title: Removal of N-acetyl groups from blocked peptides with acylpeptide hydrolase. Stabilization of the enzyme and its application to protein sequencing.
abstract: Acylpeptide hydrolase, an enzyme that removes the modified residue from N-terminally acetylated peptides, has been purified from ovine liver and developed as a tool in sequencing blocked peptides and proteins. Its instability imposes a major limitation on the use of the mammalian enzyme in protein chemistry. Coupling to Sepharose followed by intramolecular cross-linking with dimethyl-suberimidate increased its thermostability and rendered it more resistant to inactivation by either SDS or N,N-dimethylformamide. The resulting enzyme preparation is reusable and more effective at cleaving longer acetylated peptides. It is therefore useful for unblocking acetylated proteins prior to protein sequence analysis. Intact proteins and many isolated peptides are still too large to be cleaved directly, but in this paper we describe a procedure for overcoming this difficulty. The protein is fragmented and non-acetylated peptides are then absorbed out with isothiocyanato-glass. The N-terminal peptide remains in solution and is unblocked with stabilised acylpeptide hydrolase. No chromatographic separation are required. The N-terminal sequence can then be obtained by automated Edman degradation. This procedure has been successfully demonstrated on a large synthetic peptide.
authors.affiliation: Pharmacia LKB Biochrom Ltd, Cambridge, England.
authors.forename: T C
authors.initials: TC
authors.lastname: Farries
authors.forename: A
authors.initials: A
authors.lastname: Harris
authors.forename: A D
authors.initials: AD
authors.lastname: Auffret
authors.forename: A
authors.initials: A
authors.lastname: Aitken
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism*; D004795:Enzyme Stability; D010446:Peptide Fragments; D010447:Peptide Hydrolases / Q000378:metabolism*; D011506:Proteins / Q000187:drug effects
chemical_list: D010446:Peptide Fragments; D011506:Proteins; D010447:Peptide Hydrolases; D000626:Aminopeptidases; C038220:acylaminoacyl-peptidase