4) You have isolated a peptide and want to find the sequence. You have a handful

Question

4) You have isolated a peptide and want to find the sequence. You have a handful of enzymes and reagents to work with, but no access to a peptide sequencer. But you're confident it can be done, so you do the following tests: (Note: assume Carboxypeptidase A cleaves all but P, R, K and requires a free carboxylic acid) a) A bulk amino acid analysis yields the following in equivalent amounts- E, I, C, M, K, Y, F b) The peptide does not react with Edman's reagent c) The peptide does not react with Carboxypeptidase A d) When you treat with Chymotrypsin, a single peptide results. This peptide reacts with Edman's reagent to produce PTH-R and Carboxypeptidase A to yield Y. e) When you treat with Staphylococcal protease, a single peptide results. This peptide reacts with Edman's reagent to produce PTH-C and Carboxypeptidase A to yield E. f) When you treat with cyanogen bromide, a single peptide results that reacts with Edman's reagent to produce PTH-l and does not react with Carboxypeptidase A. However, you find the peptide now reacts with an enzyme that cleaves lactones. g) Trypsin treatment produces two peptides. Edman's reagent reacts with both to yield PTH-M and PTH-E, respectively. Neither peptide reacts with Carboxypeptidase A. Only the peptide producing PTH-E reacts absorbs UV at 280nm What is the sequence of the initial peptide and how do you explain the results?

Explanation / Answer

B) The given peptide cannot react with EDMAN REAGENT

cysteine or Glutamic acid(E) if present at N -terminus cannot react with Edman reagent. From N-terminus it can be either of them C or E.

c)The peptide cannot react with carcoxypeptidase A, it means from C terminal the amino acid can be R or K.

Carboxypeptidase A hydrolysis the C-terminal of the peptides, Thus amino acids such as Lysine(K), Arginine(R),and Glutamic acid(E) does not reacts with carboxypeptidase A.

d) It can be Y(tyrosine).

Chymotrypsin digestion results into a single peptide, Chymotrypsin cuts at C-terminus peptide amide bond near the hydrophobic amino acids at phenylalanine, tyrosine and tryptophan so only Y(tyrosine ) is present.

e)Treatment with Staphylococcus protease cuts only at C-terminus of aspartic and glutamic acid(E)

f) Cleavage with CNBR , it cuts only at methionine,giving rise to single peptide , this will be Methionine(M). PTH-lysine does not react with carboxypeptidase.

G)Trypsin digestion , it cuts at lysine and arginine.

PTH-E peptide absorbs UV at 280 nm is the one having Tyrosine amino acid.

So the sequence might be

N- C K M EYK/R - C

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