Bio Lab Report - Altering Catecholase’s Enzyme Activity Through the Use of pH, Temperature, Enzyme PDF

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Altering Catecholase’s Enzyme Activity Through the Use of pH, Temperature, Enzyme Concentration, and Substrate Concentration ...

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Altering Catecholase’s Enzyme Activity Through the Use of pH, Temperature, Enzyme Concentration, and Substrate Concentration Kennedy Parker BY 123L-BZA

Introduction Enzymes are a macromolecule that serves as the body’s biological catalyst. They are used to speed up the rate of a chemical reaction and lower the activation energy barriers without being consumed or destroyed (Campbell and Reece, 2008). The reason for this study is to get a better understanding of how enzyme activity can be altered. Some factors that can change an enzyme’s activity are temperature, pH, substrate concentration, and enzyme concentration (Helms et al, 1998). The enzyme can either denature, which means that is has been altered and cannot function properly (Newman, 2018). The enzyme that is being used for this experiment is catecholase. Catecholase can be found in fruits or potatoes that have turned brown due to the catechol compound being oxidized (Helms et al, 1998).

Hypothesis For the temperature experiment, the null hypothesis is that temperature does not have an effect on the enzyme. The alternative hypothesis is that a damage in the temperature does have an effect on the enzyme. For the pH experiment, the null hypothesis is the buffer will have little or no effect on the enzyme’s activity, however the alternative hypothesis is that the enzyme activity is affected by a change in pH. The null hypothesis for the enzyme concentration is that the substrate concentration will not affect the enzyme activity while the alternative hypothesis is the higher your enzyme concentration, the more product will be produced. Lastly, the null hypothesis for the substrate concentration experiment is the enzyme concentration will not have an affect on the enzyme concentration and the alternative hypothesis is the higher the concentration the more product will be made.

Materials & Methods For the temperature experiment you will need, a pH 7 phosphate buffer, potato juice, catechol stock, water, ice, and a thermometer. Make sure that the cold temperature is 2 degrees Celsius, the room temperature is 24 degrees Celsius, and the warm temperature is 62 degrees Celsius. Add 3 ml of the pH 7 phosphate buffer into 3 tubes. Each test tube was placed into each temperature treatment. While those test tubes are reaching their goal temperatures, fill two test tubes with 3 mL of potato juice and place one in the ice-water bath and the other in the hot water bath and let them reach their goal temperatures. Do the same thing with the catechol stock. After 10 minutes, add 10 drops of the room temperature catechol stock and room temperature potato juice to the test tube at room temperature. Do this step with your test tube at cold temperature and the test tube at the high temperature, but make sure to add the matching temperature to the correct test tube. Allow the three test tubes to stand for 5 minutes in their respective temperature (while shaking the mixture multiple times during the 5 minutes) and record your results. For the pH experiment, you will need pH 4, pH 6, pH 7, pH 8, and pH 10. For each tube, add 3 mL of the matching phosphate buffer. Add 10 drops of the catechol and 10 drops of potato juice into each tube. Cover the tube with Parafilm and invert it several times to ensure the contents have mixed. After inverting the test tubes, remove the Parafilm and allow the tubes to stand for 3-5 minutes and continue to invert the tubes in 1- minute intervals. Record your results. For the enzyme concentration experiment: In test tube A, add 3 mL plus 20 drops of the pH 7 phosphate buffer. In test tube B, add 3 mL plus 15 drops of the pH 7 phosphate buffer and 5 drops of potato juice. In test tube C, add 3 mL plus 10 drops of the pH 7 phosphate buffer and 10 drops of potato juice. In test tube D, add 3 mL of the pH 7 phosphate buffer and 20 drops of potato juice. Cover the tube with Parafilm and invert several times to ensure the contents have

