PART I: A SIMPLE ENZYME-CATALYZED REACTION
When you cut open an apple, banana or pear, you cut through many cell walls and release the complex contents of many cells. Left exposed to the air, the white surface of the fruit will turn brown in a short time. This familiar color change is a good example of an enzyme catalyzed reaction. The brown color comes from the reaction of oxygen in the air with catechol in the fruit to form benzoquinone. Catechol (the substrate) is clear and benzoquinone (the product) is brown. This browning reaction is catalyzed by catecholase, an enzyme that occurs naturally in the fruit.
catecholase Catechol + 1/2 O2 ————> benzoquinone + H2O
(clear) (brown)
This simple browning reaction can be used to illustrate several important properties of enzymes in general. In Part I, you will make some preliminary qualitative observations on the nature of enzyme catalyzed reactions by recording how environmental factors affect the browning reaction. For example, must the fruit be exposed to air to turn brown? Does temperature affect the rate of browning? Is the browning rate changed by the presence of an acid, like lemon juice? You will be asked to use the observations you make in Part I to formulate qualitative hypotheses about how enzymes work. In Part II you will test your hypotheses quantitatively.
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7.1 Exercise 1 – Browning of fruit
In this exercise you will compare the relative amount of browning that occurs in banana slices placed under six different conditions. Methods 1. Work with a partner. 2. Set up the six experimental treatments (described below) before you obtain a piece of banana. The comparison of browning rates will be easier if all the slices begin browning at the same time.
To observe the effect of contact with air (oxygen):
Slice 1: leave open to the air at room temperature.
Slice 2: immediately wrap tightly in saran wrap.
To observe the effect of temperature:
Slice 3: place in a beaker of ice.
Slice 4: place in a petrie dish on a warming plate.
To observe the effect of acidity (pH):
Slice 5: keep moist with distilled water in a petri dish.
Slice 6: keep moist with lemon juice in a petri dish. 3. Acidity is measured in terms of a unit called pH. On the pH scale, any value below 7 is considered acidic, anything above 7 is basic, and 7 is neutral. Acids typically have pH values between 2 and 6. The stronger the acid, the lower the pH value. Use litmus paper to determine the acidity (pH) of lemon juice and water. Lemon juice pH = ______ Distilled water pH = ______ 4. From a fresh banana, cut 6 equal, unpeeled slices. Working quickly, place one slice in each of the conditions described in step 2. 5. Check the sections every 3-5 minutes for the first half hour. In Table 1 record (a) the time you first detect browning, and (b) the extent of subsequent browning (if any).
Between observations, begin Part II.
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Results Table 1: Extent of browning under different environmental conditions.
Minutes elapsed to first browning
Extent of further browning
Slice #1 (open to air)
Slice #2 (wrapped)
Slice #3 (cold)
Slice #4 (warm)
Slice #5 (distilled water)
Slice #6 (lemon juice)
Analysis and Discussion l. Look at the browning rates of slice #1 (exposed to air) and slice #2 (wrapped in plastic). a. Which one turned brown faster? Why? (With what is the catechol reacting?)
b. If your observations are not what you expected, consider this. Ripening fruit releases ethylene, a gas that speeds the ripening process. Saran Wrap Classic is made of polyvinyl and Saran Wrap with Cling Plus (formerly HandiWrap) is polyethylene. How might this affect browning rate? c. How could you test whether the wrapping material is increasing browning rate?
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2. If you use the browning rate of slice #1 (left out at room temperature) as your standard of reference (called your experimental “control”) : a. What effect (if any) did cooling have on the browning rate? b. What effect (if any) did warming have on browning rate? c. What do these observations suggest about the effect of temperature on enzyme-catalyzed reactions? — Based on these observations, what should happen to the rate of browning as temperature increases? State this in the form of a hypothesis. HYPOTHESIS A: “As temperature increases, the rate of an enzyme catalyzed reaction (browning) should ___________________________.” (Choose one: increase, decrease, or remain the same) 3. Comparing the browning rates of slice # 5 (treated with distilled water) and slice #6 (treated with lemon juice). a. What effect did lemon juice have on the rate of browning? b. What do these observations suggest about the effects of acidity on enzyme activity? — Based on these observations, what should happen to browning rate as pH increases? (Remember, the stronger the acid, the lower the pH.) State this in the form of a hypothesis. HYPOTHESIS B: “As pH increases (meaning the environment becomes less acidic), the rate of an enzyme catalyzed reaction (browning) should _________________________________.” (increase, decrease, or remain the same) These last two hypotheses are based on the qualitative observations you made in Part I. They may or may not be correct. In Part II, you will test these last two hypotheses quantitatively.