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Determining mino acid sequences of peptide and octapeptide

1. What is the amino acid sequence of the peptide?

The complete hydrolysis of an unknown nonapeptide revealed the presence of Glu, Val, Val,Gly,Lys, Lys, Tyr, Thr and Phe residues.
The first amino acid to be detected as a phenylthiohydantoin derivative on Edman degradation of the peptide was glutamic acid.
The only amino acid detected after treating the peptide with hydrazine (converts all amino acids except the C-terminus into acyl hydrazines)was threonine.
Treatment of the peptide with trypsin (cleaves on the carboxyl side of lys and arg) and chymotrypsin (cleaves carboxyl side of aromatic amino acid)gave three fragments in each case. T1, T2, T3 and C1, C2, C3 respectively. None of the trypsin fragments was identical to the chymotrypsin fragments.
C2 and T2 proved to be dipeptides;C1 and T1 were tripeptides and C3 and T3 were tetrapeptides
The N-terminal residue of T3 was shown to be phenylalanine and the C-terminus threonine.
The N-terminus of C1 was glycine and C-Terminus was the same as in T3.
C2 was shown to contain tyrosine and glutamic acid.
T1 was composed of lysine, tyrosine, and glutamic acid.
The N-terminus of T2 was valine
The N-terminus of C3 was lysine
At basic Ph the C3 fragment migrated with a net charge of +2

2. Determine the amino acid sequence of an octapeptide that was found to contain GLY, GLy, ASP,CYS,LYS,MET,ASN, and ARG. from the information given below
A. The intact peptide was treated with cyanogen bromide (cleaves on carboxyl side of methionine) and two fragments were detected. One of the fragments was reacted with phenylisothiocyanate followed by an acid work-up. The cyclic phenythiohydantoin amino acid that was released from the residue was found to contain Asparagine.
B. Another sample of the octapeptide was reacted with hydroxylamine (cleaves Asparagine-Glycine bonds) and again two fragments were detected. One of the fragments was determined to be three residues long by gel filtration chromatography.
C. The octapeptide was then treated with Trypsin (cleaves on carboxyl side of arginin andn lysine) which resulted in three fragments. One of the fragments was determined by paper chromatography to be Lysine. One of the other fragments was determined to be two residues long and when subjected to electrophoresis in the horizontal direction placed in an atmosphere of per formic acid, and eletrophoresed in the vertical direction, it was found to lie off the diagonal.
D. Finally, 2-nitro-5-thiocyanobenzoate (amino side of cysteine) was reacted with the original peptide and two fragments resulted. One of the fragments contained two residues, which gave the same results as in #3 when subjected to diagonal electrophoresis. This same two-residue fragment also sticks on a column that has diethylaminoethyl (DEAE) groups attached to an immobile resin, when the Ph is 7

Solution Preview

Peptide Primary Structure Elucidation

What is the amino acid sequence of the peptide?

RESPONSE: I'll do this first one in great detail to help you see how to work these problems., and then I'll give you hints on the second problem and let you work it through. Sound fair enough? You did ask for help getting started after all.

The complete hydrolysis of an unknown nonapeptide revealed the presence of Glu, Val, Val, Gly, Lys, Lys, Tyr, Thr and Phe residues.

The first amino acid to be detected as a phenylthiohydantoin derivative on Edman degradation of the peptide was glutamic acid.

RESPONSE: Edman degradation (treatment of peptide with PTH) reveals the N-terminus of the entire nonapeptide. The N-terminus is Glu.

The only amino acid detected after treating the peptide with hydrazine (converts all amino acids except the C-terminus into acyl hydrazines) was threonine.

RESPONSE: If all the amino acids get converted into acyl hydrazines EXCEPT the C-terminus and Thr did not get converted into an acylhydrazine, we know that Thr must be the C-terminus.

Treatment of the peptide with trypsin (cleaves on the carboxyl side of lys and arg) and chymotrypsin (cleaves carboxyl side of aromatic amino acid) gave three fragments in each case. T1, T2, T3 and C1, C2, C3 respectively. None of the trypsin fragments was identical to the chymotrypsin fragments.

RESPONSE: Although we know this to be so, we do not know the order of these three fragments in each case. What we do know, however, is that two fragments resulting from trypsin cleavage have Lys as their C-termini. How do we know this? We know this because trypsin cleaves at the C-terminal side of Lys and Arg. Since there are no Arg in the nonapeptide, we must conclude that two of the fragments have Lys as their C-terminus. In addition, since we know that Thr is the C-terminus of the entire structure, we now know the C-terminus of the three tryptic fragments: Lys, Lys, and Thr.

RESPONSE: In addition, we also know that the chymotryptic fragments have Tyr, Phe, and Thr at their C-termini. How do we know that? Chymotrypsin cleaves on the C-terminal side of aromatic residues. Tyrosine and phenylalanine are aromatic. Therefore, chymotrypsin cleaves next to Tyr and Phe. Of course, we also know that Thr is the C-terminus of the entire peptide.

RESPONSE: Therefore, you can draw this information out so far.

C2 and T2 proved to be dipeptides; C1 and T1 were tripeptides and C3 and T3 were tetrapeptides.

RESPONSE: We'll use this information as we get going. But let's work on the tryptic fragments first.

The ...

Solution Summary

Expert provides very detailed explanation of how to determine amino acid sequences of both peptide and octapeptide. The first question is a step-by-step detail of how to solve the problem, while the second allows room for the student to use learned knowledge from the first question to complete it. Hints are provided in order to solve these problems in the future.

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