase) into each
test tube. In this experiment, the temperature of the mixture and time taken for
incubation are held constant, whereas test tube 1 is fixed as the control instrument
in which sucrose solution is not added into it. With the presence of sucrase
enzyme, the sucrose solution will be catalyzed and broken down into glucose and
fructose which are both reducing sugar. The experiment is continued by carrying
out the Somogyi-Nelson method in order to estimate the concentration of reducing
sugar present in each test tube. In the Somogyi-Nelson method, the mixture in the
test tube reacted with Reagent A+B and arsenomolybdate and their respective

absorbance reading is taken by using the spectrophotometer at 510 nm. Then, a
graph of reducing sugar against pH is drawn, absorbance reading is used as the
instrument to estimate the concentration of reducing sugar for this experiment.

Based on the graph obtained, it can be observed that the graph is displayed in
a smooth bell curve shape from pH5 to pH7. The result of the experiment showed
that at pH5, the absorbance reading is 1.5974 Au, at pH6, the absorbance reading is
1.7920 Au. At pH7, the absorbance reading is 1.5860 Au whereas at pH8, the
absorbance reading rose back to 1.6620 Au. It can be seen that the absorbance
reading at pH 6 is the highest, indicating that the optimum pH of enzyme sucrase is
pH6. At optimum pH (pH6), the sucrase enzyme functioned best and the rate of
sucrase enzyme activity is maximum. The number of substrate (sucrose) binded
precisely to the active site of enzyme sucrase is the highest at optimum pH, the
number of enzyme-substrate complexes formed is the highest. Hence, the number
of products formed, which is the reducing sugar (glucose and fructose) is the
highest at pH6.

On the other hand, at pH 5, the enzyme activity is low because pH 5 is not the
optimum pH for sucrase enzyme, sucrase enzyme undergoes alteration in structure
either by additional or breakage of existing bonds. Ultimately, the chemical
makeup of the sucrase enzyme changed which caused the active site to change,
hence the substrate (sucrose) can no longer fit into the active site of sucrase
enzyme. The sucrase enzyme activity at pH 5 is inactive, the concentration of
enzyme-substrate complexes formed is low, concentration of products (glucose and
fructose) is low, hence the absorbance value is low. However, the alteration of
sucrase enzyme is reversible as when the pH value increased from pH 5 towards
pH 6, the sucrase enzyme activity increased. At pH 7, the absorbance reading
dropped, indicating the sucrase enzyme activity decreased. This is because pH 7 is
an extreme pH for sucrase enzyme, it undergo denaturation process irreversibly.
Therefore, the substrate (sucrose) cannot bind to the active site of sucrase enzyme,
enzyme-substrate complexes cannot be formed, products (glucose and fructose)
cannot be formed, absorbance value is low.

When the pH increased from pH 7 to pH 8 , the absorbance value increased.
Some errors might occurred in the experiment which caused this result to be
inaccurate because theoretically the absorbance value should decrease when pH
increased from 7 to 8. Errors that might have occurred during the experiment
included the surfaces of the disposable cuvettes are not cleaned properly using
tissue papers because any contaminants on the surface of cuvette will block the
light rays of spectrophotometer from passing through the solution in the cuvettes.

Hence, the absorbance reading taken is inaccurate. Besides, error might occurred
when the level of eyes are not directly perpendicular to the bottom of the concave
meniscus on the scale of the pipette, hence the volume of solutions taken might be
inaccurate. Theoretically, the optimum pH of the sucrase enzyme (invertase) is pH
5.8, the optimum pH obtained from the experiment is pH 6, hence the result is
considered successful.

C) The Effect Of Temperature

Procedure:
1. Five test tubes are prepared according to the table below.

