Enzyme Lab Final PDF

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Enzyme Lab Report (FINAL) Aaron Chen BIOL 101L-433 TA Daniela Munoz

UNC Honor Pledge: I certify that no unauthorized assistance has been received or given in the completion of this work. Experiments described were performed by me and/or my lab group and this write-up is entirely my own creative work.

Signature: ______________________________

Introduction Everything that happens within the human body is carried out by the work of enzymes, which is a substance produced by a living organism that acts as a catalyst to bring about a specific biochemical reaction. The purpose of enzymes is to speed up chemical reactions in breaking down or building up a molecule. Enzymes are very important in several systems throughout the human body in order to expedite different chemical reactions that are necessary to maintain the bodily processes of life. They are also known to be a catalyst, which are substances that speed up the rate of a chemical reaction. Early scientist made an attempt to change substances from one form to another. In doing so they discovered there was a certain substance that increased the speed in this change. They called this substance a catalase. An enzyme can be identified typically with the suffix of “-ase.” The enzyme catecholase for example, is commonly found in fruits and other plants. When a plant comes across an injury, they begin the healing process, as would most living things. When the inside of the fruit or vegetable is exposed to oxygen, the catechol is oxidized (electrons removed from). Benzoquinone is produced and acts as an antiseptic for the fruit. The reaction is as follows: Catechol + ½ O2 (in the presence of catecholase)  Benzoquinone + H2O. The substrate in an enzymatic reaction is that what the enzyme is acting upon, and the resulting molecules are called products. The rate of a chemical reaction increases as the substrate concentration increases. Enzyme may also be affected by the levels of heat or speed. However, enzymes can also become saturated when the substrate concentration is high. The catecholase enzyme solution in this experiment was taken from white potatoes. The process of extracting the catecholase enzyme solutions began when the skin of the potato was peeled off because potato skin does not contain catechol and this way is good for

making sure that the experiment’s data would not be obstructed. We used chilled potatoes and chopped it into small pieces and added into a chilled blender. The potatoes were chopped to allow more surface area and less volume to extract the solution easily. The chilled potato was added into 500 mL of chilled distilled water, and blended for three 10-second bursts. The reason for chilling all the components was to slow down all cellular mechanism, which in this case to prevent the production of benzoquinone. Water was distilled to ensure there weren’t any inorganic elements added to the solution. The distilled water also allowed it to have a hypotonic solution, which busted the potato cells also aided in extracting the catechol. The potatoes were blended in short-intervals in order to avoid denaturing the enzyme. Blending the potatoes simulated the “injuries” of the potatoes; releasing catechol exposed to the oxygen, and formed the catecholase enzyme. The solutions were then strained through the cheesecloth, and funneled into a container until over-full to prevent oxidation, which was immediately sealed and kept on ice. The cheesecloth acted as a filter-to-filter out the pulp or the “meat” of the potatoes, which separated the enzyme catecholase. The solutions were immediate sealed to prevent oxygen from reacting with the catechol from the blended potatoes. Enzymes also required a cofactor in order to bind the substrate and catalyzed the reaction. Cofactors are non-protein chemical compound, and are mostly derived from vitamins. Our hypothesis of this enzyme experiment is Mg2+ is the cofactor that slows down the enzyme (catecholase) reaction with substrate (catechol). Cofactors can be divided into two groups, organic cofactors and inorganic cofactors. Organic cofactors include Flavin, or heme, and inorganic cofactors includes metal ions such as Mg2+, Cu+, or Mn2+. Potential cofactors were removed from solutions, and measured a change in enzyme activity. EDTA was used to bind Calcium and Magnesium; PTU was used to bind Copper; Citric acid is a weak organic acid that

