*This is a group Project but everyone have to write their own final report. Below will be I have one report and just need to change the wording so it doesn’t get flag for plagiarism.
Materials and Method
1. Lab reports should be written in the past tense and in a passive voice (no we, I, our, you, etc.)
2. At the end of the introduction, be certain to state a Null Hypothesis and an Alternate Hypothesis from each section.
The intent behind this experiment was to determine the optimal conditions for the
enzyme alkaline phosphatase (ALP) to perform a catalytic reaction. The effects of
pH, enzyme concentration, and temperature were tested. To test the effects of each
condition on catalytic rate, the effected enzymes were placed into a cuvette then
placed in a spectrophotometer to measure absorption. The greater the absorption
recorded indicates the greater rate of reaction in the enzyme. For the first experiment,
enzymes were placed into three solutions of pH ranged from acidic to base. A neutral
pH balance was found to provide the enzymes with optimal conditions for a reaction
to occur. The second experiment determined temperatures effect on catalytic reaction
and the optimal temperature for the reaction to occur. The results followed the same
circumstance as the results for pH. That is, the optimal temperature for enzyme
reactions was 32 degrees Celsius, room temperature. The enzymes exposed to 32
degree Celsius saw steady increase of absorption across all tests, as opposed to
temperature treated enzymes. The finale experiment examined the effects of enzyme
concentration on its catalytic rate. It was determined that an enzyme concentration of
500 microliters (the highest concentration tested) encouraged a higher rate of reaction
from ALP. The experiments revealed that pH balance, temperature, and concentration
do have an effect on the rate of ALP reaction.
Multiple reactions are constantly occurring within the body to allow molecular
change and the preservation of life. These reactions require a certain level of
activation energy (EA) to initiate these changes. Without assistance, many of the
molecules won’t reach the threshold for change to occur (Urry et al, 2020 ). Enzymes
are molecules that lower the threshold for activation and allow the molecules they
interact with to change into a molecule able to perform work.
Enzymes are able to lower EA required for a reaction to occur by optimizing the
reacting molecules shape or environment. Enzymes will bend these molecules into the
shape they need to reach activation, arrange them into the proper order, or create a
microenvironment suitable for activation to occur (Urry et al 2020). Enzymes are
extremely important because they allow life to be possible. Without them life would
cease, because organisms wouldn’t be able to acquire enough energy to initiate the
reactions required for the change of energy.
Additionally, Enzymes will perform better when exposed to optimal conditions. The
experiment performed tested Alkaline Phosphatase (ALP) an enzyme common to the
human body and p-nitrophenyl phosphate(pNPP) a substrate that activates with ALP.
The experiments performed sought to determine the optimal conditions for ALP to
perform. The optimal condition found in the lab translates to the optimal conditions
our bodies should be exposed to, to have optimal reactions and possibly better health.
For the first experiment, the null hypothesis was: A change in pH environment will
have no effect on enzymatic reactions. The alternative hypothesis was: The pH
environment will have an effect on the optimization of enzymatic reactions. For the
next experiment, the null hypothesis was: The concentration of the enzyme will not
affect its rate of reaction. The alternative hypothesis was: The concentration of the
enzymes will affect the rate of reaction. For the third and last experiment the null
hypothesis was: Temperature will have no effect on the rate of enzymatic reactions.
The alternative hypothesis was: Temperature will affect the rate of enzymatic
Part 1. The effect of pH
Solution E was prepared for four cuvettes by adding 6.5 mL of substrate pNPP to
6.5 mL of distilled water and distributing the solution among the cuvettes. Distilled
water is used to create a baseline for the other solutions to venture from. One cuvette
was used as a control and did not have its pH altered. Test stripes were used to verify
the pH of the three solutions that would be applied. Once verified they were added to
the Solution E. The acidic pH solution of 1.9mL hydrogen chloride(HCI) was added
to one cuvette, a neutral pH solution was created by adding 1.9 mL of distilled water
to Solution E. The basic pH solution was created by adding 1.9 mL of sodium
carbonate (Na2CO3) to the last cuvette of solution E. Solution D (enzyme ALP high
concentration) was not added to the cuvettes until the solution was ready to be placed
into the spectrometer. It was vital that all the solutions were homogenous in color to
ensure the accuracy of the absorption readings (Nguyen 2020).
