You wish to determine the sequence of a 7 residue peptide. Analysis after comple

Question

You wish to determine the sequence of a 7 residue peptide. Analysis after complete digestion indicates the presence of the following amino acids: Asp, Glu, Phe, Pro, Trp and Val. when reacted with Edman Reagent, a PTH derivative similar of Trp is produced. When reacted with chymotypsin, three peptides of 1, 1 and 5 amino acids are produced. When reacted with Endopeptidase Lys-c (Lys cleaving enzyme), one peptide of 7amino acids produced. When reacted with Staphylococcal protease, three peptides of 1, 3 and 3 amino acids are produced When the buffer for the Staphylococcal protease reaction was changed to bicarbonate buffer, three peptides of 1, 3 and 3 amino acids are also produced. When reacted with Trypsin (Arg, Lys cleaving enzyme), one peptide of 7 amino acids was produced. Chemical fragmentation with hydroxylamine at pH 2.5 produces two peptides of 3 and 4 amino acids. What is the sequence of the peptide? Information about the fragmentation patterns are listed in the notes for chapter 5.

Explanation / Answer

There are 6 amino acids given: Asp, Glu, Phe, Pro, Trp and Val.

'Trp' is present in the end. Edman's degradation gives the N' terminal amino acid.

Three peptides are produced on cleavage with chymotrypsin. So, 2 sites of chymotrypsin are there. Chymotrypsin cleaves at the ends of aromatic amino acids (one is 'Phe' and the other is 'Trp'). In total, there are 7 amino acids in the protein. At N-terminal Edman' degradation has already revealed 'Trp'.

So, the sequence becomes:

N'-Trp (1st cut) ---------- (4 amino acids)-----------------------------Phe--(2nd cut)--(//)-C'

// signifies any other amino acid.

Now, acid hydrolysis reveals three fragments of sizes of 1, 3 and 3 amino acids. Acid hydrolysis will occur at C-terminal ends of acid.

N'-Trp--Val-- Glu--(1st cut)--Asp--(2nd cut)--Pro--Phe--(//)-C'

When cleaved with Trypsin, one peptide of 7 amino acids was obtained. Trypsin cleaves at C-terminal of 'Arg' and 'Lys'. Please note that the in the initial information given, basic amino acids are not there in this protein. And a single fragment is revealed from trypsin hydrolysis, so you can conclude that at the C-terminal end, the 7th amino acid of this protein is Lysine or Arginine.

So, the final protein sequence becomes:

N'-Trp--Val-- Glu---Asp--Pro--Phe--(Lys/Arg)-C'

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