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    Amino Acid Problems

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    9. An organism of unknown origin produces a potent inhibitor of nerve conduction which you wish to sequence. Amino acid analysis 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.)

    10. 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.

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    Question #9

    An organism of unknown origin produces a potent inhibitor of nerve conduction which you wish to sequence. Amino acid analysis 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.)

    Response:

    PTH reacts with the N-terminal amino acid residue. Therefore, we know that Ala is the N-term amino acid residue.

    Trypsin cleaves peptides on the C-terminal side of the basic amino acid residues lysine and arginine. Therefore, we know the following since a tripeptide and tetrapeptide are formed:

    N-Ala--aa--Lys-C and N-aa--aa--aa--aa-C

    Chymotrypsin cleaves on the C-terminal side of hydrophobic amino acid residues like Phe.

    Therefore, we know that the hexapeptide structure ends with Phe at the C-term:

    N-Ala--aa--Lys--aa--aa--Phe-C and Ala

    Therefore, if we fill in the rest we can put together the entire structure as:

    N-Ala--Ala--Lys--Ala--Ala--Phe--Ala-C

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    Question #10

    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 ...

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