Discussion:
Amino Acid Problems
1. An organism of unknown origin produces a potent inhibitor of nerve conduction which you wish to sequence. Amino acidanalysis shows the peptide's composition to be 5 Ala, 1 Lys, 1 Phe. Reaction of the intact peptide with phenylisothiocyanate (Edman's reagent) followed by treatment with trifluoroacetic acid liberates a phenylthiohydantoin (PTH)-derivative of alanine. Trypsin cleavage of the intact peptide gives a tripeptide and a tetrapeptide, with compositions 3 Ala, 1 Phe and 1 Lys, 2 Ala. Reaction of the intact peptide with chymotrypsin yields a hexapeptide plus free alanine. What is the inhibitor's sequence? (Justify your answer by explaining how each treatment would produce the observed products.)
2. A solution which contains a mixture of three tripeptides -- 1) Tyr-Lys-Thr, 2) Asp-Met-Ala, and 3) Asp-Cys-Arg -- is spotted in the middle of three strips of filter paper. Each strip is then subjected to paper electrophoresis at a different pH: 2.0, 6.0, and 13.0. Sketch the patterns of separation you would expect for these tripeptides at each of these pHs, indicating their positions relative to one another and to the anode (i.e., + electrode), spot of application (i.e., origin), and cathode (i.e., - electrode). See Table 4.1 in the Garrett and Grisham text for pKas. Hint: the separation is based on the net charge carried by the peptides.