In the Laboratory
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Simple Recipes for Prebiotic Soup A High School or Undergraduate Chemistry Laboratory
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Marisol Martinez-Meeler, Nika Aljinovic, and Dorothy Swain† Department of Chemistry, Santa Fe Community College, Santa Fe, NM 87508
Is it possible for scientists to outline a plausible scenario for the development of living cells from simple molecules based on experimental evidence? In 1953, Stanley Miller demonstrated that methane, ammonia, hydrogen, and water will spontaneously form amino acids in the presence of a continuous electrical spark, a concoction referred to as “prebiotic soup” (1, 2). A schematic diagram of Miller’s apparatus is shown in Figure 1. Miller used a vacuum pump to evacuate the system and then ammonia, methane, and hydrogen gases were added from cylinders. Water was added via the bottom chamber and heated so that it circulated within the apparatus. An electrical spark was introduced between the two electrodes, which provided the energy necessary to fragment the gas molecules. After operating the apparatus for a week, Miller found a gooey residue at the bottom of the apparatus. He analyzed the residue and found that it contained a high level of amino acids. The clear demonstration of biological molecules spontaneously forming under plausible early-Earth conditions stimulated many researchers to design their own prebiotic-soup experiments. Since 1953, Miller and other scientists have devised new prebiotic-soup recipes (3– 9) that have contributed to the lively debate (10–26) surrounding life’s origins. The experiment presented here was designed to bring that debate into the introductory chemistry classroom by describing a simple, inexpensive apparatus and set of procedures that can be used in a low-tech laboratory to help beginning chemistry students discover the joys of prebiotic cooking.
electrodes were inserted into the holes until there was about a 0.5-cm gap between them and then they were fixed into place with epoxy and sealed with silicone. Holes for the glass tubing were drilled into both bottle lids. After bending the
electrodes
H2O NH3 H2
CH4
condenser water out
to vacuum pump
water in
H2O containing organic compounds
boiling H2O
Experimental Procedure When Stanley Miller ran his prebiotic-soup experiments in the 1950s, he made use of specialty glassware, vacuum pumps, gas cylinders, pressure gauges, and a high-voltage power supply: equipment that is not standard-issue in high school and community-college chemistry labs. The experiments in this article make use of a simplified reactor described in the American Biology Teacher (27). A schematic diagram is shown in Figure 2.
Building the Reactor The simplified reactor was constructed by inserting stainless-steel electrodes into a polypropylene chemical-storage bottle and glass tubing into the lids of two polypropylene bottles. These processes required a drill, glass tubing, a Bunsen burner, 1-mm diameter steel electrodes, and epoxy and silicone to fix and seal the attachments into place. Holes for the electrodes were drilled in one bottle, 180⬚ apart from each other and about 4 cm from the bottom of the bottle. The † Current address: Coquille Valley Middle School, Coquille, OR 97423.
Figure 1. Schematic diagram of the apparatus Stanley Miller used for prebiotic soup experiments in 1953 (1, 2).
natural gas supply
dc power source
NH3(aq)
reaction vessel
ⴚ ⴙ flasher
ⴙ ignition coil H2O ⴙ ⴚ ground
Bunsen burner
condenser Figure 2. Schematic diagram of the simplified reactor for prebioticsoup experiments described in the American Biology Teacher (27).
JChemEd.chem.wisc.edu • Vol. 80 No. 6 June 2003 • Journal of Chemical Education
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In the Laboratory
tubing into the shapes indicated by the diagram, the tubing was inserted into the holes, glued with epoxy, and sealed with silicone. The bottle with the electrodes was referred to as the “reactor”. The bottle without the electrodes served as a holding-container for the concentrated aqueous ammonia NH 3(aq) (sometimes labeled as ammonium hydroxide, NH4OH, on commercial containers). The electrical part of the apparatus was constructed using a variable 12-V dc power supply, a 12-V automobile-ignition coil, a 12-V automobile-ignition condenser, and a 12-V signal flasher. The following electrical connections were made as shown in Figure 2: the signal flasher and ignition condenser were attached in parallel between the positive pole of the power supply and the primary positive pole of the ignition coil; the negative pole of the ignition coil was attached directly to the negative pole of the power supply; the secondary positive pole of the ignition coil was attached directly to one of the electrodes in the reactor; and the other reactor electrode was grounded (we attached ours via a banana plug to the ground in an electrical outlet). This electrical setup converts the steady 8–9 V output of the power supply to an intermittent 20–30 kV discharge across the gap of the electrodes.
