Application of immobilized aminopeptidases to the sequential hydrolysis of proline-containing peptides

Publisher Summary This chapter describes application of glass-bound Clostridial aminopeptidase (CAP) and aminopeptidase P (AP-P) to the sequential hydrolysis of proline-containing polypeptides. Peptide bonds associated with proline residues—present in a polypeptide chain—are unique in their susceptibility to hydrolysis by aminopeptidases. CAP isolated from Clostridium histolyticum is able to cleave N-terminal proline residues from polypeptide chains at rates comparable to the cleavage rates of other N-terminal amino acid residues. The enzyme does not cleave N-terminal residues if followed by proline. Useful enzymatic preparations are obtained by coupling AP-P and CAP to amino-glass beads by the glutaraldehyde method. The resistance of cytochrome C to hydrolysis and the ordered release of amino acids during the hydrolysis, demonstrated for several peptides in the chapter, shows that the preparations are free of interfering enzyme activities. The resistance of X-Pro bonds to hydrolysis by CAP and the absolute specificity of AP-P for N-terminal amino acids, which are followed by a proline residue make it possible to degrade polypeptides up to any proline residue present in the peptide chain, resulting in peptide fragments with Pro or X-Pro at the amino end.