In the Laboratory
The Sweetness of Aspartame1 A Biochemistry Lab for Health Science Chemistry Courses Paul J. Stein Department of Chemistry, College of St. Scholastica, Duluth, MN 55811 Proteins control most functional aspects of cell behavior, usually by binding to a specific ligand. Examples include oxygen binding to myoglobin or hemoglobin, substrate binding to enzymes, and hormone binding to a receptor in the target cell’s membrane. In non-allosteric cases, such proteins display a hyperbolic binding plot, with saturation at high ligand concentrations. This paper describes a laboratory experiment that reinforces the universality of the concept of saturation using the binding of the ligand aspartame to the protein receptor that determines taste. Although the “sweetness” receptor has never been isolated, the similarity of the taste response to standard protein–ligand binding profiles supports its existence. Students can readily associate the receptor concepts incorporated in this lab with their everyday experience. Despite the subjectivity of the taste measurement, the results unequivocally illustrate the hyperbolic nature of protein binding, albeit to varying degrees of perfection. They also illustrate an example of a blind study, such as is required in many scientific tests. A survey of the many types of sweeteners has been presented by Ellis (1). Aspartame is the methyl ester of the dipeptide aspartyl-phenylalanine. As with previous artificial sweeteners, saccharin and cyclamates, the discovery of its sweetness was serendipitous (1, 2). When first using this laboratory exercise for classes, I was surprised by the variety of concerns expressed about aspartame. It became apparent that many students hear anecdotal information or read newspaper articles about food additives, and immediately regard the worst-case scenario as fact. For that reason, a brief summary of some aspartame studies follows. The FDA has established 50 mg/kg/day as the acceptable daily intake (ADI) level for aspartame ingestion (40 mg/kg/day by the World Health Organization), while surveys indicate that the 90th percentile of consumption is between 2 and 10 mg/kg/day (3). A single can of diet pop contains 184 mg of aspartame. Nonetheless, there have been instances where consumers linked aspartame consumption with headaches, mood swings, seizures, and other symptoms. Few of these claims are supported by experimental studies. Results of a study involving 120 women taking a single daily dose of approximately 230 mg aspartame indicate no connection between mood swings and aspartame use (4). However, this may not be the case in individuals prone to depression, who should avoid excessive aspartame consumption (5). Between 1986 and 1993, there were 265 reports to the FDA in which aspartame use was believed to be linked to seizures (6, 7). Although data are ambiguous at doses exceeding 1,000 mg/kg, studies on individuals with a history of seizures show no evidence for such a connection at lower doses (8, 9). Diet soda cans contain a warning for people with phenylketonuria. There is some concern that individuals who are heterozygous for phenylketonuria and unaware of this condition might be susceptible to some effects. A review of clinical studies of blood levels of phenylalanine, aspartate, and methanol following aspartame ingestion by phenylketonuric heterozygotes indicates no cause for safety concern by this population (10). A study involving five individuals homozygous for phenylketonuria (PKU),
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ranging in ages from 11 to 23, shows that a single can of diet pop produces only slight increases in plasma phenylalanine levels. These increases are not clinically significant (11). Aspartame is also a good model to use in emphasizing structure–activity relationships in biochemistry, a subject of a recent symposium in this Journal (12, 13). Required components for a number of artificial sweeteners suggest that hydrogen bonding, possible salt bridges, and hydrophobic interactions all contribute to the aspartame–receptor binding. Procedure
Basic Concepts Students are separated into two groups in order to run a blind taste-testing procedure. Each student is given a numbered paper cup in which to prepare an Equal® solution and is assigned an amount of Equal to use. The amount of Equal assigned to a specific cup number is predetermined randomly by the instructor. Each student prepares a 50-mL solution containing either 0.031, 0.062, 0.125, 0.250, 0.500, or 1.00 g of Equal. Each student saves a small portion of the sample in a test tube to determine the absorbance at 257 nm. This is done by the instructor or teaching assistant, who explains the basic concepts of UV spectroscopy and Beer’s law. Each student tastes 4 to 6 samples from the opposite group, depending on the number of students in a lab, then rates the taste on a 0–5 scale based on first tasting water and a 1.0 g/ 50 mL control sample. Students record the mass and A257 of their own sample, and the assigned taste rating to each tasted sample. When all the tasting is completed, the instructor collects the data and loads it into a prepared spreadsheet template. An LCDoverhead display is used to show the graphed results to the entire class. Procedural Details Since the lab involves tasting samples, I prefer using a room that is not a typical chemistry lab. If this is not possible it is important that the lab is clean and other chemicals are not available to the students. Weighing is done directly from the Equal packet into the sample cups by tapping the sides of the packet. Careful weighing is important for good results. Three-place balances are required for accurate weighing of the lower-mass samples. Tap water is dispensed from drinking pitchers and measured using a “calibrated” paper cup (marked with a 50-mL line). An unused disposable beral pipet is used to mix and dispense the aspartame solutions. Each student is assigned a plastic spoon for tasting purposes. He/she pipets five drops unto the spoon from the Equal cup, so the tasting volume is consistent. Students are instructed to take water before each sample tasted to eliminate any previous taste. The laboratory usually takes from 1 to 1 1/2 hours to complete. Alternatively, it can be set up as a classroom exercise, with the data analysis completed at a later date.