binds strongly to Copper. An experiment was performed to determine if removing one of these ions would prevent enzyme activity. EDTA was used mostly in food as a preservative to help it from being spoiled by preventing Calcium and Magnesium ions from enzymatic reaction by bacteria and fungi. PTU was used to treat hyperthyroidism, has a bitter taste to be used as a common safe diagnostic genetic taste test in humans, where it inhibits an enzyme involved in thyroid hormone synthesis. PTU also binds to copper to be used as silver polish. Citric acid occurs naturally in citrus fruits such as oranges and lemon, and is used as cleanser, disinfectants, and softens water. I hypothesis that since all the ions has a equal amount of valance charge, than each ion are just as essential in helping catecholase repair cells. Materials and Methods In the initial procedure portion of the lab, we performed an experiment to make sure that the presence of only catechol or catecholase would not produce significant amount of benzoquinone because the control is very important for the cofactor experiment. A spectrophotometer was turned on for 15 minutes for the lamp to warm up, and was set to 540nm, which is visible to the human eye as green light. A brownish orange color was seen as the result of an orange wavelength that was appeared on the bite of the potatoes signifying that the benzoquinone is reflecting it. Using this ideology, green light was used because benzoquinone absorbed it. Therefore the spectrophotometer measured the absorbance or green light as catecholase catalyzed the conversion. Catechol is colorless, so it would not absorb green light as well. A spectrophotometer was used instead of our eyes because it provides more accurate quantitative reading instead of qualitative and subjective, which is neither measureable nor reliable. If the spectrophotometer were set to read orange color, there would be little to no absorbance since benzoquinone reflects it to our eyes. The spectrophotometer was calibrated

with a blank test tube to established a minimum value of absorbency, close to zero as possible. Four test tubes were set up as shown: Tube 1 consisted of 2 mL of catechol solution, and 3 mL of distilled water. Tube 2 consisted of 4 mL of distilled water and 1 mL of enzyme solution. Tube 3 consisted of 2 mL of catechol solution, 1 mL of enzyme solution, and 2 mL of distilled water. Tube 4 consisted of only 5 mL of water. Tube 4 acted as the blank, or the calibration tube. All four tubes were labeled carefully not to block the path of the emission light, and was covered with Parafilm and inverted to shake well. Turning the tubes upside down begin the reaction because all solutions were mixed. Volume was constant to prevent inclusion of other independent variables into the experiment. The enzyme was added last so all tubes can catalyze the reaction at the same time. Each tube was wiped with Kimwipes in order to remove factors such as fingerprints that might prohibit the machine light going through the tube to get the testing result. The calibration tube was inserted to the spectrophotometer, and pressed the measure blank button to zero the machine. Tube 1 was inserted and recorded the absorbency, and followed by Tube 2 immediately, and Tube 3 followed immediately after Tube 2. After the initial readings were done for the three tubes, they were placed into a 25 degrees Celsius water bath to make sure that the heat would not affect our testing result, and it was gently shook after 5 minutes. The tubes were shook in order to ensure everything was mixed well. After 10 minutes, reset the spectrophotometer with Tube 4 after wiping with Kimwipes, and inserted Tube 1 and Tube 2 for readings. All test tubes were wiped prior to taking readings. In the cofactor experiment, five test tubes were set up and labeled properly with each test tubes containing 1 mL of enzyme except for the calibration tube (Tube 5). In addition, tubes 1-3 are the experiential tubes that contain their individual chelating agent such as EDTA, PTU, and Citric acid. Tube 1 was pipetted in 2mL of EDTA, Tube 2 was pipetted in 2 mL of PTU, Tube 3

was pipetted in 2 mL of citric acid, and Tube 4 pipetted in 2 mL of water. Tube 5 contained 5 mL of water to be used as a calibration blank. Each tube was covered with parafilm and inverted to shake every 2 minutes to mix well in order to let the chelating agent and enzymes begin their chemical reaction. All test tubes sat at room temperature for a minimum of 10 minutes; check the absorbance rate on the spectrophotometer and observe colors change. Then 2 mL of catechol was added to all four test tubes, and immediately inserted to the spectrophotometer for initial reading. You do not add in the catechol in with the other mixtures, because it will oxidize into benzoquinone when the other tubes are unprepared. Prior to each reading, tube 5 (calibration tube) was inserted to use as the blank. All tubes were placed in a 25 degrees Celsius water bath for 10 minutes, and then a second reading was measured and colors observed. Tube 4 was the control since no chelating agent was added.

