Enzyme lab report

1. Exploration
1.1. Research question
Investigating the effect of hydrogen peroxide concentration on the rate of
reaction in catalase in potato extract.

1.2. Background theory

Enzymes are globular proteins and are called biological catalysts, which have
the ability to speed up chemical reactions within cells. The enzyme has a so-
called active site into which a substrate goes, the enzyme catalyzes the
substrate and a product comes out. Each enzyme matches a specific
substrate.
Potato extract has an enzyme called catalase which catalyzes the breakdown
of hydrogen peroxide, obtaining water and oxygen as a product.
There are factors that can alter the rate of reaction of enzymes. One of them
is the concentration of substrate, the less substrate there is, the less
frequency of coalition between the substrate and the active site, the more
substrate there is, the greater the rate of reaction or coalition, but there
comes a point where the enzymes, which are limited, they are completely
saturated and it reaches its maximum rate of reaction, so it continues to work
at its maximum speed and no longer increases, this means that the graph
increases only until it reaches the maximum speed point, which is when the
graph becomes constant.

1.3. Variables

Independent variable: hydrogen peroxide concentration.

it will be diluted from low concentration to high concentration.

Dependent variable: Time for the paper disc to reach the surface.
This will be measured with a stopwatch.

Controlled variables: enzyme concentration, same amount of potato extract

absorbed by the paper discs, equal measure of the paper discs.
The amount that the paper absorbs will be measured by the time it soaks,
which is 30 seconds. The paper discs will be cut with the same perforator, so
 that the amount of enzyme is the same and the experiment will not be
affected.

1.4. Equipment
- 5 test tubes
- a marker
- 25 paper discs
- a rule (±0.1cm)
- a watch glass
- 3 x 1 ml pipettes (±0.02ml)
- 2 rods
- forceps
- beaker
- 1 stopwatch (±0.01s)
- a test tube rack
- 300ml of potato extract
- 250ml of 3% hydrogen peroxide
1.5. Method

i) First the tubes will be marked with a horizontal line with the marker 5
cm from the base and placed in the test tube rack.

ii) Then the first concentration of 1 ml of H2O2 (3%) and 4 ml of water

will be made, with a pipette 1 ml of 3% hydrogen peroxide is taken
and then put in a beaker, then with another pipette 4 ml of water are
taken and put in the same beaker, then with a rod mix the solution.

iii) This solution will be poured into the tube until it reaches the marked
line, if it does not reach the marked line, another solution with the
same amounts will be made again until it reaches the line, the same
will be done in the remaining 4 tubes.

iv) Then in a watch glass 3 ml of potato extract (containing catalase) will

be placed with another pipette, then 1 paper disc will be placed and
soaked in the extract for 30 seconds counted by the stopwatch.

v) After 30 seconds, the soaked paper disc will be removed with the
forceps and will be slightly pressed to the side of the glass to remove
the excess extract and then placed at the bottom of the tube with the
help of another rod. As soon as the paper disc touches the bottom the
time it takes for the paper disc to reach the surface will be recorded
with a stopwatch.

vi) steps 4-5 will be done with the remaining 4 paper discs.

vii) Subsequently, the 5 tubes will be washed and dried to be reused later.
 viii) Steps 2-7 will be performed for the remaining solutions: 2 ml H2O2
(3%) and 3 ml water, 3 ml H2O2 (3%) and 2 ml water, 4 ml H2O2
(3%) and 1 ml water, and finally 5 ml H2O2 (3%) and 0ml of water.

1.6. Safety and environmental considerations

Table 1: risk assessment
Hazard risk person at level of risk control new level of
risk (high, measuremen risk
medium or ts
low)

Hydrogen Hydrogen the high use gloves, medium

peroxide peroxide is experimenter face mask
irritating. and glasses
contact with for protection
skin, eyes, while
mouth managing
this
substance

water this the medium be careful low

substance experimenter while using
can fall to water to not
the ground spill it
and cause a
fall of a
person

beaker, test these lab the medium do not put low

tubes, watch tools could experimenter these tools
glass fall and near the
break, and edge of the
when trying workplace,
to pick them be careful
up they can while using
cut the skin them

2. Analysis
2.1. Data collection
After submerging the paper discs in their respective concentration, they took
a certain amount of time, presented below in table 2.

Table 2: the time taken for the discs to reach the surface for each
concentration
Time taken for the disc to reach the surface
H2O2 concentration (±0.01s)
(mol/L)
H2O2 (3%) Water (±0.01mol/L
(±0.02ml) (±0.02ml) (%) ) trial 1 trial 2 trial 3 trial 4 trial 5
1 4 0.6% 0.26 8.73 14.93 16.76 17.98 11.33
 2 3 1.2% 0.51 13.01 5.58 12.41 9.11 11.96
3 2 1.8% 0.77 11.84 9.41 30.24 17.48 18.41
4 1 2.4% 1.02 15.44 23.45 15.04 13.77 12.50
5 0 3.0% 1.28 14.06 11.37 13.69 15.14 13.74

