Biology Unit 1

Experiment # 8
January 24, 2024
Title: The effect of Substrate Concentration on Enzyme-Catalyzed Reactions

Aim: To investigate the effect of substrate concentration (hydrogen peroxide) on the rate of an
enzyme-catalyzed reaction using catalase from fresh liver tissue.

Background:
Enzymes are biological catalysts that facilitate chemical reactions within living organisms by
lowering the activation energy required for these reactions to occur. One such enzyme, catalase,
plays a vital role in breaking down hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2),
thereby preventing the accumulation of harmful hydrogen peroxide in cells. This reaction is
particularly crucial in metabolically active tissues like the liver, where catalase is abundant and
serves as a natural defense mechanism against oxidative stress. Catalase achieves this
detoxification process by binding to the substrate, hydrogen peroxide, and facilitating its
decomposition into harmless byproducts.The rate at which catalase catalyzes this reaction can be
influenced by various factors, including substrate concentration. According to the Michaelis-
Menten kinetics, enzyme activity initially increases with substrate concentration until it reaches a
point of saturation, where all enzyme active sites are occupied. Beyond this point, further
increases in substrate concentration do not increase the rate of the reaction, as the enzyme is
already working at its maximum capacity.In this experiment, we aim to investigate the effect of
substrate concentration, represented by varying concentrations of hydrogen peroxide, on the rate
of the catalase-catalyzed reaction using fresh liver tissue as the enzyme source. By systematically
varying the concentration of hydrogen peroxide and measuring the rate of the reaction, we can
elucidate the relationship between substrate concentration and enzyme activity. This
investigation will not only deepen our understanding of enzyme kinetics and substrate
interactions but also have implications in various fields such as biochemistry, medicine, and
biotechnology.
Apparatus/Materials:

1. Fresh liver tissue (chopped into small pieces)

Method:
1. 5 Test tubes were labeled as 1%, 2%, 3%, 4%, 5% to correspond to the different
concentrations of hydrogen peroxide.
2. Measuring cylinders were used to measure 10 ml of hydrogen peroxide solution for each
test tube.
3. The liver tissue was prepared by chopping it into small pieces.
4. Approximately 1-2 grams of liver tissue were added to each test tube.
5. The liver tissue and hydrogen peroxide solution were mixed by gently swirling the test
tubes to ensure even distribution.
6. The timer or stopwatch was started immediately upon mixing.
7. The time it took for the gas foam to reach 5 cm in each test tube was observed and
recorded. This indicated the rate of the enzyme-catalyzed reaction.
8. The experiment was repeated for each hydrogen peroxide concentration, ensuring fresh
liver tissue was used each time.
9. The rate of reaction was calculated by determining the reciprocal of the time taken for the
gas column to reach 5 cm (1/time).
10. A table was prepared to organize the data, including substrate concentration and
corresponding reaction rates.
11. A graph was created with substrate concentration on the x-axis and reaction rate on the y-
axis.

Results:
TABLE SHOWING THE EFFECT OF SUBSTRATE CONCENTRATION ON THE RATE OF
ENZYME CATALYZED REACTIONS

Concentration of hydrogen Time (s) Rate (1/time) (s-1)

1 30 0.033

2 25 0.040

3 18 0.056

4 15 0.067

5 12 0.083

Sample Calculation:

Using 3% hydrogen peroxide

Rate = 1/time = 1/18 = 0.056min-1

Discussion:
Catalase, an enzyme found in various organisms, including humans, plants, and microorganisms,
is crucial for breaking down hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2),
essential for cellular function to prevent harmful hydrogen peroxide accumulation. The formula
depicting the breakdown of hydrogen peroxide catalyzed by catalase is: 2H2O2 2 H2O+O2.
Particularly abundant in metabolically active tissues like the liver, catalase aids in detoxifying
harmful substances. Enzymes like catalase serve as natural catalysts, accelerating reactions by
providing an alternative route for reactions to proceed efficiently under normal conditions, each
enzyme being highly specific to certain reactions or groups of reactions. Catalase activity can be
influenced by various factors, including substrate concentration. As hydrogen peroxide
concentration increases, so does the rate of the catalase-catalyzed reaction, reaching a plateau at
higher concentrations due to enzyme saturation.In our experiment, we explored how altering
hydrogen peroxide concentration affects catalase's reaction rate using fresh liver tissue. The
graph showed an initial rate increase followed by a plateau, indicating the direct impact of
substrate concentration. Additionally, the time taken for the column to reach 5 cm decreased with
increasing concentration, indicating a faster reaction process. This investigation will deepen our
understanding of catalase-catalyzed reaction kinetics and enzyme-substrate interactions.This
phenomenon can be attributed to the greater availability of substrate molecules for binding with
the enzyme's active sites, resulting in the formation of more enzyme-substrate complexes and
consequently increased product formation per unit time. The data produced a linearly increasing
graph, reflecting the greater availability of substrate molecules for enzyme binding and
subsequent product formation. Specifically, at lower substrate concentrations like 1% H2O2, the
enzyme had fewer substrate molecules to interact with, leading to a slower reaction rate as
evidenced by longer gas foam formation times and a lower reaction rate of 0.033 s-1. However,
as the substrate concentration increased to 2%, 3%, and 4%, the reaction rates correspondingly
rose to 0.040 s-1, 0.056 s-1, and 0.067 s-1, respectively. The maximum reaction rate was
observed at the highest substrate concentration of 5% H2O2. Beyond a certain threshold,
increasing substrate concentration no longer boosts catalase activity proportionally due to
enzyme saturation, marking the maximum efficiency achievable under the given conditions.

Limitations:
 1. Environmental conditions such as temperature and pH can influence enzyme activity.
2. Using whole liver tissue means other enzymes or substances might be present, interfering
with the reaction or causing background noise in the results.

Precautions:

1. Prepare the liver tissue the same way each time, ensuring uniformity in enzyme activity.
2. Running the experiment several times for each condition increases our confidence in the
results. More trials mean we can better spot any unusual results or errors, making our
conclusions more reliable.

Sources of Error:

1. Liver quality may vary between batches, impacting enzyme activity. This can occur if the
liver is not fresh or stored correctly, causing variations in enzyme effectiveness and
resulting in inconsistent results.
2. Inadequate mixing of hydrogen peroxide and liver tissue can lead to uneven substrate
distribution. This can cause differences in reaction rates among test tubes, reducing data
accuracy.

Conclusion:

In conclusion, it was observed that as the concentration of hydrogen peroxide increased, the rate
of the catalase-catalyzed reaction initially rose before reaching a saturation point.