Biochemistry Laboratory for the Freshman Chemistry Curriculum Peter M. Faikl HQ USAFAIDFC, USAF Academy, CO 80840 Ideally, lahoratory courses in the freshman chemistry curriculum should fulfill several purposes. The laboratory experience should reinforce chemical principles discussed in the lecture part of the coursc with "real.'examples. 2. The lahoratory experience should teach the student how to observe and describe a physical phenomenon and the logical approach to solving a scientific problem. 3. If given a certain amount of freedom to experiment, a student should be able to explore a chemical phenomenon to a greater depth by trying related experiments on his or her own ( I ) . 1.
At the Department of Chemistry a t the United States Air Force Academy, we have developed a hiochemistry lahoratory for our freshman curriculum. I t is intended to introduce the students to two important biochemical lahoratory principles, the study of hiomolecules and the study of metaholism. In the study of hiomolecules, the students discover the chemistry of carbohydrates, lipids, amino acids, and proteins. When studvinr! . - metabolism, the students incubate fermenting yeast cells with glucose and ohserve the production ofcarhon dioxide. The biochemistry of starvation (withdrawal of glucose) and poisoning (addition of arsenate to the svstem) are also demonstrated in this series of experiments. The lahoratory experiments demand an understanding of most other areas of chemistry and are, therefore, recommended t o he incorporated toward the end of the course. The methods for the experiments discussed in this paper are available from us upon request. Discussion In the carbohydrate experiments, the students are asked to investigate two aspects of sugar chemistry. First, they differentiate between reducing and nonreducing sugars using the Tollen's (the "silver mirror test") andlor the Fehling's test (the "copper reduction test"). Next, the students characterize monosaccharides and polysaccharides using the iodine . ~reaction ~ - - 12.3). -~ The lipid expeiiments show the students that different t w e s of lioids are oresent in livine svstems and that one &Ajor diffirence is 'the unsaturation df the fatty acyl moieties contained within them. The examdes we use in our experiments, lard, olive oil, and sunflower oil, are very different in their fattv acid saturation and are thus well suited for this line of experimentation. In our experimental setup the instructor demonstrates the difference in melting points of these lipids, and the students are asked to determine differences in unsaturations of these lipids by observing differences in oxidation rates with KMn04. Protein chemistry is divided into the study of amino acids and the studv of intact oroteins. Amino acids that occur naturally on the skin c& he examined hy a simple fingerprinting technique using niuhydrin. Ninhydrin is very sensi~~~~~~
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Present address: institute fuer Mikrobioloaie. Toxikolosie. und Histdogie, Bundesanstait fuer ~leischforschuniE.-c.-~auiann Str. 20.8650 Kulmbach, West Germany. 944
Journal of Chemical Education
Figure 1. Reanions of proteins. This experiment demonstrates several reactions of a soluble protein, ovalbumin (egg white)wilh varlous reagents: Silver andlor lead ions bind ineversiblytoanionicaminoacid residues inW protein causing denaturation. This is shown hare in the heavy precipitates. Emanol. acetone. andlw ammonium sulfate remove water molecules from me orolein molecule~idehvdrationl . . . ~This~also causes , a reversible oreci~itation reaclion 01 me protein The protam can be redissolved by lheaddtion ol water. Notlce me dnemce bwwn this precwmle and the one caused by the heavy metal ions. Lowering me pH disrupts me salt bridges in the protein, which in turn disruptsthe teniary structure of the protein molecule. Ifthe pH is very low, i.e., by addition of a strong acid, thew salt bridges are permanently disrupted resulting in denahnation. Notice the similarity of this precipitate wilh the one caused by heavy metal ions. Raisina - me .oH usuallv inaesses i rote in sobbilitv. The aWRion of 6 M ammonium hydroxide therefore h not cause a noliceable change In the protein solution. Neutral salts in moderate concenbations also have little ewen on me solubiilty 01 proteins as shown here. ~7~~~
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tive toward any mine-containing compound; hence, it is well suited for this experiment. Recent studies (4) have shown that there are other reactions of amino acids from the surface of the skin that give other color reactions. We are currently investigating these for possible incorporation into this lahoratory experience. One of the hest and cheapest sources of a soluble nrotein for freshman exoerimentation is egg white (ovalbumin).We can serve the laboratory needs of more than 40 students with one eaa. On a microscale the students can perform a Biuret test askell as protein sulubilitv tests. Anexam~leofthesoluhilitytest is given inFigure 1 - In an experiment on plant hiochemistry&e introd&e the student t o chromatography, extractions, and chlorophylls. A piece of plant tissue can easily he impregnated into a filter paper and chromatographed in a suitable solvent such as acetone. With spinach leaves a clearly visible separation between chlorophyll A and chlorophyll B can he achieved. The metabolic experiment is designed t o show the student how living systems use chemical reactions to perform physiological functions, for example, energy utilization. The organism we use successfully on a microscale is the yeast system.
Figure 2. Yeast incubations f w the metabolic experiment. A few drops of activated yeast suspension and a few drops of the other reagents indicatedare drawn intoa micropipet. The micropipetsare then placed intoa test tube rack as shown. Actively metabolizing cells will produceethanol and carbon dioxide. Three maiw metabolic events are demonstated: 1. "Nwmal" metabolism wilh an adequate energy supply (glucose). 2. Metabolism without adequate energy supply: stalvation (glucow is removed).
3. "Abnwmal" metabolism. Th3 ~ b o l l reaction c sequence is inhibkd by arsenate (poisonfng).
look as follows: A few microliters of reagent, i.e., a glucose solution is drawn into a disposable micropipet. Subsequently, a few drops of an actively growing yeast &pension is added. This mixture is kept in a warm place until the next laboratory period (about two days). In a second and third disposable micropipet, yeast can be incubated with deionized water or a dilute arsenate solution to demonstrate the chemical basis of starvation and poisoning, respectively. An experimental setup is shown in Fimre 2. Because the micropipets have very small openings, the gas exchange is negligible; therefore, semianaerobic conditions are established for the yeast cells. In the second laboratory period the students analyze the system for C 0 2 with a Ca2+solution. This biochemistry laboratory provides a basic introduction into modern biochemistry and an appreciation for the scope of the subject. The first part of this laboratory, the study of biomolecules, is especially well suited for the transition from "regular chemistry" to biological chemistry. I t provides the instructor with the opportunity to review a number of topics covered during the earlier part of the course. The individual experimentation provides the student with evidence that biumolecules follow the same physical and chemical laws as the chemicals that he or she has already encountered earlier in the course. The metabolic part of this laboratory provides the student with the opportunity to experiment with living systems and observe "biochemistry in action". Literature Clted
When active yeast is incubated with elucose for one to three days under semianaerobic conditions one can detect the C 0 2 produced. A typical experimental protocol might
1. Braydich, M.D ; M".b,H. J..man"sniptin preparation. 2. Shriner, R. L.: Fuaon. R C.; Curtain. D. Y. The Systemfie Idenfifieofbn of Organic Compounds:Wiley: New York, 1 W pp 173-174. 3. Wenedict. S. R.J B i d Chem. 1909.5,485. 4. Aimog, J.: Zeiehner. A,: Svetlana, S.; Scharf.0.J FareReicSei. 1981,32,58&596.
Volume 66 Number 11 November 1989
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