Research question
By putting the enzymes, catalase in different pH condition (ranging from 4 to 10) and
measure the time taken to collect certain amount (100cmᵌ± 0.5cmᵌ) of oxygen, how are
students able to see the effect of different pH on the rate of catalase activity?
Aim
To determine the optimum pH for liver enzymes, catalase.
Introduction
This experiment measures the rate of reaction between the enzyme catalase and the
substrate molecule hydrogen peroxide (H2O2). The liver enzymes, catalase will break down
the hydrogen peroxide (H2O2) and form products which are the water molecules and oxygen.
The reaction happens as follow:
2H2O2 (aq) ➔ H2O (l) + 2O2 (g).
To determine optimum pH for the catalase activity, students can measure the rate of
reaction between the substrate molecule (hydrogen peroxide, H2O2) and the catalase at
different pH level. Students can measure the time taken for a certain amount (100cmᵌ) of
oxygen gas produced to be collected in a gas syringe, and plot the data on a graph to see the
catalase activity at different pH level. Optimum pH is when the rate of reaction is at the
highest point on the curve.
Background knowledge
Enzymes
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Biological catalyst produce by living cells that catalyze biochemical reactions in living
organisms
Speed up the reaction by reducing activation energy Ea required
Made of globular proteins therefore have specific three dimensional structure
(conformation), which determines the properties of enzymes.
Two hypothesis that explains the enzyme action:
i)
Lock and key model substrate fits exactly to the active site of enzyme
ii)
Induced fit model active site is flexible and changes shape to bind to substrate molecules
Enzymes are sensitive to pH changes therefore a small change in pH value will
severely affect the enzyme activity. As pH of medium changes to a large extent, the
conformation of globular protein of the enzyme changes, distorting the structure of
active site, thus substrate can no longer fit into the active site.
According to the www.bbc.co.uk/bitesize/, the optimum pH varies greatly across
different enzymes. For example, intestinal enzymes have an optimum pH of 7.5
whereas enzymes in the stomach have an optimum pH of 2, therefore enzymes in
stomach can work better in acidic conditions.
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Other factors affecting the enzyme activities are temperature, substrate and enzyme
concentration, ionic strength etc.
Liver enzyme, catalase
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Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long. It
contains four porphyrin heme (iron) groups that allow the enzyme to react with the
hydrogen peroxide.1
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Catalyzes the reaction of the decomposition of hydrogen peroxide (2H2O2 (aq) ➔ H2O
(l) + 2O2 (g).)
Found mostly in mammalian tissues. Catalase break them down to prevent the
accumulation of peroxide that is produced by metabolic reactions, in which could
possibly damage the body tissue. 2
Catalase has a high turnover number (number of substrate molecules that are
converted by an enzyme to form product molecules in one minute) of 6 million: 6
million molecules of H2O2 into H2O and O2 in one minute.3
Optimum pH for human catalase is around 7. 4 Though the optimum pH varies across
the range of 4-11 for other catalases depending on their species. 5
Active site of enzyme can be influenced by the acidity and alkalinity of the
surrounding. If it is an acidic condition (pH lower than 7), enzymes will gain more H⁺
ions, causing a repulsion between substrate and enzyme molecules. Whilst in an
alkaline condition where it is a high pH (>7), enzymes have too many OH⁻, causing
active site to repel substrate. 6
The optimum temperature for human catalase is 37°C. 7
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1
Boon EM, Downs A, Marcey D. "Catalase: H2O2: H2O2 Oxidoreductase"
(http://biology.kenyon.edu/BMB/Chime/catalase/frames/ cattx.htm). Catalase Structural Tutorial Text. .
Retrieved 2007-02-11
2
Britannica School, s.v. "catalase," accessed December 7, 2014,
3
Goodsell DS (2004-09-01). "Catalase"
(http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb57_1.
html). Molecule of the Month. RCSB Protein Data Bank. . Retrieved 2007-02-11.
4
Maehly A, Chance B (1954). "The assay of catalases and peroxidases". Methods Biochem Anal. Methods of
Biochemical Analysis 1: 357–424. doi:10.1002/9780470110171.ch14. ISBN 978-0-470-11017-1. PMID 13193536
5
"EC 1.11.1.6 - catalase" (http://www.brenda
enzymes.org/php/result_flat.php4?ecno=1.11.1.6&Suchword=&organism[]=&
show_tm=0). BRENDA: The Comprehensive Enzyme Information System. Department of Bioinformatics and
Biochemistry, Technische Universität Braunschweig. . Retrieved 2009-05-26.
6
7
Mah Chee Wai, Dr.Tina Lim Swee Kim. Nexus SPM Biology . Selangor Darul Ehsan, Malaysia: Sasbadi Sdn. Bhd.,
2012.
Aebi H (1984). Aebi, Hugo. ed. "Catalase in vitro". Meth. Enzymol.. Methods in Enzymology 105: 121–126.
doi:10.1016/S0076-6879(84)05016-3. ISBN 0-12-182005-X. PMID 6727660.
