Formal lab report (3)

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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

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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

5 Table 1: Average and Standard Deviation of Effect of PH ALP Activity Enzyme Activity (A/s) pH Average SD Acidic -1.67E-06 2.89E-05 Neutral 1.28E-04 5.77E-06 Basic 3.53E-04 2.3094E-05 Figure 1 This graph shows the effect of PH levels: acidic,neutral,and basic. The enzyme activity changes the pH. The line is the class average of the activity. The acidic level was the lowest, and it increases to the basic level due to enzyme activity. Table 1 The enzyme activity average and standard deviation of acidic, neutral, and basic.

6 Table 2: Average and Standard Deviation Effect Of Concentration on ALP Activity Enzyme Activity (A/s) Enzyme Concentration Average SD Low 2.17E-04 7.59E-05 Medium 6.04E-04 1.22E-04 Medium-High 1.39E-03 6.51E-04 High 2.72E-03 0.000734713 Figure 2 This graph shows the concentration of enzyme activity: low, medium,Medium-High. Both the concentration and enzyme activity are increasing from medium to high. Table 2 The enzyme activity average and standard deviation of the enzyme concentration are low, medium, and medium-high.

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7 Table 3: Average and Standard Deviation Effect Of Temperature On ALP Activity Enzyme Activity (A/s) Temperature Average SD 4°C 1.45E-04 5.77E-06 20°C 1.50E-04 0.00E+00 32°C 2.58E-04 5.20E-05 60°C 1.41E-04 4.2339E-05 Figure 3 This graph shows the effect of temperature on ALP activity. The temperature increases to 32 °C and then starts to decrease after that temperature. Table 3 The enzyme activity average and standard deviation increase as the temperature increases.

8 Discussion: In Figure 1, the optimum pH for ALP activity was basic because of the increase in the average enzyme activity. The data for the acidic pH (-1.67E-06), The neutral Ph (1.28E-04), and the basic Ph (3.53E-04).The null hypothesis for pH states that the pH will not affect the rate of enzyme activity, which was rejected, and the alternative states that the pH will affect the rate of enzyme, which was supported. Figure 2 it tested the effect of enzyme concentration from low to high, and based off the graph you could see the enzyme activity increase. The low concentration( 2.17E-04), The medium concentration (6.04E-04), The medium-high concentration (1.39E-03), and the high concentration (2.72E-03). According to (lee,2022) In organisms, oxidative stress increases the activity of antioxidant enzymes, but excessive oxidative stress inhibits the activity of antioxidant enzymes, which can lead to apoptosis. The ALP was more affected in the medium enzyme concentration, making the medium the optimum. The null hypothesis states that the enzyme concentration will not affect the rate of enzyme activity, which was rejected, and the alternative hypothesis states that the enzyme concentration will affect the rate of enzyme activity, which was supported. In Figure 3, the temperature had an increase going up to 32° C and then started to decrease after 32°C.The enzyme activity was 4°C(1.45E-04),20°C(1.50E-04), 32°C(2.58E-04),and 60°C(1.41E-04).The optimum for temperature ALP is 32° C (2.58E-04). According to (Nakanishi,2011) states that “The results demonstrate that low temperatures induced decreasing -glucosidase inhibitory activity, while the induction of newly grown shoots by the scission of branches induced increasing -glucosidase inhibitory activity.’’The null hypothesis states that temperature will not affect the rate of enzyme activity, which was rejected, and the alternative hypothesis states that temperature will affect the rate of enzyme activity, which was supported. Some errors that could have been caused in the experiment is that the

9 micropipette may have had air bubbles, and adding too much or too little concentration to the solution could have been a human error, too. Literature Cited Lynggaard, L. S., Rank, C. U., Hansen, S. N., Gottschalk Højfeldt, S., Henriksen, L. T., Jarvis, K. B., Ranta, S., Niinimäki, R., Harila-Saari, A., Wolthers, B. O., Frandsen, T. L., Heyman, M., Schmiegelow, K., & Albertsen, B. K. (2022). Asparaginase enzyme activity levels and toxicity in childhood acute lymphoblastic leukemia: a NOPHO ALL2008 study. Blood advances, 6(1), 138–147. https://doi.org/10.1182/bloodadvances.2021005631 Nakanishi, H., Onose, S., Kitahara, E., Chumchuen, S., Takasaki, M., Konishi, H., & Kanekatsu, R. (2011). Effect of environmental conditions on the α-glucosidase inhibitory activity of mulberry leaves. Bioscience, biotechnology, and biochemistry, 75(12), 2293–2296. https://doi.org/10.1271/bbb.110407 Lee, D. W., Song, J. A., Park, H. S., & Choi, C. Y. (2022). The effects of low pH and high water temperature on oxidative stress and cell damage in juvenile olive flounder Paralichthys olivaceus: comparison of single and combined environmental conditions. Fish physiology and biochemistry, 48(5), 1251–1264. https://doi.org/10.1007/s10695-022-01112-1 Vimalraj S. (2020). Alkaline phosphatase: Structure, expression and its function in bone mineralization. Gene, 754, 144855. https://doi.org/10.1016/j.gene.2020.144855

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10 Nguyen, J., Mertzlufft, D., Koerner, S., Corneille, K., Daniels- Abdulahad, M., Raja, A., & Schmitt-Lavin, E. (2022). Biology 1500 Laboratory Manual (3rd ed.). Morton Publishing Company.

Formal lab report (3)

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