OVERVIEW In this lab we will explore the reaction between the enzyme lactase and its substrate lactose (i.e. its target molecule). Lactase hydrolyzes lactose to form the monosaccharides glucose and galactose. Review the sections on disaccharides, the glycosidic bond, and enzymes in the text before attempting the pre-lab SAFETY & DISPOSAL onpg is a potential irritant. Be sure to wash your hands after the lab. BACKGROUND Enzymes play a fundamental role in the human body: they speed up reactions that would normally go slowly (or not at all) in the mild conditions typically present in human cells. Enzymes are catalytic, which means that they are not consumed in the reaction. Rather, they are regenerated to their original state, and can therefore continue to speed up the same reaction over and over again. Enzymes are also highly specific, which means that they speed up only one reaction (or one class of reactions). This specificity is due to the high degree of chemical complementarity between the substrate and the enzyme s active site. The chemical arrangement of the active site ensures that only molecules that match the polarity, charge, and shape of the target substrate are converted to product. Kinetics is the study of how fast chemical reactions occur; an enzyme assay is a method used to measure the rate of a reaction in the presence of an enzyme. Enzyme assays are used regularly in clinical situations to determine whether a particular enzyme is working properly in a patient, malfunctioning, or absent. A variety of enzyme assays are available for the diagnosis of common clinical ailments. For instance: Elevated levels of alkaline phosphatase may indicate the presence of bone cancer (osteosarcoma). Low levels of chymotrypsin in the intestine can indicate chronic pancreatitis. Different forms of lactate dehydrogenase (LDH) are found in skeletal muscle cells than heart muscle cells. If a clinical assay reveals elevated levels of the heart LDH in blood, this is an indication that some heart tissue may have been damaged during a heart attack. In this lab, you will assay a human enzyme called lactase (also known as β-galactosidase). This enzyme is found in the intestine, where it breaks down milk sugar (lactose) into its monosaccharides, galactose and glucose. Like all enzymes, lactase is specific: it only binds and splits disaccharides that have a galactose and a glucose connected via a β(1 4) linkage as shown in the figure below. You or someone you know may suffer from lactose intolerance, a condition in which painful bloating and cramps occur after ingesting dairy products. This condition is caused by the lack of the enzyme lactase in that person s intestine; without lactase, the lactose molecules remain intact and are converted into gases (e.g. H 2, CO 2, CH 4) by bacteria in the small intestine. Although most humans (~75%) are lactose intolerant to some Page 1
degree, its prevalence varies widely by ethnicity. One way to diagnose a lactase deficiency is to obtain a sample of the intestinal mucosa via endoscopy, and then perform a lactase assay. Fortunately, the symptoms of lactose intolerance can be overcome by simply adding lactase supplements to one's diet. Of the various commercial sources of lactase, in this lab we will use Lactaid tablets as a source of the enzyme for our assays. The goal of any enzyme assay is to determine the velocity of the reaction in the presence of the enzyme. The velocity is usually defined as the rate of product formation (usually in μmol) as a function of time (seconds or minutes). amount of product formed velocity = unit of time To perform an assay, the substrate is first added to an aqueous buffer. Once the enzyme is added, the concentration of the product is measured at regular time intervals. A representative graph is shown to the right As the reaction continues, formation of product slows and then stops. This leveling off effect illustrates the importance of measuring the rate near the beginning of the reaction when the relationship between time and product formation is linear. The slope in the graph's linear region is called the initial velocity, v 0. The products of the lactase reaction, galactose and glucose, are not easily measured because they are colorless. To simplify the assay, it is common to use an alternate substrate that produces a colored product. The concentration of a colored product can be readily measured by a simple spectrophotometer such as a SpectroVis. In Section 1 you will measure the velocities of lactase catalyzed reaction using an inexpensive substrate called o-nitrophenyl-β-galactoside (onpg), which is colorless. When lactase hydrolyzes onpg, the products galactose and o-nitrophenol are produced. Both galactose and the acid form of o-nitrophenol are colorless; however the base form of o-nitrophenol, called o-nitrophenolate or onp, is yellow in color. Since the pk a of o-nitrophenol is 7.2, the yellow o-nitrophenolate product will form at ph 7.4, the ph of this experiment. HO O O O 2 N ph 7.4 HO O + pka = 7.2 HO O 2 N H + O - O 2 N onpg (colorless) galactose (colorless) o-nitrophenolate onp (yellow), λ max = 420 nm Page 2