Hypothesis
The optimum pH for the liver enzyme, catalase is around pH 7. Therefore, when enzymes are
under the pH of 7, the time taken for 100cmᵌ of oxygen to be collected should be the
shortest because this is the pH when enzymes are working at their full capacity, breaking
down the hydrogen peroxide into water and oxygen at the fastest speed, hence the highest
rate of reaction. When the pH is lower than 7 (acidic), enzymes gain more H⁺ ions, whilst if
the pH is higher than 7 (alkalinity), enzymes will gain more OH⁻ ions. Both conditions
decrease the enzyme activity due to the repulsion between the active site8 and the substrate
molecules, hydrogen peroxide, making the substrate molecule unable to fit into the active
site. Furthermore, at the extremes of pH, enzymes will be denatured due to the change in
the tertiary structure and the active site of the enzymes. Substrate cannot fit into the active
site, enzymes thus no longer able to catalyse the reaction. The graph for the rate of reaction
of enzymes working at different pH should look as follow:
Figure 1. Graph for the rate of enzyme activity at different pH and with the optimum pH
falling around at the neutral pH of 6 or 7.
Variables
Independent Variable: pH buffer solution
Enzymes are sensitive to pH changes therefore a small change in pH value will severely affect
the enzyme activity. Different enzymes work best at different pH condition, resulting in
different enzyme activity, hence the time taken to collect 100cmᵌ of oxygen and the rate of
reaction will be different. A too acidic or too alkaline medium will denature the enzyme,
8
Mah Chee Wai, Dr.Tina Lim Swee Kim. Nexus SPM Biology . Selangor Darul Ehsan, Malaysia: Sasbadi Sdn. Bhd.,
2012.
damaging its globular (tertiary) structure and the structure of active site, damaging the
properties and the function of enzymes.
Dependent variable: Time taken to collect 100 cmᵌ of oxygen
Catalase catalyses the reaction of the decomposition of hydrogen peroxide (2H2O2 (aq) ➔ H2O
(l) + 2O2 (g)). As rate of reaction can be measured through how fast reactant changes into
product, students can measure the time taken to collect 100cmᵌ of oxygen at different pH
condition as a measure of the rate of reaction.
Constant variable:
i)
Types of enzymes used
Different enzymes have different properties, therefore, will affect the rate of
reaction occurred. 9Enzymes are specific where they only catalyze one specific
reaction as only substrate with a particular shape is able to fit into the active
site. Therefore in this case, only the hydrogen peroxide molecule is able to fit
into the active site of catalase and be broken down by it, forming water and
oxygen.
ii)
Volume of liver enzyme
Volume of liver enzyme must be kept the same as it will affect the concentration
of the enzyme molecules that exist within the solution. A greater concentration
of enzymes will increase the reactivity between the enzymes, catalase with the
hydrogen peroxide molecules as more reactant particles were exposed for
collision. Hence, increasing the effective collision frequency and the rate of
reaction.
iii)
Volume and concentration of hydrogen peroxide
An increase in volume and concentration of hydrogen peroxide will increase the
reaction as there are more reactant particles per unit volume, hence increasing
the collision frequency and the effective collision frequency, thus the rate of
reaction.
iv)
Temperature of solution
Temperature is another factor that will affect the enzyme activity therefore it
has to be kept the same. An increase in temperature will increase the kinetic
energy of particles and the effective collision frequency between them, hence
9
Mah Chee Wai, Dr.Tina Lim Swee Kim. Nexus SPM Biology . Selangor Darul Ehsan, Malaysia:
Sasbadi Sdn. Bhd., 2012.
the rate of reaction. Though if the temperature exceeds its optimum, the
enzymes will be denatured due to a change in the tertiary structure that results
from broken chemical bonds (too much vibration). This will stop the enzymes
from to catalyzing the reaction.
Materials
1. pH Buffer (4,5,6,8,10)
2. Catalase- Liver enzymes
3. Hydrogen peroxide (H2O2)
Apparatus
1.
2.
3.
4.
5.
6.
7.
Conical flask
1 x 10ml syringe (±0.1ml)
2 x 5ml syringe (± 0.1ml)
100ml Gas syringe (± 0.5ml)
Stop watch (±1s)
Rubber bunk
Connecting tube
Procedure
1. Add 5ml of catalase (liver enzymes) into the conical flask.
2. Add in 5ml of pH4 buffer solution and 10ml of hydrogen peroxide together into the
conical flask.
3. Bung the conical flask with a rubber bunger attached with a gas syringe.
4. Once conical flask is bunged, start timing with the stopwatch until 100cmᵌ of oxygen
gas is collected.
Figure 1. Model of experiment. (*Reaction mixture is the Hydrogen peroxide, Liver enzymescatalase & pH buffer solution)
5. Observe and record down the time.
6. Repeat step 1 to 5 for different pH (4,5,6,8,10) buffer solution and do it in many
trials.
Safety concerns
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Put on goggles to prevent any hydrogen peroxide from contacting with the eyes in
which would cause irritation
Wear lab coats to prevent any stain hydrogen peroxide that can cause the whitening
of skin and stinging sensations.
Handle the glass apparatus carefully to prevent them from breaking
Raw data
Time taken to collect 100cmᵌ of oxygen
(±1second)
Trial 1
Trial 2
Trial 3
pH
value
4
5
6
8
10
5.62
8.06
13.5
12.32
13.74
49.2
36
1.04
9.48
6.91
Processed data
pH
value
Average
4
5
6
8
10
24.38
21.90333333
6.756666667
9.57
12.78
Standard deviation
22.41309439
13.97172263
6.293125879
2.706122688
5.453741835
18.32
21.65
5.73
6.91
17.69