mixed. Add 10 drops of catechol to each tube. Cover the test tube again and invert to mix the content. Let the tubes stand for 3-4 minutes without the Parafilm on the tube (mix the contents in 1- minute intervals). Record your results. For the substrate concentration experiment: For test tube 1, add 5 mL plus 47 drops of the pH 7 phosphate buffer and 1 drop of catechol. For test tube 2, add 5 mL plus 46 drops of the pH 7 phosphate buffer and 2 drops of catechol. For test tube 4, add 5 mL plus 44 drops of the pH 7 phosphate buffer and 4 drops of catechol. For test tube 8, add 5 mL plus 40 drops of the pH 7 phosphate buffer and 8 drops of catechol. For test tube 16, add 5 mL plus 32 drops of the pH 7 phosphate buffer and 16 drops of catechol. For test tube 24, add 5 mL plus 24 drop of the pH 7 phosphate buffer s and 24 drops of catechol. For test tube 32, add 5 mL plus 16 drops of the pH 7 phosphate buffer and 32 drops of catechol. For test tube 48, add 5 mL of the pH 7 phosphate buffer and 48 drops of catechol. Cover each tube with Parafilm and invert them to mix the contents. Add 30 drops of diluted potato juice extract to each tube. Cover the tube and mix the contents. Incubate the mixtures for 5 minutes without the Parafilm (mix each tube at 1-minute intervals). Record the color for each tube as your observations.

Results In Table 1 it is shown that when the temperature increased and decreased there was only a tint, so there was not enough product being made. In Table 3, there was an increase each time. It is clearly shown in Table 4 that the higher amount of catechol drops you added to your mixture, the more product was produced. That is why you can see an increase in the color intensity.

Table 1. Effect of Temperature on Enzyme Activity

Temperature Treatment

10 degrees

24 degrees

50 degrees

Intensity of Color

Celsius +

Celsius ++

Celsius +

The color intensifies as the enzyme is close to its optimum temperature. The color represents when the enzyme is functioning or working properly. Table 2. Effect of pH on Enzyme Activity KEY pH

pH 4

pH 6

pH 7

pH 8

pH 10

Intensit

0

++

++

++

0

0 = No Color +, ++, +++ = increasing intensities of color

y of Color

As the enzyme reaches its optimum pH, the color intensifies. The color represents when the enzyme is functioning or working properly. Table 3. Effect of Enzyme Concentration on Enzyme-Catalyzed Reactions

Enzyme

A

B

C

D

0

+

++

+++

Concentratio n Intensity of Color

As the enzyme concentration increased so did the intensity of color. Table 4. Effect of Substrate Concentration on Enzyme Activity

Substrate

1

2

4

8

16

24

32

48

0

+

+

++

++

++

+++

+++

Concentratio n Intensity of Color

As the substrate concentration increases, so does the intensity of color.

Discussion Catecholase’s activity can be altered by temperature and pH. If they are too high, the enzyme will not do anything, and if they are too low, the enzyme will not react or take a long time to react. When the substrate and enzyme concentration are high, the product will come out. The data supports all of my alternative hypotheses that were given in the introduction. The only thing that could have been changed throughout all of the experiments is that the temperature experiments were ran at the same time because it would’ve been easier to compare the color change.

Conclusion Temperature, pH, enzyme concentration, and substrate concentration can influence an enzyme’s activity. If the temperature increases it can possibly denature the enzyme, thus causing the reaction not to occur. If the temperature decreases, the reaction does occur, it just causes the rate of the reaction to be a little slower. pH can stop an enzyme’s activity if it is too high or too low because it’ll denature. The higher your enzyme and substrate concentration, the more product it can produce. This information is important to us because it gives us a better understanding how enzymes in our body can be impacted and why it is important that our body maintains homeostasis because if it doesn’t it can lead to important enzymes to be denatured.

References Campbell, Neil., Jane Reece. 2008. Biology, 8th ed. Don O’Neal ed. Benjamin Cummings Publishing, Menlo Park, California. pp. G-1. / Helms, Doris., Carl Helms., Robert Kosinski., John Cummings. 1998. Biology in the Laboratory, 3rd ed. Judith Wilson ed. Freeman Publishing, New York, New York. pp. 10-1-10-14. Newman, Tim. “Enzymes: Function, Definition, and Examples.” Medical News Today, MediLexicon International, 11 Jan 2018, www.medicalnewstoday.com/articles/319704...