David F. Dever Mocon Junior College Mocon, Georgia 31206
Allied Health Chemistry Laboratory Amino acids, insulin, proteins, a n d skin
As the chemical education profession continues in its reappraisal of itself, some of those involved with the education of nurses, dental hygienists, physical therapists, etc., realize that some of what they have been offering is not very interesting, less to-the-point than they thought, and, especially in the laboratory, quite anaesthetic. This has been manifest by the lessening, or even the exclusion of chemistry from such programs as Associate Degree Nursing in some parts of the country. We could take issue with accrediting bodies who allow this or criticize faculties who design curricula without chemistry but for one fact: We have made their decisions easy for them by giving the least attention to this part of chemistry, placing it low among our fiscal and personnel priorities, and by selecting experiments which are the cheapest, the least challenging, and designed to be foolproof; this makes them uniuterestine. unexciting. unmemorahle. and amonc! the first to he cut $en a curri&um must be trimmed. In keenine with recent attention directed to Allied Health ~bucation' this article is the first of a series which attempts to make the laboratory associated with a onequarter or one-semester inorganic/organic/hiochemistry course more germane to the allied health student. The boundary conditions which we set for ourselves include the following: (1)the experiment can be completed by the chemically naive student withm a 3-hr period, including clean-up, (2) the experiment will have specific applicahility to health chemistry, (3) where possible, the students themselves will be part of the experiment by supplying samples and specimens or testing materials via their own physiognomies, (4) molecular models which the students construct themselves will be an intimate part of the pedagogy, and (5) in place of a lab manual, a description of the experiment requiring substantial homework effort is circulated. The experiment described has been tested on more than 2M) students over a 2-yr period in labs of 24-32 student sections. The prose description of the lab which is puhlished for the student and listed here has been refined to the point where, in our experience, it is almost impossible for the student to go wrong. We publish the prose description verbatim because it has undergone extensive revision and has been brought to the point that any omission would make it less effective with the naive student. We feel that part (5) above is vital to the program: This method of involving the student in the experiment breeds apprehension and anticipation. The "Oh well, let's go to the lab and look on the blackboard so we'll know what we are going to do today" attitude cannot exist here. We feel that one of the reasons that these experiments are so successful with the naive student is that he cannot hut come prepared. Of almost equal importance is the use of molecular models wherever possihle. I t is the student's most direct connection with what is going on in the beaker, test tube, or flask. The expense is justifiable to the student since no lab manual need be p u r c h a ~ e d . ~ It is felt that this experiment is valuable to the education and training of allied health students (nurses, dental hygienists, etc.) for the following reasons: (1)the students 338 / Journal of Chemical Education
leam paper chromatography, (2) they construct molecular models of amino acids, (3) they leam to distinguish between D and L forms, (4) they observe coagulation and hydrolysis of a protein, (5) their attention is focused on the primary and tertiary structure of proteins, and (6) they work both with materials extracted from their own bodies and found in a clinical setting rather than "off the stock-rwm shelf." Notesto the Instructor
The students execute this experiment via the following steps: (1) pre-lab library preparation, molecular model assembly, and paper polypeptide assembly, (2) observation of coagulation and hydrolysis of insulin, (3) extraction of amino acids from their skin, (4) chromatography of insulin, insulin-hydrolysate, and skin extract to identify any amino acids, (5) examination and comparison of molecular models and amino acids, (6) construction of paper models, which indicate primary and tertiary structure of insulin and lysozyme from previously assembled polypeptide chains. The laboratory assignment is dittoed as shown below. In section A, a different amino acid is circled on each sheet so that each student in the lab will construct a different one; if there are more than 20 in the lab section, acids can be circled and asterisked, giving the students the opportunity to compare D and L forms. The article referred to is one of many sources showing the structures of the amino acidsf D and L forms are clearly shown in Krogmann as well as other references.' In section B, paper shapes resembling the structures of the amino acids are cut out and strung together "paperdoll" fashion in tri or tetrapeptide sections by each student and brought to lab. The student is responsible for those sections of insulin or lysozyme specified by the numbers filled in the blanks; amino acid sequences are widely availahle.5 Since same students in allied health programs have previous hospital experience (LPN's, LUN's, etc.), they recognize and are impressed with the use of clinical materials in the chemistry laboratory. Coming's :St325 works well in hydrolyzing the insulin; starter kits containing 1-g samples of 20 amino acids are put up by several vendors.= Heimer's solvents' are recommended here, particularly pH = 4; development times of less than 5 min are common. If lab schedules are cramped for time, the use of 3 X
'2YC Conference, April, 1973,Dallas. aBenjamin/Ma~zen36970or Cenca 91507 is adequate for this. 3Doty,P. W., Sei.Amer., Sept. (1957). 'Krogmann, David W., "Molecules, Measurements, Meanings," W. H. Freeman & Co., San Francisco, 1971, p. 2. 5Phillips, D. C., Sei. Amer., Nov. (1966) and Rauth, J . I., Eyman, D. P., and Burton, D. J., "A Brief Introductiqn to General, Inorganic, and Biochemistry," W. B. Saunders Co., Philadelphia, 1971,p. 329. =NutritionalBiochemical Co.. 26201 Mites Rd.. Cleveland. OH. 44128 or Sigma Chemical Co., B& 14508 St. ~ouis; Ma.63178. THeimer, E. P., J . CHEM. EDUC., 49,547 (1972).