Operating the Reactor The entire apparatus was operated in a fume hood. About 50-mL boiling water was added to the reactor, and about 250-mL aqueous ammonia was added to the holdingbottle. The lids were screwed on to the bottles. Rubber tubing was used to attach the glass tubing on the reactor to a Bunsen burner and the glass tubing on the holding-bottle to a natural gas outlet. Natural gas was gently bubbled through the aqueous ammonia and was burned as it emerged from the Bunsen burner. The system was flushed in this way for at least thirty minutes to remove air from the system and to replace the air with one of the several plausible prebiotic atmospheres suggested in the literature: CH4, NH3, and H2O, a reducing atmosphere. Once the system was flushed, the rubber tubing was clamped off on both sides and the natural gas was turned off. At this time the power supply was turned on, and an audible clicking from the signal flasher was noticeable. By dimming the lights, it was possible to see an intense blue spark flash across the electrodes intermittently (every twenty seconds or so, at first). After about three days, the power supply was turned off and the reaction mixture analyzed. There was a pale brown color to the water in the reactor and a brown flaky buildup on the electrodes. Analyzing the Reaction Mixture The solution was poured from the reactor into a beaker, where it was concentrated by heating over a hot water bath (a rotary evaporator, if available, could also be used) and then analyzed for amino acids. Ninhydrin (28) was used to test for amino acids. Ninhydrin is a spray-on reagent that will turn purple in the presence of amino acids (29), amines, carbonates, oxalates, and sulfides. The balanced equation for the reaction of ninhydrin with amino acids is shown in Scheme I. As a control, a dilute solution of aqueous ammonia was used that had undergone the same process of concentration over a hot water bath as the prebiotic soup (after bubbling 666
some CH4 through the ammonia solution): this was a system with all of the chemicals but no spark. For a different control, the entire apparatus could be run without adding CH4 to the system (just adding 50 mL of boiling water to the reactor and 250 mL of aqueous ammonia to the holding-bottle, then sealing off the tubing without flushing and operating the system for three days with air replacing the CH4): this would be a system that had been sparked with all of the chemicals except methane. It is important for the control to have had some exposure to NH3(aq) since NH3(aq) is the most likely source of a false positive ninhydrin test. Using clean medicine droppers, a couple of drops of the reaction mixture were placed on one-half of a filter paper and a couple of drops of control on the other half. Using a pencil, a clear line was drawn down the middle of the filter paper, and the halves were labelled. The samples were allowed to air dry. Under the hood, a thin, even layer of 1% ninhydrin solution was sprayed onto the dry filter paper and the filter paper was allowed to dry again. The dry filter paper was heated in a 110 ⬚C oven for five minutes. Upon removing the paper from the oven, an obvious purple color was seen on the reaction-mixture side and either no color or a fainter purple color was seen on the control side. This difference in purple color was evidence for the formation of amino acids in the reactor. Variations on this experiment are described in the Supplemental Materials.W They include changing the composition of the gas mixture and adding various suggested catalysts for the polymerization reaction of amino acids into peptides and proteins. Instructors may also wish to consider using an alternative stain such as fluorescamine (30, 31) in place of ninhydrin. Fluorescamine specifically detects primary amines by forming fluorescent-reaction products (390-nm excitation, 475-nm emission). Instructors may also wish to expand the experimental procedure by performing chromatography (1, 2, 27) on the prebiotic-soup mixture.
O O
+
H2N CH C
OH
2
OH
OH
R
O ninhydrin (colorless)
amino acid (colorless)
O
O N H O
O (purple)
+ RCHO
+
CO 2
+
3H2O
Scheme I. Generalized reaction of ninhydrin with amino acids.
