PART II: EFFECTS OF ENVIRONMENTAL FACTORS ON RATE OF ENZYME-CATALYZED REACTIONS
In this section you and a partner will design and carry out experiments to test the two hypotheses you made at the end of Part I concerning the effects of temperature and pH on enzymes. The quantitative techniques you will be using will allow you to measure rates of the browning reaction far more accurately than before. The experiments are intended to give you first hand experience with the methods of scientific inquiry. They also reveal some interesting features of enzyme-catalyzed reactions. The Spectrophotometer The Spec 20 is a device that measures how “dark” (opaque) a liquid is. The Spec 20 shines a beam of light through the liquid in a test tube and measures how much of the light is absorbed as it passes through the test tube. Measurements are expressed in terms of “absorbance” on a scale from 0 to 2.
You will be using the Spec 20 to measure the rate of the browning (benzoquinone formation) under different conditions. As more and more (clear) catechol is converted to (brown) benzoquinone, less and less light will be able to pass through the test tube, and the percentage of light absorbed will increase. We will be using absorbance to measure the amount of benzoquinone produced under different temperatures and acidities (pH’s).
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Follow this overall, 3-step procedure whenever you need to use the Spec 20. For exercises 2 and 3, you will need to use the Spec 20 to measure rates of reaction. Use these three steps as a reference when you do exercise 2 (page 9) and exercise 3 (page 12). 1. Prepare a reference solution (or “blank”)
The starting solution of enzyme and substrate is not perfectly clear and so absorbs some light even before the browning reaction begins. To correct for this fact, you will be using a “blank,” a test-tube filled with the starting solution as a reference. You use the amount of light absorbed by the blank as your reference point. The Spec 20 can be adjusted to treat the amount of light absorbed by the blank as “zero”. Any additional light absorbed can then be attributed to newly formed benzoquinone. To make a blank:
a. Label a small test tube with the letter “B” for “blank”. This test tube will hold the reference
solution for setting the “0% absorbance” level on the Spec 20. b. Use a pipette and pipette pump to measure 1 ml of potato extract and 6 ml of distilled
water into the tube. c. Cover the tube with a small square of parafilm and invert to mix.
2. Calibrate the Spec 20 (i.e., standardize the internal light level): a. Set the wavelength knob on the Spec 20 to 540 nanometers, a wavelength in the “green” portion of the light spectrum. We use 540 nanometers in this experiment because benzoquinone (the product) absorbs this wave length better than any other. b. With the sample compartment empty and the cover closed, set the transmittance to zero by using the Zero Control (left hand) knob or by following the directions posted next to the Spec 20. c. Wipe off the “blank” tube with a Kimwipe (to remove light-absorbing fingerprints) and insert it into the sample compartment and close the cover.
d. Set the absorbance reading to zero by using the Absorbance (right hand) knob, or by following the posted instructions. Calibrated in this way, the Spec 20 will measure only increases in absorbance above the reference level established by the “blank”. 3. To measure the absorbance of your samples in Exercises 2 & 3: a. Remove the blank. (Blanks get old fast. When setting up the testube series you will be using for each experiment, make a fresh blank at the same time.) b. Insert the sample tube into the sample compartment. c. Close the cover of the sample compartment. d. Read “absorbance ” off the lower dial. e. Remove sample tube and repeat steps 3b-d with each sample.
In Part II, you and your partner should work together on both the temperature experiment (Exercise 2) and the pH experiment (Exercise 3).
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7.2 Exercise 2 – Effect of temperature on enzyme-catalyzed reactions
For Exercise 2, you will be setting up test tubes containing potato extract (a good source of the enzyme catecholase), water, and catechol (the substrate). Even though potato contains catechol, you will be adding extra substrate (catechol from a commercial supplier) to make the reaction go faster. There are several different ways to speed up an enzyme catalyzed reaction. One way is to add more enzyme. Another way is to increase the concentration of the substrate on which the enzyme is working. In this experiment, we will be making the reaction go faster by adding extra substrate (namely catechol). We are not adding extra enzyme. There is plenty of the catecholase enzyme in the potato extract already. Set up all your tubes with everything in them (see below) except the catechol. When everything is ready add the catechol last, so all the reactions start at the same time. 1. Obtain 6 test tubes and a test tube rack. With a wax pencil, mark the tubes near the top with your initials and numbers 1 through 5, plus B (for “blank”, the tube that will hold the reference solution used to calibrate the Spec 20.) 2. With a pipette, measure into each of the 5 tubes: 1 ml of potato extract (a rich source of the enzyme catecholase) and 4 ml of water. Avoid picking up any of the particulate matter (cloudy with particles) that has settled to the bottom of the potato extract flask. Any cloudy material will throw off the measurement of browning rate. 3. To make a “blank,” put 1 ml potato extract and 6 ml of water into the sixth tube. The extra 2 ml of water makes up for the 2 ml of catechol that you will not be adding to the control blank. Cover all 6 tubes with parafilm, invert to mix, and stand the tubes in rack. 4. Do not add catechol yet. Put one sample tube into each of the 5 different water baths in the Central Study Area. In Table 2 record the actual temperatures of each of the baths. 5. BEFORE ADDING THE CATECHOL to your samples, use a thermometer to make sure the temperature of the solution inside your test tube has actually reached the temperature of its water bath. This should take 3-7 minutes. 6. Plan ahead! Read steps #7-9 completely before proceeding. Try to run the 5 tubes simultaneously, or closely together in the same sequence, so that reaction times in the 5 samples will be comparable. 7. Add 2 ml of catechol solution to each of the 5 sample tubes. You will need to remove the tube from the water bath, remove the parafilm, add the catechol, put the parafilm back on, and invert tube to mix the contents. Return each tube to its bath for 5 minutes. 8. Use the blank (test tube “B”) you made earlier to recalibrate the Spec 20. 9. Exactly 5 minutes after adding the catechol, remove each sample tube from its water bath, dry it with a Kimwipe, insert the tube into the sample holder of the Spec 20, and measure absorbance. Quickly repeat for the other 4 tubes, one at a time, in numerical order. Record these values in Table 2.