Peptide bond chemical fragmentation

The most commonly utilized chemical cleavage agent is cyanogen bromide (it cleaves the peptide bond on the carboxyl side of methionine residues). V8 protease, produced by certain staphylococci, along with trypsin are two of the more commonly used proteolytic-based fragmentation agents. 

Cleaving the Polypeptide Chain Several methods can be used for fragmenting the polypeptide chain. Enzymes called proteases catalyze the hydrolytic cleavage of peptide bonds. Some proteases cleave only the peptide bond adjacent to particular amino acid residues (Table 3-7) and thus fragment a polypeptide chain in a predictable and reproducible way. A number of chemical reagents also cleave the peptide bond adjacent to specific residues. 

In order to perform a native chemical ligation, two prerequisites have to be fulfilled. First, the C-terminal fragment (2) has to contain a Cys residue (which in principle could also be Ser, although it is less reactive) at the N-terminus. The N-terminal fragment (1) possesses a C-terminal thioester. The reactivity of this activated carboxyl group can be tuned by proper selection of the thiol substituent. For example, aromatic groups are more reactive than alkyl groups. The mechanism of peptide bond formation encompasses two steps. In a first reversible reaction the... 

Sequential analysis can be accomplished by using the Edman technique. Treatment of an intact polypeptide with phenylisothiocyanate derivatizes the N- amino acid leaving the rest of the peptide intact for further Edman degradation. Large chains must be fragmented into shorter peptides, more easy to work with chemically. Cleavage of peptide bonds at specific amino acid residues is accomplished using enzymes such as trypsin (Lys, Arg), chymotrypsin (aromatics), and carboxypeptidase (C-terminus amino acids). 

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