Question

1.  Partial hydrolysis of lysozyme yielded an octapeptide that was later identified as the N-terminal segment of the protein.  From the following information, determine the sequence of this octapeptide.

a.  The following amino acids were identified after complete hydrolysis of the octapeptide:

            Arginine (Arg)                         Glycine (Gly)               Phenylalanine (Phe)

            Cysteine (Cys)                        Leucine (Leu)              Valine (Val)

            Glutamic Acid (Glu)                Lysine (Lys)

b.  Treatment of the octapeptide with dinitroflurobenzene (Sanger reagent) followed by complete hydrolysis gave lysine (Lys) labeled with two dinitrophenyl (DNP) groups.

c.  Treatment of the octapeptide with trypsin gave lysine (Lys), a tripeptide, and a tetrapeptide – the sequence of the tripeptide was found to be Cys-Glu-Leu.

d.  Hydrolysis of the tetrapeptide from part c with chymotrypsin gave a two dipeptides:

                         Dipeptide 1:  Contains arginine (Arg) and glycine (Gly)

                         Dipeptide 2:  Contains phenylalanine (Phe) and valine (Val)

Write the sequence of amino acids in the octapeptide and explain how this sequence was determined.

2.  Information about the structure of an octapeptide is given below.

a.  Hydrolysis of the peptide gives the following amino acids in equal amounts:  Ala, Asp, Glu, Met, Lys, Phe, Tyr, and Val.   

b.  Treatment of the peptide with 2,4-dinitrofluorobenzene followed by hydrolysis yields Asp labeled with a 2,4-DNP group.

c.  When the peptide is treated with carboxypeptidase, the concentration of Val increases rapidly.

d. Hydrolysis of the peptide with trypsin gives a tripeptide (A) and a pentapeptide (B).

e.  The tripeptide (A) is cleaved by chymotrypsin to give Phe and a dipeptide with a pI of about 5.6.

f.  The pentapeptide (B) is cleaved by chymotrypsin to give a dipeptide and a tripeptide.  Reaction of the tripeptide with cyanogen bromide gives Tyr.

Write the sequence of amino acids in the octapeptide and explain how this sequence was determined.

Answer 1

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Step 1- identify the N terminal amino acid and C terminal amino acid, sangers reagent is used for the labeling of the C terminal peptide .

Step 2- identify the peptides obtained by the treatment with trypsin, cleaves at lys and arg

Step 3- identify the peptide obtained by chymotrypsin, cleaves at bulky aromatic amino acid ( phe, tyr, trp)

Step 4- compare the data obtained by different steps.