Journal of Chemical Education • Vol. 80 No. 6 June 2003 • JChemEd.chem.wisc.edu
In the Laboratory
Laboratory Requirements The laboratory requirements for this experiment include: •
Fume hood with a natural gas outlet and electrical outlets available
•
Variable 12-V, dc power supply
•
About 10 insulted wire connectors with alligator clips on both ends
•
Insulated wire connector with an alligator clip on one end and a banana plug on the other
•
Drying oven (110 ⬚C)
•
Centrifuge (optional)
•
Rotary evaporator (optional)
Hazards The potential for electrical shock is present during this experiment. Only well-insulated connecting wires should be used, and none of the wires or electrodes should be handled while the apparatus is running. The concentrated aqueous ammonia has a strong, noxious, choking odor. Containers of aqueous ammonia should be handled carefully with protective gloves and should be only opened under the fume hood. The ninhydrin solution should only be handled under the fume hood while wearing protective gloves. Ninhydrin is a potential carcinogen and the ethanol solvent is an inhalation hazard. The epoxy glue and silicone used to construct the apparatus should only be used under the fume hood. Inhalation of epoxy fumes can result in lightheadedness and giddiness. Acknowledgments This research was funded by the National Science Foundation’s Project Emerald, NSF grant #CHE-9974883. The authors gratefully acknowledge Dana Brabson for providing the impetus that got this project started, Julie Harris for her cheerful and efficient management of administrative details, Seth Abrahamson for loaning us the power supply and advising us on practical electronics, and Leonard Henio for helping us to troubleshoot the electrical setup. W
Supplemental Material
Teacher notes, a detailed student lab procedure, a summary of current literature debates, and an extensive bibliography are available in this issue of JCE Online.
Literature Cited 1. Miller, S. L. Science 1953, 117, 528–529. 2. Miller, S. L. J. Am. Chem. Soc. 1955, 77, 2351–2361. 3. Keefe, A. D.; Miller, S. L.; McDonald, G.; Bada, J. L. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 11904–11906. 4. Ferris, J. P.; Hill, A. R., Jr.; Liu, R.; Orgel, L. E. Nature 1996, 381, 59–61. 5. Lee, D. H.; Granja, J. R.; Martinez, J. A.; Severin, K.; Ghadiri, M. R. Nature 1996, 382, 525–528. 6. Yao, S.; Ghosh, I.; Zutshi, R.; Chmielewski, J. Nature 1998, 396, 447–449. 7. Ertem, G.; Ferris, J. P. Origins Life Evol. Biosphere 1998, 28, 485–499. 8. Imai, E.; Honda, H.; Hatori, K.; Brack, A.; Matsuno, K. Science 1999, 283, 831–833. 9. Nelson, K. E.; Levy, M.; Miller, S. L. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3868–3871. 10. Horgan, J. Sci. Amer. 1991, 264, 117–125. 11. Orgel, L. E. Sci. Amer. 1994, 271, 76–83. 12. de Duve, C. Amer. Sci. 1995, 83, 428–437. 13. Lazcano, A. Lecture Notes in Artificial Intelligence 1995, 929, 105–115. 14. Travis, J. Sci. News 1996, 149, 278. 15. Wilson, E. K. Chem. Eng. News 1998, 76 (49), 40–44. 16. Pigliucci, M. Skeptical Inquirer 1999, 23 (5), 21–27. 17. Senior, K. The Lancet 2000, 355, 814. 18. Travis, J. Sci. News 2000, 157, 363. 19. Bahn, P. R.; Fox, S. W. Chemtech 1996, 26, 26–29. 20. Wachterhauser, G. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4283–4287. 21. de Duve, C. Gene 1993, 135, 29–31. 22. Levy, M.; Miller, S. L. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 7933–7938. 23. Miller, S. L.; Bada, J. L. Nature 1988, 334, 609–611. 24. Bada, J. L. The Sciences 1995, 35, 21–25. 25. Kasting, J. F. The Sciences 1995, 35, 5–6. 26. Rawls, R. Chem. Eng. News 1999, 77 (51), 29–32. 27. Dubowsky, N.; Hartman, E. M., Jr. Amer. Biol. Teacher 1985, 47, 51–53. 28. West, R. J. Chem. Educ. 1965, 42, 386–387. 29. Campbell, M. Biochemistry, 2nd ed.; Saunders College Publishing: Philadelphia, 1995. 30. Weigele, M.; DeBernardo, S. L.; Tengi, J. P.; Leimgruber, W. J. Am. Chem. Soc. 1972, 94, 5927–5928. 31. Udenfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.; Leimgruber, W.; Weigele, M. Science 1972, 178, 871–872.
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