Once all the solutions were created, solution D would be added to the specific
cuvette. The cuvette being tested would be inverted to ensure proper mixture and
placed within the spectrometer set at 405 nm (max absorption of pNPP). Every 30
seconds over the course of 5 minutes, the tested cuvette was removed, inverted,
reinserted, and had its absorption recorded. This process was performed for each
Part 2. The effect of Enzyme Concentration
An Alkaline buffer solution and the substrate pNPP was mixed and placed in four
cuvettes. The Alkaline buffer is used to maintain a habitable environment for the
substrate to reside. It would provide similar conditions to that of the human body.
This solution was called solution F. One cuvette of solution F was used as a control.
The first cuvette was infused with 100 microliters of low concentration ALP using a
micropipette. The solution was placed into the spectrometer for 5 minutes, recording
results every 30 seconds. The second cuvette received 400 microliters of low
concentration ALP and tested. The third cuvette received 200 microliters of ALP, and
the last received 500 microliters of high concentration ALP (Nguyen 2020).
Part 3. The effect of Temperature
Again, solution F was used in four cuvettes for this experiment. As well as 100
microliters of low concentrate ALP placed in four centrifuges. The cuvettes of
solution F were placed into the centrifuges and placed into environments of 4° C
(refrigerator), 23°C (room temperature), 32°C (water bathe), and 60°C (water bathe).
After setting for at least 20 mins. The cuvettes were ready for testing. Before placing
the cuvettes in the spectrometer, re-blank the spectrometer to 405 nm and condesation
was wiped from the cuvettes to maintain accuracy (Nguyen 2020). After re-blanking
the spectrometer, the cuvette solutions were mixed with their respective solutions of
ALP as they were ready to be tested. The absorbance was read and recorded every 30
seconds, over the course of 5 minutes. Finally, average enzyme activity was
calculated with the equation: Enzyme activity= Absorbance/Time. As pNPP reacts
with ALP, the mixture becomes a deeper shade of yellow. As time passes and the
solution becomes more yellow, we are able to determine the rate of enzyme activity
from the amount of light absorbed by the solution.
Part1. The effect of pH
The normal range for pH within the body is from 7.35 to 7.45, meaning the optimal
environment for the enzyme ALP would be slightly above neutral. When the ALP
was placed in the neutral environment it had an average reaction rate of 1.37E-03.
Which was significantly higher than its reaction rate when exposed to basic and
acidic environments. When exposed to acidic environments it is drastically less
reactive compared to higher pH levels ( Bhatti et al, 2002). This is because, at lower
pH’s the enzymes lose ions and are broken down into monomers. When the pH is
raised, the monomers that generate the enzymatic proteins are able to bond and carry
out their reactions. Following the conclusions from the data collected the alternative
hypothesis would be accepted and null rejected. The pH environment that surrounds
the enzyme ALP will affect its optimization. Possible errors that could have occurred
by not allowing the enzymes to incubate for a long enough period. The data suggests
that, as time elapsed, enzymes in the basic pH rose steadily behind the enzymes
within the neutral pH. It is possible that given enough time, the enzymes placed in the
basic environment may have reached a greater average reaction rate. In the future,
research can be done to determine the length of time required to allow enzyme
reactions to reach optimal levels in various pH.
Part2. The effect of enzyme Concentration
Figure 4. shows the average reaction rate steadily trending upward as it reaches its
highest concentration. This was the concentration of 500 microliters of enzyme ALP.
Which had an average absorbance of 0.769.
VI. References (I’VE INCLUDED 2 OF THE 4 REFERENCES)
Bhatti, A., Alvi, A., Walia, S., & Chaudhry, G. (2002). pH-dependent modulation of alkaline
phosphatase activity in serratia marcescens. Current Microbiology, 45(4), 245-9.
Aragon, J. J., & Sols, A. (1991). Regulation of enzyme activity in the cell: Effect of enzyme
concentration. FASEB Journal, 5(14), 2945-2950.
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