Catecholase 281 29 PDF

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Enzyme Lab Report Biol 101L Section: 401

Name: Rui (Sarah) Wang Honor code: I pledge that I have neither given nor received unauthorized assistance on this assignment and it is entirely my own creative work.

Introduction Enzymes make reactions in cells to go at a speed that is necessary to maintain life. Some materials can be changed from one to another and the rating of change can be faster depend on the presence of certain chemicals. These chemicals are known as catalyzes, which to speed up a reaction without being changed or consumed. Enzymes are proteins that acts as catalysts in our cells, and increase the rate of a reaction by lowering its activation energy. The reaction normally produces the brown color seen in damaged plant parts, or fruits The substrate for a catecholase reaction is catechol and dioxygen, producing benzoquinone and water. Catechol + ½ O2

catecholase

benzoquinone + H2O.

Enzymes regulate the rate at which chemical reactions proceed without it being altered in the process. An example of this would be the breakdown of sucrose in to glucose and fructose. The reaction would occur spontaneously. Enzymes also aid in digestions of food by breaking down large nutrient molecules such as proteins or carbohydrates into small molecules. Enzymes typically end in the suffix of “-ase”. 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. In other factors, enzyme may be affected by heat or a particular speed. However, enzymes become saturated when the substrate concentration is high. In a reaction of catecholase enzyme, which occurs mostly in plants and fruits such as apples or potatoes when it gets cut open, oxygen will oxidize the exposed catechol, producing water. This oxidation of catechol results in the product benzoquinone, which acts as an antiseptic for the plant. The catecholase enzyme solution in this experiment was taken from white potatoes.

The process of extracting the catecholase enzyme solutions began by a chilled potato and the skin of it was peeled off because potato skin does not contain catechol and this way is good for making sure that the experiment is clear. 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 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 potatoes were blended in intervals to avoid denaturation of 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, which was sealed and kept on ice. The cheesecloth acted as a filter-to-filter out the pulp or meat of the potatoes, which left the enzyme catecholase. The solutions were immediate sealed to prevent oxygen from reacting with the catechol from the blended potatoes. Enzymes 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 slow done 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 sued 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 citrous fruits such as oranges and lemon, and is used as cleanser, disinfectants, and softens water.

Materials and Methods In the lab, we performed an initial procedure as a control to make sure that the presence of only catechol or catecholase would not produce significant amount of benzoquinone because this 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 read at 540nm, which was seen from our eyes as green light. The brownish orange color of benzoquinone was the result of an orange wavelength that was reflected off from the benzoquinone. 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 and will 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 was 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.

Three test tubes were set up as shown: Tube 1 was added 2 mL of catechol solution, and 3 mL of distilled water, Tube 2 was added 2 mL of catechol solution, 1 mL of enzyme solution, and 2 mL of distilled water, and Tube 3 has only 5 mL of water. Tube 3 acted as the blank, or the calibration tube. All three tubes were labeled carefully not to block the path of the emission light, and was covered with parafilmed and inverted to shake well. Turning the tubes upside down begin the reaction because all solutions were mixed, and agitated. Volume was constant to prevent inclusion of other independent variables into the experiment. The enzyme was added last so all tubes can catalyze at the same time. Each tube was wiped with Kimwipes in order to remove factors such as finger prints that might prohibit the machine light going through the tube to get the testing result. The tubes were prior to reading to prevent oil or excess contents from interfering with the light from the spectrophotometer. 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. After the initial readings were done for the two tubes, they were placed into a 25C water bath to make sure the heat would not affect our testing result, and gently shook after 5 minutes. The tubes were shook to ensure everything was mixed well. After 10 minutes, blanked the spectrophotometer with Tube 3 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, four test tubes were set up and labeled properly with each test tubes containing 1 mL of enzyme. Tube 1 was pipetted in 2mL of EDTA, Tube 2 pipetted in 2 mL of PTU, Tube 3 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 parafilmed and inverted to shake every 2 minutes to mix well in order to let the chelating agent and enzymes be

well combine. All test tubes sat at room temperature for a minimum of 10 minutes; check with 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. Prior to each reading, tube 5 was inserted to use as the blank. All tubes were placed in a 25C 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 In the initial procedure, a color change was observed in Tube 2 due to the addition of the 1 mL of enzyme solution (Table-1), which benzoquinone was produced, and a dark orange/brown color was shown. Tube 1 formed a slight yellow color but it was not as apparent as Tube 2’s color since it does not have the enzyme added in. Test tube 1 was the control in this experiment. Both tube contained the same amount of catechol, but differs in the amount of distilled water added to the tube, and the enzyme addition.

Table-1 Tube

Absorbency at time 0

Absorbency at 10

Color Change

1 2

-0.017 1.32

Minutes 0.015 1.725

Light yellow Browndark orange

0

brown none

3

0

Table-2 Tube

Absorbency at 10

Absorbency at 20

Color Change

1

Minutes 1.598

Minutes 1.124

Light browndark

2 3 4

0.920 0.803 1.216

1.029 0.843 1.638

brown Same Same Light browndark

5

0

0

brown Same

Graph-1 1.8 1.64

1.6

1.6 1.4

1.22 1.2

1.12 1.03 0.92

1

0.80.84

0.8

Absorbency at 10 Minutes Absorbency at 20 Minutes

0.6 0.4 0.2 0 1

2

3

0 0 5

4

Graph-2 2 1.73 1.5

1.32

1 Absorbency at time 0 Absorbency at 10 Minutes 0.5 0.02 0

-0.5

-0.021

2

0 3 0...