Section on Edman degradation in Chapter 3 of your textbook (p. 98- 100). should

Question

Section on Edman degradation in Chapter 3 of your textbook (p. 98- 100). should help you answering the following questions. Upon complete hydrolysis, a peptide yielded gly + ala + 2 cys + arg + glu + ile + thr + phe + val + NH^+_4. Reduction of the original peptide with mercaptoethanol. followed by alkylation of the cysteine residues with iodoacetate (to prevent the re formation of disulfide bridges) yielded two smaller peptides (A and B). during hydrolysis glutamine (Gln) is converted into glutamate (Glu) + KH^+_4. Check table 3 - 6 in your textbook to find the specificity of trypsin and chymotrypsin Question: Suggest a likely structure of the original peptide (primary structure of the two small peptides, and possible covalent bonds between them) from the following data: Peptide A: Contained ala + gly + cys + glu + arg + ile + KH^+_4 Carboxypeptidase A (enzyme that cleaves the C - terminal residue in a peptide) liberated isoleucine. Treatment with the Edman reagent (phenylisothiocyanate. P1TC) yielded the phenylthiohydantoin derivative of glycine (PTH-glycine). See Figure 3 - 27 and corresponding text in your book for details on the Edman degradation. Treatment with trypsin yielded two peptides. The first peptide contained glu + ile + NH^+_4. The second contained gly + ala + cys + arg Peptide B: Contained thr + val + cys + phe Carboxypeptidase A liberated valine Chymotrypsin liberated valine and a tripeptide containing cys + thr + phe The Edman degradation yielded PTH-threonine

Explanation / Answer

Upon complete hydrolysis peptide yielded

Gly + ala + cys + cys + arg + glu + ile + thr + phe + val + NH4+

Peptide A contained ala + gly + cys + glu + arg +ile + NH4+

i) Carboxypeptidase A cleaves peptide bond at C terminus. Which liberated ile.

Possible structure = C – ile

ii) Edman reagent cleaves amino acid from amino terminal.

Possible structure = C – ile –x – x – x – x – gly-N

iii) Treatment with trypsin yielded two peptides.

Trypsin cleaves peptide chain at C terminal at lysine or arginine

Possible structure = C – ile – glu + NH4+ – arg – cys – ala – gly – N

Peptide B contained thr + val + cys + phe

i) Carboxypeptidase A cleaves peptide bond at C terminus. Which liberated val.

Possible structure = C – val

ii) Chymotrypsin liberated val and a tripeptide.

Chymotrypsin cleaves at carboxyl side with large hydrophobic amino acids(tyr,trp,phe)

Possible structure = C – val – phe –

iii) Edman reagent cleaves amino acid from amino terminal, which liberated PTH-threonine

Possible structure = C – val – phe – cys – thr – N

Structure

C – ile – glu + NH4+ – arg – cys – ala – gly – N

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                      C – val – phe – cys – thr – N

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