Protein Chemistry

We've tried many specific exercises that address protein chemistry. Some we've used are useful in some respects, but aren't instructive in the way I like labs to mesh with lecture because they don't make simple connections with proteins' molecular properties. I designed much of the following and I like the outcome better.

Various manuals include the biuret, xanthoproteic acid, Hopkins-Cole, and Millon's tests. I dropped the Hopkins-Cole and Millon's tests because the former doesn't add much to the xanthoproteic acid test and its chemistry is "not completely understood" and the latter employs mercury which I'd rather avoid.

R Group Tests: Xanthoproteic Acid Test. Students are told that this is a chemical test for specific functional groups in amino acid side chains that gives a color with only two of the twenty amino acids, tyrosine and tryptophan. Depending which is present, formation of a yellow or orange color is a positive test. They are to add 5 drops of concentrated nitric acid to 10 drops of two amino acid solutions, either 0.02% tryptophan or 0.005% tyrosine (may need a bit of NaOH to dissolve); tubes should be quite dry since even a few drops of water may dilute the nitric acid enough to prevent nitration. After heating in a boiling water bath for five minutes, students add 25-30 drops of 3 M NaOH and note the final color formed. Tyrosine should be yellow and tryptophan orange.

Protein Tests: Biuret Test. Students are told this is a general test for proteins, not amino acids, and that a similar color is formed with all proteins. They are to put 10 drops of water, a 2% amino acid mixture (e.g. Difco's Casamino Acids), 2% egg albumin, or 2% gelatin in four separate tubes and add 5 drops of 3 M NaOH to each. Finally, 2 drops of 1% copper sulfate (pentahydrate) are added and the results are noted. The water forms a light blue solution, the amino acids a darker blue, and both proteins a purple solution. We either make the albumin solution from powdered crude egg albumin, with lots of stirring, or simply dilute raw egg whites with 5.5 volumes of water. Either way, the solution needs filtering through, for example, cheesecloth and Kimwipes and is still somewhat cloudy. Gelatin of course can be dissolved in boiling water, but is much faster to dissolve if you let it hydrate overnight in a refrigerator.

Xanthoproteic Acid Test. This test is performed on 10-drop samples of albumin and gelatin exactly as described above.

Cystine Test. In this test, a black precipitate indicates the presence of disulfide-bonded cysteine in proteins. Students put 10 drops of 2% gelatin or 2% albumin in separate tubes, then add 5 drops of 3 M NaOH and 2 drops of 0.1 M lead nitrate. They then heat the tubes for 5 minutes in a boiling water bath and record observations.

Determination of Protein Shape: This test depends on the simplistic assumption that fibrous molecules make a more viscous solution than compact, globular ones. I have contrived simple viscometers using either burets or (for smaller volumes) Pasteur pipets. The burets or pipets were connected so that they drained through a capillary tube.  For burets (the kind with removable Teflon stopcock and glass tip), I replaced the usual glass tips with 2.5-inch lengths of glass capillary (approx. 5.5 mm o.d.; I don't know the bore diameter, I found this stuff lying around the stock room). For the pipets, I used a standard disposable 25-microliter glass capillary.  Then I made marks on the buret or pipet such that water takes 30-40 seconds to drain from one mark to the other. Students are told that this drain time depends on the viscosity of the solution (intuitive) and that the viscosity depends, in part, on the shape of the molecules in the solution. (As an analogy, I ask them to compare eating spaghetti and Spaghettios.)

In separate devices, they were to determine the drain times for water, 2% albumin, and 2% gelatin. After subtracting the time for water from both protein solutions, they see the effect of the dissolved proteins (about 5 seconds for albumin, 20-30 seconds for gelatin). I'm not enough of a physical chemist to know in what respect this is a gross simplification, but I'm not naive enough to think it isn't. At least, it is a simple way for students to see and measure a macroscopic consequence of the different shapes of gelatin and albumin, which is the goal of the test. I think it's pretty cool. One possible problem is that albumin inevitably coagulates and if a glob of it got into the capillary it could slow the flow. I haven't seen this happen, but I keep an eye open for it.

Protein Denaturation: There are of course many ways to denature albumin (including, it seems, looking at it too hard), but I only want to use those that illustrate the importance of weak bonds in globular proteins' tertiary structures. Ten drops of 2% albumin or 2% gelatin are treated with six drops of methyl ethyl ketone or of 3 M HCl, or were placed briefly in a boiling water bath. In all cases, albumin became insoluble but gelatin was unaffected. We looked at this from the perspective that methyl ethyl ketone presumably disrupts hydrophobic interactions, the acid disrupts ionic bonds, and heat disrupts hydrogen bonds. Sadly for the point of the exercise, neither urea nor NaOH causes albumin to precipitate; I guess you can't have everything. Students were instructed to equate denaturation with coagulation, which of course isn't general but works okay here.  Lately, I have also been including treatment with an ice-water bath to make the point that cold does not denature proteins, so I can lead later into the idea that we use cold to preserve food not by denaturing microbial proteins, but by slowing microbial chemistry.

After recording all their observations, students were asked about (1) the presence or absence of tryptophan, tyrosine, and cysteine in albumin and gelatin, (2) the color of and the structural requirement for a positive biuret test, (3) the molecular structure, fibrous or globular, of albumin and gelatin, and (4) their conclusions from denaturation tests about whether albumin or gelatin are compact, folded globular proteins.

In the xanthoproteic acid test, albumin gives a distinctly orange color, whereas gelatin gives yellow. For question (1) above, students were told that gelatin is said to be incomplete because it lacks tryptophan, and they should respond that their observations were consistent and that, further, gelatin must have tyrosine to produce a yellow color. The concentrations of amino acids in the first test above should be adjusted, if necessary, to match the colors obtained with the intact proteins. I thought about adding a final question asking them to summarize everything they learned from this exercise about the chemistry of albumin and gelatin. It might be a nice finale if it doesn't seem too redundant.

 

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Revised 8/31/06