Test Tube 1(Control) 2 3 4 5
4 ml 4 ml 4 ml 4 ml
Sucrose 0 ml 2 ml 2 ml 2 ml 2 ml
1 ml 1 ml 1 ml 1 ml
Buffer, pH 5.0 2 ml 0°C 30°C 45°C 65°C

Water 5 ml 1 ml 1 ml 1 ml 1 ml
8 ml 8 ml 8 ml 8 ml
Incubation Room 0°C 30°C 45°C 65°C

temperature Temperature

for 10 minutes

Enzyme 1 ml

Total volume 8 ml

Incubation Room

temperature temperature

for 10 minutes

2. After 10 minutes, 2.0 ml of the reaction mixture is pipetted out from each
test tube and is placed inside new test tubes.

3. The concentration of reducing sugar is estimated by using Somogyi-Nelson
method.
3.1 1.0 mL of Reagent ( A+B ) is added to each test tube.
3.2 The test tubes are boiled in a water bath for 20 minutes and are cooled.
3.3 1.0 ml of arsenomolybdate reagent is added to each test tube and mixed
well.
3.4 6.0 ml of distilled water is added to each test tube and mixed well. Each

test tube is left for 5 minutes.
3.5 The absorbance at 510 nm is read. Test tube 1 is used as blank.

Somogyi- Nelson method

Test Tube Sample A+B Arseno- Water (ml)
Reagent Molybdate

(ml) (ml)

1(Control) 2.0 ml 1 Boil for 1 6
2 2.0 ml 20 1 6
3 2.0 ml 1 1 6
4 2.0 ml minutes.
5 2.0 ml 1 Cool.

1 16

1 16

4. The graph of reducing sugar against temperature is drawn. The optimum
temperature of this enzyme is determined.

Results: 1 2 3 4 5
0 30 45 65
Test tube Room
Temperature 0.4660 0.9840 0.8150 0.6900
Temperature
(°C) 0.0000

Absorbance
Value

Figures:

Observation of samples after boiling 20 minutes

Observation of samples after 1.0 ml Arsenomolybdate is added

Graph:

Discussion:
This part of the experiment is conducted to determine the effect of

temperature on sucrase enzyme activity. The experiment is started by putting
sucrose, buffer solution with pH 5.0 and water together into five test tubes. Then,
the test tubes are placed respectively at 5 different incubation temperatures for 10
minutes in order to allow the temperature of the substrate solutions to achieve
equilibrium with incubation temperature. In this case, water baths are used for
incubation temperatures 30°C, 45°C and 65°C. Meanwhile, ice bath has been used
to achieve the 0°C incubation temperature. Buffer with pH 5.0 is used in this
experiment to maintain a relatively constant pH during the enzyme assay. As the
pH is constant throughout the experiment, thus it will not affect the rate of sucrase
enzyme activity and the experiment can be focused studying on the effect of
temperature towards the sucrase enzyme activity.

The experiment is continued by adding the sucrase enzyme into each test tube
and the test tubes are incubated at each different temperature for 10 minutes,

thereby enzymatic reaction is started. Afterwards, Somogyi- Nelson method is used
to measure the concentration of reducing sugar by using reagent A+B (alkaline
copper tartrate) and arsenomolybdate. This method utilized the reducing properties
because of the presence of potential aldehyde or keto-groups in reducing sugar
such as glucose and fructose. The reducing sugars that are heated with alkaline
copper tartrate will reduce the copper from cupric to cuprous state, thus cuprous
oxide or copper(I) oxide is formed, appearing in yellow colour. Then, cuprous
oxide is treated with arsenomolybdate forming molybdenum blue. Consequently,
determination of concentration of reducing sugars is based on the absorbance
reading at 510 nm by using spectrophotometer.

Based on the graph of reducing sugar against temperature, it can be observed
that graph is shown in a rough bell curve shape from the temperature 25°C to 65°C
while the maximal rate of reaction is at temperature 30°C which showed the
highest amount of reducing sugar (0.9840 Au). Therefore, the optimum
temperature for sucrase can be defined as 30°C. In addition, the higher temperature
generally causes more collisions among the molecules and therefore increases the
rate of a reaction. More collisions happen due to the increase in velocity and
kinetic energy. As the molecules carry more kinetic energy, thus the energy
difference between the threshold energy and the energy of the molecules will
decrease, that is, the activation energy will decrease.