Task: Enzyme Concentration and Reaction Rate INSTRUCTIONS Your instructor will prepare the enzyme and onpg solutions. Initial setup Operation of the Vernier SpectroVis Plus Spectrophotometer for kinetic assays Trial 1 1. Obtain about 8 ml of phosphate buffer in a small beaker. 2. Rinse two SpectroVis cuvettes with deionized water and shake dry. 3. Use a glass pipette to transfer 3.00 ml of phosphate buffer (ph 7.40) to each cuvette. 4. Use the 1000-µL micropipette to transfer 200 µl (0.200 ml) of the onpg (Substrate) solution to each cuvette. 5. Mix the contents of each cuvette thoroughly using a small piece of Parafilm as a cap. 6. One cuvette will be used in Trial 1 and the other will be used in Trial 2. 7. Open the LoggerPro3 program on the computer by clicking on the icon on the Desktop. 8. Connect the SpectroVis to the computer via the USB cable. 9. Calibrate your SpectroVis using one of the cuvettes you prepared above as the blank. Place the cuvette in the cuvette slot. Make sure that it is inserted correctly with the clear sides in the light path. Choose the Experiment menu: select Calibrate select Spectrometer Follow the prompts to complete the calibration. You may skip the warm up step if the spectrometer has been on for several minutes. Click OK. 10. Click on the rainbow icon to configure the SpectroVis. Change the collection mode to Absorbance vs. time and select 420 nm as the wavelength that will be used to monitor the production of onp, the product. Click OK. 11. Click on the clock/ruler icon (Data Collection). Ensure the mode is Time Based. Check Continuous Data Collection, which overrides the duration input and allows you to stop the assay whenever you choose by pressing the stop button. For the Sampling Rate, enter 10 seconds/sample. Make sure that Sample at Time Zero is not checked. Click done. 12. Obtain a small piece of Parafilm that will be used to cover the cuvette during mixing. 13. Place a fresh disposable tip on the 1000-µL micropipette, adjust the volume to 150 µl (0.150 ml), and transfer 150 µl of buffer to the cuvette. 14. The following steps must be completed within 10 seconds, so please review them before performing the trial. a. The enzyme catalyzed reaction begins when the enzyme solution is added to the substrate solution in the cuvette. b. Remove the cuvette from the cuvette slot and add the enzyme solution. Click on Collect (data) as soon as possible after the enzyme solution has been added to the cuvette. c. After the enzyme solution is added, the contents of the cuvette are immediately mixed thoroughly by placing a small piece of Parafilm on top of the cuvette as a cap and inverting 2-3 times. d. The cuvette is placed back into the SpectroVis as soon as possible (within 10 seconds). The absorbance vs. time will be displayed on the screen. e. The data will be collected until you click Stop. Page 3
Trial 2 15. Place a fresh disposable tip on the micropipette and obtain 150 µl of the enzyme solution. Trial 1 will begin when you add the 150 μl of enzyme solution to the cuvette as described in #14 above. 16. Follow each of the steps described in #14 and collect the Absorbance data till the values on the display stabilize (i.e. less than 0.005 change in 20 sec). 17. Click on Stop to stop collecting data. 18. Select the <Experiment> menu, and choose <store data from previous run>. 19. Remove the cuvette from the SpectroVis. Do not discard the solution because the absorbance will be measured later and recorded in #30 and on the lab report. 20. Obtain a small piece of Parafilm that will be used to cover the cuvette during mixing. 21. Use the 2 nd cuvette to calibrate the SpectroVis according to the instructions in #9. 22. The following steps must be completed within 10 seconds, so please review them before performing the trial. a. The enzyme catalyzed reaction begins when the enzyme solution is added to the substrate solution in the cuvette. b. Remove the cuvette from the cuvette slot and add the enzyme solution. Click on Collect (data) just after the enzyme solution has been added to the cuvette. c. After the enzyme solution is added, the contents of the cuvette are immediately mixed thoroughly by placing a small piece of Parafilm on top of the cuvette as a cap and inverting 2-3 times. d. The cuvette is placed back into the SpectroVis as soon as possible (within 10 seconds). The absorbance vs. time will be displayed on the screen. e. The data will be collected until you click Stop. 23. Adjust the volume of the micropipette to 300 µl and obtain 300 µl of the enzyme solution. Trial 2 will begin when you add the 300 μl of enzyme solution to the cuvette as described in #22 above. 24. Follow each of the steps described in #22 and collect the absorbance data till the values on the display stabilize (i.e. less than 0.005 change in 20 sec). 25. Click on Stop to stop collecting data. 26. Remove the cuvette from the SpectroVis. Do not discard the solution because the absorbance will be measured later and recorded in #30 and on the lab report. DATA & ANALYSIS: QUALITATIVE 27. Select the true statement about the concentration of substrate (onpg) in Trial 1 and Trial 2. a. In Trial 1, the onpg concentration was higher than in Trial 2. b. In Trial 1, the onpg concentration was lower than in Trial 2. c. The onpg concentration was the same (held constant) in both trials. 28. Select the true statement about the concentration of enzyme (lactase) in Trial 1 and Trial 2. a. In Trial 1, the lactase concentration was higher than in Trial 2. b. In Trial 1, the lactase concentration was lower than in Trial 2. c. The lactase concentration was the same (held constant) in both trials. Page 4
