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Chapter: Biochemistry: Protein Purification and Characterization Techniques

Cleavage of the Protein into Peptides

Proteins can be cleaved at specific sites by enzymes or by chemical reagents.

Cleavage of the Protein into Peptides

Proteins can be cleaved at specific sites by enzymes or by chemical reagents. The enzyme trypsin cleaves peptide bonds preferentially at amino acids that have positively charged R groups, such as lysine and arginine. 


The cleavage takes place in such a way that the amino acid with the charged side chain ends up at the C-terminal end of one of the peptides produced by the reaction (Figure 5.16). The C-terminal amino acid of the original protein can be any one of the 20 amino acids and is not necessarily one at which cleavage takes place. A peptide can be automatically identified as the C-terminal end of the original chain if its C-terminal amino acid is not a site of cleavage.

Another enzyme, chymotrypsin, cleaves peptide bonds preferentially at the aromatic amino acids: tyrosine, tryptophan, and phenylalanine. The aromatic amino acid ends up at the C-terminal ends of the peptides produced by the reaction (Figure 5.17).


In the case of the chemical reagent cyanogen bromide (CNBr), the sites of cleavage are at internal methionine residues. The sulfur of the methionine reacts with the carbon of the cyanogen bromide to produce a homoserine lac-tone at the C-terminal end of the fragment (Figure 5.18).


The cleavage of a protein by any of these reagents produces a mixture of peptides, which are then separated by high-performance liquid chromatogra-phy. The use of several such reagents on different samples of a protein to be sequenced produces different mixtures. The sequences of a set of peptides produced by one reagent overlap the sequences produced by another reagent (Figure 5.19). As a result, the peptides can be arranged in the proper order after their own sequences have been determined.



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