In the Laboratory
Working with Enzymes—Where Is Lactose Digested?
W
An Enzyme Assay for Nutritional Biochemistry Laboratories Sandi R. Pope, Tonya D. Tolleson, R. Jill Williams, Russell D. Underhill, and S. Todd Deal* Department of Chemistry, Georgia Southern University, Statesboro, GA 30460
The Department of Chemistry at Georgia Southern has traditionally offered a sophomore-level course in biochemistry specifically for our American Dietetic Association–approved “Foods and Nutrition” major. Historically, this course was taught as a typical short course in biochemistry, covering many topics in minimal detail with a similarly designed laboratory experience. In an effort to better serve the student clientele of the course, the curriculum was recently redesigned to focus on the nutritional aspects of biochemistry. To complement this new focus for the course, a new laboratory component was also created. This new lab experience is designed to utilize foodstuffs and their chemistry to allow students to investigate biochemical principles relevant to nutrition. Many of the experiments used in this new lab are adaptations of labs previously published in this Journal. The first of these experiments is the evaluation by redox titration of the vitamin C content of fresh green peppers and peppers cooked by various methods, as originally described by Johnson (1) and modified by Deal and Pope (2). This is followed by an extensive investigation of the biomolecules (specifically casein and lactose) in skim milk as most recently summarized by Petersen (3). These experiments occupy a large portion of the academic quarter and illustrate several important techniques as well as classes of biomolecules. However, we felt that the lab needed to incorporate some enzyme chemistry to give the students a more comprehensive picture of biomolecules. While experiments involving the isolation, purification, and kinetics of enzyme systems are readily available (4 ), none of them focus on the nutritional role of enzymes—a theme we desired to maintain throughout the laboratory experience. To address this gap in the laboratory experience, we have developed an experiment that we call “Working with Enzymes— Where Is Lactose Digested?” The experiment fits very nicely into the previously described laboratory scheme because it continues the study of milk and its components. In the experiment, students assay the activity of the enzyme lactase in a solution of 5% lactose (“synthetic milk”) at pH values of approximately 1, 6, and 8. Once the activity (or lack thereof) has been determined for all the pH values, the students can easily determine where lactase is located in the digestive system when given pH values at various spots along the tract. In the experiment, students use the synthetic milk to alleviate the complications associated with protein coagulation or interference from other compounds in real milk. The lactase is obtained from Lactaid tablets or similar generic tablets available at most pharmacies. We have found this to be an inexpensive, readily available, and dependable source *Corresponding author. W Supplementary materials for this article are available in JCE Online at http://jchemed.chem.wisc.edu/Journal/Issues/1998/ Jun/abs761.html.
of the enzyme. In addition, the tablets are easy to handle, since the students simply crush them using a mortar and pestle, suspend the resulting powder in distilled water, and filter out the solids to prepare the working solution of the enzyme. The assay for enzyme activity is performed using paper chromatography as previously described by Ivany and Heimer (5) or the glucose oxidase/horseradish peroxidase system developed by Bateman and Evans (6 ). The activity of the enzyme is assayed at 15-minute intervals over a period of one hour. Depending on the system used to detect activity, the experiment can be completed in just over that hour or it may take an additional hour or so (to develop and visualize the chromatography sheet). In this experiment, the students are exposed to laboratory techniques involving enzymes and they learn some of the basic principles of enzyme assays. Since the lab is actually an elementary pH assay, students discover the idea of optimum pH for enzyme activity, which can then be further developed in the classroom. Since our course is taught on a basic level, we have chosen a very narrow focus for this experiment; however, the techniques utilized lend themselves readily to more complex assays and kinetic studies of lactase such as investigations of optimum temperature, substrate specificity, and detailed kinetics. A related example of a kinetic study was published in this Journal by Russo and Moothart (7 ). The materials necessary to perform this experiment are readily available in most chemistry departments or are easily obtainable. As mentioned above, Lactaid or generic lactase tablets are available over the counter from most pharmacies. The paper chromatography version of the experiment uses inexpensive materials (chromatography paper and a developing solvent), but a longer time is required to complete the experiment and the results are variable. The glucose oxidase/ horseradish peroxidase version of the experiment requires some slightly more expensive and less common reagents, but the experiment is very quick and the results are reliable. Supplementary Materials Supplementary materials for this article (student handouts and instructor notes) are available in JCE Online at http://jchemed.chem.wisc.edu/Journal/Issues/1998/Jun/ abs761.html. Literature Cited 1. 2. 3. 4.
Johnson, E. R. J. Chem. Educ. 1988, 65, 926–927. Deal, S. T.; Pope, S. R. J. Chem. Educ. 1996, 73, 547. Petersen, Q. R. J. Chem. Educ. 1996, 73, 848–849. See for example: Stenesh, J. Experimental Biochemistry; Allyn and Bacon: Boston, 1984; Section V. 5. Ivany, J. W. G.; Heimer, E. P. J. Chem. Educ. 1973, 50, 562. 6. Bateman, R. C., Jr.; Evans, J. A. J. Chem. Educ. 1995, 72, A240–A241. 7. Russo, S. F.; Moothart, L. J. Chem. Educ. 1986, 63, 242.
JChemEd.chem.wisc.edu • Vol. 75 No. 6 June 1998 • Journal of Chemical Education
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