From the graph shown above, the amount of reducing sugar in terms of
absorbance is zero at room temperature (around 25°C) because test tube 1 is used
as blank and since there has no sucrose inside test tube 1, so there is no sucrase
enzymatic reaction. On the contrary, the presence of substrate (sucrose) in test
tubes 2,3,4, and 5 allowed the enzymatic reaction to occur and as a result,
absorbance value can be obtained by using the spectrophotometer. Other than that,
by comparing the amount of reducing sugar at the temperature 0°C (0.4660 Au),
45°C (0.8150 Au) and 65°C (0.6900 Au), the lowest rate of sucrase enzymatic
reaction is at temperature of 0°C, followed by 65°C and 45°C. This means that the
sucrase enzyme undergoes both cold and heat denaturation. Denaturation occurs
when an enzyme loses its shape and structure, leading to the loss of enzyme
activity. The substrate (sucrose) cannot bind to the active site of the sucrase

enzyme when the enzyme undergoes denaturation, hence enzyme-substrate
complex cannot be formed.

One of the precautionary steps that can be taken during the experiment is
storing the sucrase enzyme solutions at 4°C- 20°C. For instance, enzymes should
be kept in ice baths to avoid the deactivation of enzymes. By doing this, shelf life
of the enzymes can be enhanced. Theoretically, the optimum temperature of
sucrase enzyme activity is 40°C , however the result obtained from the experiment
showed that the optimum temperature is 30°C, some errors might happened during
the experiment including the mixture of solutions might not mixed well which
caused part of the solution to be more concentrated than the other part, hence the
absorbance reading is inaccurate as the dispersion of light ray that pass through the
solution in cuvette is not uniform.

CONCLUSION

In a summary, through this experiment, students are able to determine the effect of
pH and temperature on sucrase enzyme activity by measuring the amount of
reducing sugars (glucose and fructose) present in molybdenum blue through the
Somogyi-Nelson method. Based on the results obtained from part A of the
experiment, it can be concluded that the amount of glucose (reducing sugar)
present in a given sample is directly proportional to the absorbance reading.
Besides, from the graph of reducing sugars (absorbance values) against pH in part
B, it showed a smooth bell curve shape that indicated sucrase enzyme activity
decreased in extreme pH but the rate of enzyme activity increased when pH is
increased towards the optimum pH value. The optimum pH obtained from the
experiment for sucrase enzyme is pH 6 which complies with the theoretical value.
While from the graph of reducing sugar (absorbance values) against temperature in
part C, it can be concluded that sucrase enzyme activity decreased at extreme
temperatures. The graph is displayed in a rough bell curve shape and the
experimental value of optimum temperature for sucrase enzyme is 30°C. It does
not comply with the theoretical value, some errors might happened in the
experiment which caused the result to be inaccurate. Basically, the objectives of the
experiment are achieved, the optimum pH value and temperature for sucrase
enzyme are both determined.

REFERENCES:

1. NotesHippo.Com. (2022, March 26). Nelson Somogyi Method for Glucose
Estimation. Retrieved from:
https://noteshippo.com/nelson-somogyi-method-of-reducing-sugars-principl
e-procedure-and-calculation/

2. Biology Wise. (2019). Effect of pH on enzyme activity. Retrieved from:
Effect of pH on Enzymes - Biology Wise

3. Bio-Resource. (2011, April 26). Determination of Reducing Sugars by
Nelson-Somogyi Method. Retrieved from:
http://technologyinscience.blogspot.com/2011/04/determination-of-reducing
-sugars-by.html

4. Creative Enzymes. (2022). Effect of Temperature on Enzymatic Reaction.
Retrieved from:
https://www.creative-enzymes.com/resource/effect-of-temperature-on-enzy
matic-reaction_50.html

5. David L.Nelson & Michael M.Cox. (2021). Principles of Biochemistry, 8th
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