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Downstream processing of an industrial process yielded a highly purified bioactive protein. This protein was subjected to cleavage by trypsin. Chromatographic separation of products resulted in 4 peptides (P, Q, R, S) with the following amino acid sequences
P. phe-val-met-val-arg
Q. ala-ala-try-gly-lys
R. val-phe-met-ala-gly-lys
S. phe-gly-try-ser-thr
Chemical cleavage of the same protein with cyanogenbromide and chromatographic separation resulted in three peptides (i, ii, iii) with the following sequences
(i) ala-gly-lys-phe-gly-try-ser-thr
(ii) ala-ala-pry-gly-lys-phe-val-met
(iii) val-arg-val-phe-met
The order of the peptides that gives the primary structure of the original protein is
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- P, Q, R, S
- Q, P, R, S
- Q, R, P, S
- R, Q, P, S
Correct Option: C
Cyanogen bromide hydrolyzes peptide bonds at the C-terminus of methionine residues. This reaction is used to reduce the size of polypeptide segments for identification and sequencing. The aspartate residue (Asp 189) located in the catalytic pocket (S1) of trypsins is responsible for attracting and stabilizing positively charged lysine and/or arginine, and is, thus, responsible for the specificity of the enzyme. This means that trypsin predominantly cleaves proteins at of the amino acids lysine and arginine except when either is bound to a N-terminal proline. Although large-scale mass spectrometry data suggest cleavage occurs even with proline. Trypsins are considered endopeptidases, i.e., the cleavage occurs within the polypeptide chain rather than at the terminal amino acids located at the ends of polypeptides.
