Formal lab report (3)
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Biology
Date
Feb 20, 2024
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1
The Effect Of Ph,Enzyme concentration, and temperature ALP activity
Abstract
The purpose of this lab experiment was to examine the different Ph levels, enzyme
concentrations, and temperature that is affected by the rate of alkaline phosphatase (ALP). An
enzyme is used in human digestion and clinical tests to test different diseases. This experiment
was divided into three sections for each variable, calculated by the absorbance/time. The first
measure was the optimum Ph level of ALP, which was the basic level (A/s=(3.53E-04). The
second part of the experiment tested the optimum effect of enzyme concentration, which was
medium (A/s=6.04E-04). The enzyme activity and concentration both increased. The highest
concentration was high (A/s=2.72E-03), and the lowest was low (A/s=2.17E-04). The last part of
the experiment tested the optimum for temperature, which was 32°. The temperature started to
increase from 4° to 32°, then after 32°, it decreased. The three variables, pH, enzyme
concentration, and temperature of the experiment, did affect the enzyme activity rate of ALP.
Introduction
Enzymes comprise protein and amino acids; they also produce living organisms to catalyze
reactions. Biological reactions need enzymes to speed up the rates of chemical reactions in cells,
and for this to happen, it has to be lowered by the activation energy for the process to start.It is
important to study enzymes because it is a part of biological life and could be used for medical
treatments. For example, Asparaginase is an enzyme treatment for anyone with lymphoblastic
2
leukemia. Lymphoblastic leukemia is mostly found in children and teenagers.
Asparagine
is an
amino acid that breaks down ammonia, which is toxic in the cells. According to
(Lynggaared,2022), Measurements of Asparaginase enzyme activity levels(AEA) are considered
the best way to monitor clinical efficacy of Asparaginase. It also states that the treatment
effectiveness of Asparaginase is based on the usage of the nonessential amino acid, asparagine
because the lymphoblasts are dependent on the exogenous sources of asparagine. The nature of
this experiment was to test the ALP enzyme for pH, temperature, and enzyme concentrations to
see what was the best condition and effect for the enzymatic activity. Alkaline phosphatase
(ALP) is an enzyme that hydrolyzes reactions in a phosphate group, and the par
nitrophenol-phosphate (pNPP) is there for the color change from colorless to yellow. The null
hypothesis states that pH will not have an effect on enzyme activity, and the alternative is that pH
will have an effect on enzyme activity. For the enzyme concentration, the null hypothesis is the
enzyme concentration will not have an effect on enzyme activity and the alternative is the
enzyme concentration will have an effect on enzyme activity. For temperature, the null
hypothesis is the temperature will not have an effect on enzyme activity, and the alternative is the
temperature will have an effect on enzyme activity.
Materials and Methods
In the experiment, we used solutions A-F to estimate the optimum pH for ALP activity.
Solution A was alkaline buffer, Solution B paranitrophenol phosphate (0.003, M), solution C
had a low concentration of enzyme ALP, solution D had a high concentration of enzyme ALP,
solution E had B/dH2o 6.5mL, and solution f had both of solution A and B (15mL). Solution A
3
was not used in the first part of the experiment because the pH needed to be measured at
different levels for the effect of enzyme activity to be seen.
Part 1:Estimating the optimum pH for ALP activity
First, we labeled the cuvettes 1-4. Cuvette 1 was the control that only had 3 ml of solution E
and 2 ml of distilled water. Cuvette 2 was the acidic condition with only 1.9 ml of HCI and 3 ml
of solution E. Cuvette 3 was the neutral condition that had 3 ml of solution E and 1.9 ml of
distilled water. Cuvette 4 was the basic condition that had 3 ml of solution E and 1.9 of
Na2CO3.When all of the solutions were mixed, it was placed on the pH paper to make sure the
solutions were correct. Cuvette 1 was placed in the spectrophotometer to be blank at 405 nm.
The spectrophotometer was used to measure the absorbance of pH that could have been affected.
Cuvette 2 then had 0.1 of solution D added, which we had to invert and wipe the cuvette before
placing it in the spectrophotometer. When placing it in the spectrophotometer, the absorbance
was recorded, and this was repeated for each cuvette for 30 seconds within the 5 minutes.
(Nguyen et al., 2022)
Part 2:Determining the effect of temperature on catalytic rate
In part 2 of the experiment, we labeled the cuvettes 1a-4a with 3 ml of solution F and 100 ul of
solution C using the micropipette. The microcentrifuge and cuvettes were incubated in their
environment for 20 minutes. Both cuvette 1a and microcentrifuge were at 4°C and were placed in
the refrigerator. Cuvette 2a and microcentrifuge were at 23°C, which was at room temperature.
Cuvette 3a and microcentrifuge were at 32°C, which was placed in the water bath, and cuvette 4a
4
and microcentrifuge were at 60°C placed in the water bath. Then, solution c was added inverted
and wiped to be placed in the spectrophotometer to read the absorbance every 30 seconds within
the 5 minutes. (Nguyen et al., 2022)
Part 3:Determining the effect of enzyme concentration on catalytic rate
In part 3 of the experiment, we labeled the cuvette 1b-4b. Solution C was the low concentration,
and solution D was the high concentration. The micropipette was used to get 100 ul of solution c
in cuvette 1b and placed in the spectrophotometer, which was already blanked. The absorbance
was recorded in 30 seconds within the 5 minutes. This was repeated for the other cuvette, but for
cuvette 2b, it used solution c 400 ul, cuvette 3b used solution D 200 ul, and cuvette 4b used
solution D 500ul.
(Nguyen et al., 2022)
Results
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Potato
extract
(mL)
Tube #
2d
3d
4d
5d
6d
7d
dH₂O
(mL)
0
0
0
0
0
0
Catechol
(mL)
6
6
6
6
6
6
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4mL pH 2
4mL pH 4
4mL pH 6
4mL pH 7
4mL pH 8
4mL pH 10
1
1
1
1
1
1
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(0 mins)
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Start Time: 3:24
A: 0.008
Start Time: 3:34
A: 0.023
Start Time: 3:36
A:
-0.006
Start Time: 3:36
0.018
A:
Start Time: 3:36
A:
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Start Time: 3:36
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water bath)
-0.030
A:
Take reading at: 3:44
A: 0.154
Take reading at: 3:46
A: 0-105
Take reading at: 3:46
A:
0-132
Take reading at: 3:46
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Take reading at: 3:46
A: 0.018
Take reading at: 3!46
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Q16) Use the difference between 2nd and 1st absorbance to support your conclusion for the optimum pH.
Subtract Omin
from 10min
absorbance
-0.04
0.0012
0.082
0.138
0.056
0.007
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Samples: lysine, cysteine, methionine
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Size
Size
Crude
Anion
Cation
2)
Exclusion
Exclusion
Lysate
Exchange
Exchange
1
2
Total Protein
Concentration
15.2
6.6
2
3.75
4.7
(mg/mL)
Final Sample Volume
60
30
20
4
3
(mL)
Enzyme Specific
Activity
0.43
1.7
7.25
18.3
12.5
(units/mgprotein)
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protocol did you effectively purify your enzyme? Select all that apply.
a) anion exchange correct
b) cation exchange correct
c) size exclusion 1 correct
d) size exclusion 2
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Rate of fizzing (1-10)
Boiled potato
Raw potato
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1
Raw liver
10
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ALl the Livers were the same mass. The liver was placed in test tubes and hydrogen peroxide was then added to each.
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Liver Condition Rate of fizzing
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11
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