2.2. Data processing

The average time was calculated with the google sheets program, with the
formula:
¿ AVERAGE( coordinates of the first∧final trial)
example:
¿ AVERAGE(C 13:G 13)
but that can also be obtained with the formula:

mean time=
∑ of the time∈all trials
number of trials
example:
(8.73+14.93+16.76 +17.98+11.33)
=13.95
5
The standard deviation was calculated with the google sheets program, with
the formula:
¿ STDEVP(coordinates of the first∧the final trial)
example:
¿ STDEVP(C 13 :G13)
The relative rate of activity wa calculated according to the formula:
relative rate of reaction=100 ÷ mean time
example:
100 ÷13.95=7.17

table 3: mean time taken for the discs to reach the surface for each
concentration, standard deviation and relative rate of activity for each
concentration
Time taken for the disc to reach the surface
H2O2 concentration (±0.01s)
relative
(mol/L) standard rate of
(±0.01mol mean deviatio reaction
(%) /L) trial 1 trial 2 trial 3 trial 4 trial 5 time (s) n (s-1)
0.6% 0.26 8.73 14.93 16.76 17.98 11.33 13.95 3.85 7.17
1.2% 0.51 13.01 5.58 12.41 9.11 11.96 10.41 3.09 9.61
1.8% 0.77 11.84 9.41 30.24 17.48 18.41 17.48 8.07 5.72
2.4% 1.02 15.44 23.45 15.04 13.77 12.50 16.04 4.30 6.23
3.0% 1.28 14.06 11.37 13.69 15.14 13.74 13.60 1.38 7.35

Figure 1: time taken for the paper discs to reach the surface vs H2O2 concentration.
Standard deviation shown by the error bars.
Figure 2: relative rate of reaction vs H2O2 concentration

3. Evaluation
3.1. Conclusion

In graph 1 it can be seen that at the beginning with a low substrate

concentration of 0.26 mol/L the reaction takes about 13.95 seconds, then by
increasing the concentration to 0.51 mol/L the reaction speed increases,
decreasing the reaction time by 3.54 seconds, that is 10.41 seconds.
By increasing the concentration to 0.77mol/L, the reaction time increases
considerably in 7.07 seconds, reaching 17.48 seconds, here the reaction
speed decreases.
If the concentration continues to increase to 1.02 mol/L, the reaction time
decreases by 1.44 seconds, reaching 16.04 seconds, that is, the reaction rate
increases.
Finally, increasing the concentration to 1.28 mol/L, the time it takes to react
decreases by 2.44 seconds, reaching 13.60, that is, its speed increases
again.

The trend line is increasing and proposes that as the substrate concentration
increases, the amount of time increases; that is, the speed decreases.

Graph 2 is a more simplified graph with which the results can be compared.
graph 2 describes the relative reaction speed of catalase vs. the
concentration of H2O2 and it is observed that first there is a straight line at
the first increase in concentration, which is interpreted as directly proportional,
the highest point or the maximum speed is given when the substrate
concentration is 0.51 mol/L, which would be the saturation point at which the
 enzymes work at their maximum speed and therefore, as the concentration
increases to 0.77, 1.02 and 1.28, the speed should remain constant.
However, this does not happen in graph 2, instead after reaching the
maximum speed, it decreases and increases again.

The error bars in graph 1, when the concentration is 0.77 mol/L, suggest that
the values were much more dispersed, so the information obtained is very
unstable.
The information obtained is much more stable when the concentration is 1.28
mol/L.
For concentrations of 0.26; 0.51; 1.02 the information obtained is slightly
unstable.

3.2. Evaluation

Carrying out the first two tests (with the first 2 concentrations) with fresh
potato extract and the remaining 3 tests with the same potato extract but after
a day, that is, not fresh, may have affected the experiment.

The temperature may have affected the experiment.

Catalase is known to work best at temperatures of 37.5'C and does not work
as well at temperatures of 30 or lower or 45 or higher.
Upon arrival at the experimentation site (the second day of experimentation
with the remaining 3 concentrations) and starting the experiment, the potato
extract was cold, so it did not work as it should at laboratory temperature
(20'C), as time progressed the potato extract was heated or took the
temperature of 20'C and worked at laboratory temperature, for this reason
there is a great decrease in the reaction rate in graph 2 that then rises.

The pipettes may not have measured the amount of substrate and water well.

The timer and the experimenter's view may have been out of sync.

By taking the paper disks two paper disks could have been stuck together,
pretending to be one, then by immersing the paper disks in the potato extract,
this double paper disk could have absorbed more catalase from the potato
extract than a only one paper disc which could have altered the
experimentation, considering that only one paper disc could be immersed for
each concentration test.

3.3. Improvement Suggestions

Do all the tests on the same day with fresh catalase.

Wait for the potato extract to reach laboratory temperature (ambient
temperature: 20'C) in order to start the experimentation.
Use graduated cylinders to measure the amount of substances.
 Synchronize the view and the stopwatch as best as possible.
Verify that only one paper disk is taken when choosing the paper disks.

4. References
https://www.google.com/search?
q=rate+of+reaction+affected+by+substrate+concentration&rlz=1C1GCEA_enFI911FI911&sx
srf=APq-WBuLS0Elcg4a5qyv80t-
IG3geH9nXw:1645741712572&source=lnms&tbm=isch&sa=X&ved=2ahUKEwjPqq3RsZn2A
hVro4sKHekSBsMQ_AUoAXoECAEQAw&biw=1366&bih=657&dpr=1#imgrc=cQ77OxM3qKI
woM

http://csef.usc.edu/History/2016/Projects/J0513.pdf

https://www.biologydiscussion.com/enzymes/factors-affecting-enzyme-activity-6-factors/
11207