Enzymatic Cleavage of Polypeptides

Abstract

There are many commercially available enzymes that may be used as tools to study protein structure. These include exopeptidases (such as carboxypeptidases and pyroglutamate aminopeptidase), which may be used to study C-terminal and blocked N-terminal sequences, and endopeptidases, which may cleave the polypeptide chain at any point, and so be used to generate peptides. This chapter describes the use of endoproteinases. The number and nature of peptides generated by a proteinase of good specificity is characteristic of a protein, since it reflects the protein’s sequence. The peptide map has long been used as a check on a given protein’s purity and integrity. Traditionally, the term “peptide map” is applied to the chromatogram or pattern of peptides resolved by a method such as HPLC or capillary electrophoresis (see Chapters 10 and 11), but the principle is the same in a more recent development in which the “map” is a list of peptide masses, determined by mass spectrometry In this approach, described in Chapter 17, a protein of unknown identity is cleaved to peptides, the masses of these determined by mass spectrometry and this list of masses compared with a database of peptide mass “maps” from known protein sequences. Individual peptides may also be purified and subjected to various sequencing techniques as described elsewhere in this volume, the purpose being to identify the parent protein or sites of modification. Chemical methods of proteolysis have also been developed (see Chapter 6), which can usefully complement enzymatic methods, but these may be unsuited to some purposes in that they may destroy biological activity.