Journal of Chemical Education • Vol. 74 No. 9 September 1997
In the Laboratory 5.00
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malized to give the highest absorbance reading the same value as the highest taste rating. This allows both sets of data to be viewed on the same graph. A regression analysis is done on the normalized A257 results. The spreadsheet has a preset graph showing the results for the class, as in Figure 1, which in this case reports the averages obtained from six lab sections. Figure 2 illustrates the variance from one lab section to another. Even in the worst cases the data are clearly hyperbolic when compared to the linear absorbance results. Each 1-g packet of Equal contains 36.7 mg of aspartame2 (the remainder is dextrose and maltodextrin). Determining the reciprocal of the y-intercept of a plot of 1/(taste rating) vs. 1/(amount of equal) yields the maximum taste rating that could be perceived. Comparison to the class results indicates that the 1 g/50 mL control sample is about 86% of the maximum sweet response.
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Conclusion Figure 1. Graph showing the linearity of the averaged absorbance data and the hyperbolic character of the averaged taste rating results from six different lab sections.
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Given sufficient sampling numbers, the perception of sweetness of various aspartame solutions shows a saturation response characteristic of protein–ligand interactions. This laboratory exercise can be used to introduce students to the concept of protein receptors, and it reinforces the basic concept of saturation binding to proteins. The linear absorbance increase provides a helpful contrast between Beer’s law and standard dose–response curves when protein binding is involved.
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Figure 2. This graph shows the individual taste rating results from six different lab sections.
Modification for Fewer than 14 Students Students are not separated into groups but the solutions are randomly renumbered while the absorbance measurements are being conducted. Students may well taste their own samples, but they will not know which is which. Students generally taste six rather than four cups to produce a larger sample. Results Students complete a work sheet that indicates the code for the assigned sample, the mass assigned, the actual mass reading, and the absorbance at 257 nm. They also record the code and taste rating for each sample tasted. The spreadsheet used to display the data for the class contains a column for each concentration of aspartame tasted. The taste and absorbance data determined by the class are entered and averaged. The averaged absorbance data are nor-
1. A preliminary version of this work was presented as a poster at the 26th annual Great Lakes Regional ACS Meeting, May 1993, Marquette, MI. 2. This information was obtained from the Nutrasweet Company. A mass of 38 mg per packet (a 3.5% error) was calculated based on the student absorption results and using the molar absorptivity of phenylalanine (195.1 M {1cm {1 [14]).
Literature Cited 1. Ellis, J. W. J. Chem. Educ. 1995, 72, 671–675. 2. Roberts, R. M. Serendipity: Accidental Discoveries in Science; Wiley: New York, 1989. 3. Butchko, H. H.; Kotsonis, F. N. J. Am. Coll. Nutr. 1991, 10, 258– 266. 4. Pivonka, E. E. A.; Grunewald, K. K. J. Am. Diet. Assoc. 1990, 90, 250–254. 5. Walton, R. G.; Hudak, R.; Green-Waite, R. J. Biol. Psychiatry 1993, 34, 13–17. 6. Maher, T. J.; Wurtman, R. J. Environ. Health Perspect. 1987, 75, 53–57. 7. Tollefson, L. Regul. Toxicol. Pharmacol. 1993, 18, 32–43. 8. Tollefson, L.; Barnard, R. J. J. Am. Diet. Assoc. 1992, 92, 598– 601. 9. Sze, P. Y. Neurochem. Res. 1989, 14, 103–111. 10. Filer, L. J.; Stegink, L. D. Diabetes Care 1989, 12, 67–74. 11. Mackey, S. A.; Berlin, C. M., Jr. Clin. Pediatr. 1992, 31, 394– 399. 12. Kinghorn, A. D.; Kennelly, E. J. J. Chem. Educ. 1995, 72, 676– 680. 13. Walters, D. E. J. Chem. Educ. 1995, 72, 680–683. 14. Practical Handbook of Biochemistry and Molecular Biology; Fasman, G. D., Ed.; CRC: Boca Raton, FL, 1989.
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