Result The tubes contents were modified for this experiment as we were instructed to do so (Table 1 and Table 3). In the initial procedure, Tube 3 seems to have the most reaction as it changed to the darkest color after all the contents were put into the tube. Following after is, Tube 1 as it is not as dark as Tube 3, but darker than Tube 2. Tube 2, which only contains water and enzyme didn’t really have much effect; therefore the cloudy color must be due to the enzyme’s original color (Table 2). This data seems to suggest the darker the color, the more rate of absorbance it has (Graph 1). For the experiments in testing the co-factors on the other hand, the lower the rate, the more impact that co-factor has on the reaction since cleating agents were used to prevent them from doing their job (Graph 2). Tube 4 which replaced chelating agents with

water has the highest rate therefore acts as the control. The clearer the tube is, the more likely the ion it suppressed has a impact on the enzymatic reaction, exception to the calibration tube. Discussion My hypothesis was clearly false, as some ions or cofactors had more rates of absorbency than others. This data suggested that copper is the most crucial to the enzymatic reaction as it had the lowest absorbency when bound to prevent reaction. Cofactors such as copper, calcium, and magnesium were tested by using chelating agents to bind them to prevent them from catalyzing the enzymatic reaction. As a result, their efficiency entirely depended on low absorbance rate. Since Tube 4 was the control, we would have thought that it would have the highest absorbance rate as the enzymatic reaction there were no chelating agents binding it. This may have been a possible error such as miscalculation on the portion of the mixture such as having lest EDTA chelating agents, that Tube 1 had fingerprints. There may also be possible errors such as inaccurate timing mixing the catechol into the solution or the water bath. If this is not the case, we would suspect that there are cofactors of the enzyme that could actually slow down the enzymatic reaction rather than speeds it up. The rate of absorbance was calculated by subtracting the absorbency at 20 minutes to the absorbency at 10 minutes. I concluded this experiment may have possible errors, but suggests that copper bound by Citric acid is the most effective for the enzymatic reaction of benzoquinone.

Appendix: Table 1- Initial Procedure Tube Contents Tube 1 2 mL catechol 2 mL EDTA

Tube 2 1 mL enzyme 4 mL dH20

Tube 3 1 mL enzyme 2 mL catechol 2 mL Citric Acid

Tube 4 5 mL dH2O

Table 2- Initial Procedures Changes Tube

Absorbency at 0

Absorbency at 10

Color Change

1 2 3

Minutes 0.005A 0.2190A 0.218A

Minutes 0.223A 0.225A 0.437A

Dark brown Cloudy Darkest, apple juice

4

0.000A

0.000A

color Clear

Table 3- Testing Co-factor Tube Contents Tube 1

Tube 2

Tube 3

Tube 4

1 mL enzyme 2 mL catechol 2 mL EDTA

1 mL enzyme 2 mL catechol 2 mL PTU

1 mL enzyme 2 mL catechol 2 mL Citric Acid

1 mL enzyme 2 mL catechol 2 mL dH2O

Table 4- Co-factors Changes at Timed Intervals

Tube 5 (calibration)

5 mL dH2O

Tube

Absorbency at 10

Absorbency at 20

Color Change

1

Minutes 0.081A

Minutes 0.223A

Light browndark

2 3 4

0.120A 0.106A 0.183A

0.141A 0.113A 0.269A

brown Cloudy Clear Cloudy Light browndark

5

0.000A

0.000A

brown Clear

Graph 1- Initial Rate of Enzymatic Reaction

Rate of Absorbence 0.25

0.2

Rate of0.15 Absorbency

Rate of Absorbence

0.1

0.05

0 1

2

3

4

Graph 2 - Testing Co-factors 0.3

0.25

0.2

Rate of Absorbency in (A) Absorbency at 10 Minutes Absorbency at 20 Minutes

0.15

0.1

0.05

0 1- EDTA

2- PTU

3- Citric Acid

4- Water

5-(Cali)

Citation Page: Stegenga, Barbara. "Enzymes." Laboratory Exercises for Biology 101. Fall 2015-Spring 2016 ed. N.p.: Hayden McNeil, n.d. 83-89. Print....