29. Examine your data. The chemical reaction occurred more quickly in Trial ( 1 / 2 ) because it contained a ( higher / lower ) concentration of ( substrate / enzyme ). 30. Insert the cuvette from Trial 1 into the SpectroVis and read the absorbance value (in the window in the lower left side of the screen.) Record the value below. Now insert the cuvette from Trial 2 into the SpectroVis and read the absorbance. Record the value below and transfer the values for Trial 1 and Trial 2 to the lab report form. Trial 1 Trial 2 31. Select the true statement about the final absorbance values of the solutions produced in trials 1 and 2. a. The final absorbance value of Trial 1 was much higher ( > 10% difference ) than in Trial 2. b. The final absorbance value of Trial 1 was much lower ( > 10% difference ) than in Trial 2. c. The two solutions have approximately the same absorbance value ( < 10% difference ). 32. The intensity of the color in the solutions produced in Trial 1 and Trial 2 is most strongly correlated to the concentration of ( galactose / onp ) produced in the reaction. The maximum final concentration of this compound is limited by the amount of ( lactase / onpg ) present in the initial solution. This question is not about rate, so assume infinite time if that helps. QUANTITATIVE 33. In LoggerPro3, select <File> and then <Export> as CSV file. 34. Save this file to the CHM 230 Kinetic Data folder on the computer desktop. 35. Minimize LoggerPro3 and find and double-click on the file that you just saved. The file should now open in Microsoft Excel. 36. a) Delete Column C. This will delete the time values from Trial 1. The times (in seconds) should be in column A, the absorbance values (Abs.) from Trial 2 in column B, and the absorbance values (Abs.) from Trial 1 in column C. b) Highlight the first row that includes numerical data. Insert a new row above this row. Enter the value of zero in column A, zero in column B, and zero in column C. This is the data point that corresponds to zero velocity at zero concentration of substrate. 37. Highlight the data in columns A, B, & C and <Insert> <Chart> (x-y scatter plot) in Microsoft Excel. This plots the absorbance values from the two trials as two series vs. time (in seconds) on the x-axis. a) The independent variable is ( time / absorbance / (concentration of product). b) The dependent variable is plotted on the ( x-axis / y-axis ). 38. Label your axes (include units), provide an appropriate title, and use a legend to clearly distinguish Trial 1 and Trial 2. If titles for the plot and axes do not show, you will have to click on the + button to the upper right side of the plot and add them. Have your instructor check your kinetic plot and initial your lab report after he/she approves your plot. 39. The linear range is a region of a plot where the dependent variable is directly proportional to the independent variable (i.e. if the value of x doubles, then the value of y doubles as well). Absorbance is directly proportional to time near the ( beginning / end ) of each trial. Page 5
40. The slope of the data in the linear range provides us with information about the initial rate (v 0) of the chemical reaction. Although we could easily estimate the slope by picking two data points from within the linear range, this method ignores all of the other data points and usually leads to an imprecise answer. To properly determine the slope of the linear range, we need to add a linear trendline to each series of data; the equation of this line is given in the form of y = mx + b. Right click on a data point in one of the series. Click <add a linear trendline>. Select <display the equation on chart> and the <display R-squared value on chart>. Click <OK> Repeat these steps for the other data series. You probably notice that the trendlines do not fit their plots very well. The R 2 value indicates how well the independent variable predicts the dependent variable. A perfect relationship will give an R 2 value equal to 1.00, but this rarely occurs when working with real data. If there was absolutely no relationship between the two variables, the R 2 value would equal zero. Your R 2 value is lower than it should be because Excel is using ALL of the data from your data series to calculate the trendline. Since we are only interested in the linear range of the plot, delete all data that is not in the linear range for each data series. a) Overall, the R 2 value ( increases / decreases / remains the same ) when data points outside the linear range are deleted. b) Record on the lab report the linear equation and R 2 value for the trendline for Trial 1. c) Record on the lab report the linear equation and R 2 value for the trendline for Trial 2. 41. The slope of each plot is directly proportional to the initial velocity of the reaction, v 0, as shown in the equation below. v 0 0.182 1 µmol = slope Transfer the answers to the following questions to the report form. a. What are the units of the slope based on your plots? b. Rearrange the equation above to solve for v 0. c. Calculate the v 0 for Trial 1 and Trial 2. Trial 1 v 0 = Trial 2 v 0 = d. What are the units of the initial velocity for your calculated values? Page 6
CONCLUSION 42. What can you conclude about the relationship between enzyme concentration and the velocity of the reaction? 43. Explain why your conclusion above makes sense by providing a particulate-level (i.e. molecules, atoms, collisions) description as to why Trial 1 and Trial 2 had different rates of reaction. APPLICATION: Other enzymes catalyze similar reactions to that of lactase. Use what you have learned in this lab to answer the following questions about a related enzyme, which we will call Enzyme Q. 44. Enzyme Q catalyzes the hydrolysis of sucrose (table sugar) into glucose and fructose. What is the likely name of Enzyme Q? 45. The chart shows the results of four assays of Enzyme Q. Please match each experiment description with one or more trials shown below. Trials may be used once, more than once, or not at all. a. This trial used a fructose solution instead of a sucrose solution. b. These two trials contained the same concentration of substrate. c. This trial had the highest concentrations of substrate AND enzyme. d. This trial used distilled water in place of the enzyme solution. 120 Reaction Velocity of Enzyme Q with Sucrose 100 Amount of glucose (µmol) 80 60 40 20 Trial 4 Trial 3 Trial 2 0 Trial 1 0 5 10 15 20 25 Time (minutes) Page 7