lar models to the center table for comparison of the several shapes and the difference between the D and L forms. Remove ehromatogram no. 2 at the appropriate time. After comparing molecular models, spray chmmatograms 1 and 2 with ninhydrin (use hood) and place in 100DCoven for 1-2 min. Rinse tank twice and refill with solvent to proper level; develop chromatograms 1 and 2 from the oven and note the positions of color on the chmmatograms; do not expect extract or hydmlysate to he completely separated. You will, however, be able to detect the presence and absence of several amino acids from the skin extract and hydrolyzed insulin by comparing the migration distances of the several spots of the mixture with those spots of the separate amino acids. Remove ehromatogram no. 3 when the solvent front is in. fmm the top. While it dries, bring your peptide chains to the hallway and connect them into the two n and B chains of insulin heing mindful of the proper order; connect the two large chains by cysteines as shown in the reference. Yaur instructor will certify this construction, after which you should construct lysozyme in the same manner. As soon as your section of the lysozyme has been inserted into the chain, return to the laboratory and spray chromatogram no. 3 and insert it in the oven. Begin elean-up at this time. After chromatogram no. 3 has been removed from the oven, return to the hallway for final inspection of the protein chains. Possible Goofs: You may spot too heavily, you may forget to rinse the developing jar between runs, and you may fill the developing jar ahove the level afthe spots on the chromatogram sheet. Note: Obtain a urine-specimen bottle and take it home with you; next week you will need a morning-void sample for lab. If you miss this ~ m t e i nlab, call in and report your chain numbers and amino acid name. Comments on Laboratory Preparation
3-in. tlc sheets will result in developing times of 110 s using the solvents mentioned. Experiment: Amino Acids, Proteins, Skin, Insulin, Etc.
lntroduction In this experiment you will compare molecular models of amino acids, connect chains of amino acids into polypeptides, and form cross-links where necessary to form proteins. You will extract amino acids from your skin, and insulin will be hydrolyzed by HC1; you will then analyze the extract and t h e hydrolysate in a n attempt t o find out which amino acids are present. Considerable library and home preparation is necessary for this experiment. Pre-Lab Assignment A. .-ls?embly of Am~no-AridMolecular Models. From the molerulnr model kit which you have, assemble the mode1 ot'rhr amino arid c~rcledbelow. Hrmg it to lab comp1vrr.d. leueine serine glutamine alanine lycine threonine glutamic acid arginine tryptophan glycine methionine asparagine phenylalanine tyrosine histidine aspartic acid proline dine isoleucine cysteine For details of the structure of each of these amino acids, see Doty "Proteins" in the reprint packet. In all cases (except those whose names are marked *) make the L form. For these details, see Figure 1.2 of the l a b procedures, pages 1-5 of Krogmann on reserve in library. B. Assembly of Chains. Cut out amino acid symbols from sheets issued. Assemble section of protein chain indicated below on string or thread. Bring to l a b for final assembly.
T h e pre-lab assignment requires the detailed attention of the instructor prior to its being handed out t o students. The instructions ahove are dittoed with the spaces in B left blank a s shown. T h e total number of amino acids in both chains is 180. T h u s each student in a 24-station l a b would have seven or eight symbols strung together "paper-doll" fashion when she comes t o lab. The one-tos&n section would he tied to the eight-to-fifteen section and so forth. T h e shapes of t h e amino acid models can be sketched by a committee of students, dittoed and distrihuted t o the students; t h e shapes should, of course, reflect roughly the shape of the R-group. As t h e instructor fills in the numbers for the amino acid chains, h e also circles a different amino acid in part A. He should asterisk several of them so they can he compared t o the L forms. A particularly impressive demonstration involves placing a D and L form side-hy-side separated by a mirror (A 12 X 16-in. mirror is large enough for the model kits recommended.) Lifting and replacing the mirror shows the geometric relationships clearly. A convenient technique for teaching students t o distinguish between D and L forms of amino acids is t o give them the following instructions
1) Insulin; textbook (use index) your section -to-
inclusiveof the-chain. 2), Lvsozvme: , . see Phillim' "The Three Dimensional Structure of an Enzyme Molecule" in reprints. Yaur section-to-inclusive.
