Effects of pH on enzyme-catalyzed reactions
The pH (acidity) of the environment can affect the molecular bonds that maintain the shape of an enzyme’s “active site,” the site on the enzyme molecule responsible for binding to the substrate or substrates. A pH value of 2 is highly acidic, 7 is neutral, and 11 is highly basic. For Exercise 3, set up test tubes containing potato extract (a good source of the enzyme catecholase), catechol (the substrate), and 5 different pH buffer solutions. Set up all 6 test tubes with everything in them (see below) except the catechol. Add the catechol last, so all the reactions will start at about the same time. 1. Obtain 6 test tubes and use a wax pencil to mark all the tubes near the top with your initial and either numbers 1 through 5 or “B” for the calibration blank. 2. Fill the 5 tubes as follows: Sample 1: 1 ml potato extract, 4 ml buffer for pH 3 . Sample 2: 1 ml potato extract, 4 ml buffer for pH 5. Sample 3: 1 ml potato extract, 4 ml buffer for pH 7. Sample 4: 1 ml potato extract, 4 ml buffer for pH 9. Sample 5: 1 ml potato extract, 4 ml buffer for pH 11. Blank: 1 ml potato extract, 4 ml buffer for pH 7, plus 2 ml distilled water. The 2 ml of water is a substitute for the 2 ml of catechol that will be added to the other test tubes but not to the blank tube. You will need only one blank, because all 5 of the pH buffer solutions are clear and have identical absorbency properties. 3. Cover each tube with parafilm and invert to mix. Stand all 6 tubes in the test tube rack. 4. Now add 2 ml of catechol to the 5 sample tubes, put the parafilm back on, and again invert the tube to mix the contents. You do not need to uncover the tube for the reaction to proceed; the solution has plenty of dissolved oxygen. Keep the test tubes at room temperature. DO NOT PUT THE TEST TUBES IN THE WATER BATH! In this experiment we are measuring the effects of pH, not temperature. 5. If you see “cloudiness,” it means either than you mistakenly picked up particulate matter from the bottom of the flask, or that an unwanted precipitate is forming. A cloudy white precipitate often forms if the pH gets too low (pH = 3). A grayish black precipitate sometimes forms if the pH gets too high (pH = 11). When precipitates are formed by reactions that have nothing to do with “browning,” they can distort your data, causing you to overestimate the rate of browning at very low and very high pHs. So record any color changes and include them in your lab report. 6. About a minute before step 6, use your blank to calibrate the Spec 20. 7. Allow the browning reaction to proceed for exactly 5 minutes. Then insert the sample tubes, one at a time in numerical order, into the Spec 20 and record the absorbances in Table 3. If the reaction ran longer than 5 minutes before you took your measurement, record the exact number of minutes and use
40
this same time interval when you measure the other sample tubes. Record the time in Table 3. Note any color changes in the test tubes. 8. If you experience measuring delays, you will need to control for the fact that the reaction product continues to form while you are waiting to measure it. After measuring the absorbance in tubes 1, 2, 3, 4 and 5, measure them again in reverse order (5, 4, 3, 2, 1) and use the average absorbance for each pH. Results Table 3: Effect of pH on extent of browning
Sample pH Absorbance after ( ) minutes
Any color changes? (See #5 above)
Blank * zero
1 3
2 5
3 7
4 9
5 11
9. Wash all test tubes and return them to the rack. 10. Plot your values for absorbance as a function of pH on Graph 2. Graph 2: Effect of pH on the browning rate