This assignment is due on Monday March 12, 2018. All work is to be submitted typ

Question

This assignment is due on Monday March 12, 2018. All work is to be submitted typed. Please submit the sequences that you determine for this peptide in the single letter format, and then in the three letter format. I.) Determine the sequence of a polypeptide that on AA composition analysis demonstrated the peptide to have: (a.) a total of 17 AAA's., (b.) The AA composition is : A(4), C(2), D, E, F H, I, L(2), R, S, T,Y: Il.) Further analysis of the peptide revealed the following: (a.) This polypeptide on treatment with Sanger's Reagent yielded the derivative DNP-E; Treatment of the intact polypeptide with Carboxypeptidase "C" yielded H, and another peptide. (b.) Treatment of the intact peptide with Chymotrypsin, cleavage sites are [F,W,M,Y,L ]; yielded the following fragments (1.) An 8-Fragment peptide that yielded DNP-A on treatment with Sanger's reagent, and H, on treatment with Carboxypeptidase "C (2.) a fragment of D-A-L (3) L (4) F (5) E-A-S-Y (c.) Treatment of the intact peptide with Thermolysin, gave the following fragments: Use the above information to determine the sequence of the peptide:

Explanation / Answer

Ans. The polypeptide has 17 amino acids and its composition is A(4), C(2), D, E, F, H, I, L(2), R, S, T and Y.

a. On treatment with Sanger's reagent it yielded DNP-E and on treatment with carboxypeptidase-C, it yielded H and another peptide.

Sanger's reagent react with N terminal amino acid. Since on reaction with Sanger's reagent it produces DNP-E, the N-terminal amino acid is E.

Treatment of polypeptide with Carboxypeptidase C releases the C-terminal amino acid. Here, it releases H. Therefore, the C-terminal amino acid is H

The amino acid is

E _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ H

b. Treatment of intact peptide with Chymotrypsin yielded an 8-fragment peptide, D-A-L, l, F and E-A-S-Y.

The 8-fragment peptide on treatment with Sanger's reagent DNP-A and on treatment with carboxypeptidase C, it releases H.

So, the 8-fragment peptide has A as the N-terminal and H as the C terminal.

So, the amino acid sequence can now be written as

E _ _ _ _ _ _ _ _ A _ _ _ _ _ _ H

Treatment of intact peptide with Chymotrypsin also gives fragment E-A-S-Y

It is already established that the N-terminal ammino acid is E and the C-terminal amino acid is H. So, we can put the amino acids present in the fragment E-A-S-Y near the N-terminal. This is possible because there is only one Y amino acid which is present in the fragment E-A-S-Y. That is, A-S-Y will follow the N-terminal E amino acid.

EASY _ _ _ _ _ A _ _ _ _ _ _ H

Treatment of intact polypeptide also gives, D-A-L, L and F. So, these amino acids are present in the first gap of the sequence written above.

c. Treatment of the intact polypeptide with thermolysin gives the fragments I-R-T, L, L, EASY, A, F and ATCH.

Thermolysin cleaves at the N-terminal side of I, L, V, A, M and F.

Since the polypeptide has only one H in it, the fragment ACTH can be placed in the sequence.

EASY _ _ _ _ _ A _ _ _ ACTH

It is already establised that the fragments D-A-L, L and F are present in the first gap of the sequence (see under b)

Therefore, the fragment I-R-C will be present in the second gap of the amino acid sequence.

EASY _ _ _ _ _ AIRCACTH

This is correct because thermolysin cleaves both before I and A

The other fragments include L, L, A, A and F.

But, the gap has the amino acids DAL, L and F

Since thermolysin do not cleave before D and the fragment has individual A and L, the DAL fragment has to be placed as follows

EASY _ _DALAIRCACTH

THe remaining amino acids L and F in either of the position because Thermolysin cleaves on the N-terminal side of both the amino acids L and Fand Chymotrypsin (which also gave individual AA fragment L and F) cleaves on the C side of both L and F.

Therefore, the sequence is either

EASYLFDALAIRCACTH

or

EASYFLDALAIRCACTH

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