title: A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations.
abstract: We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).
authors.affiliation: Department of Chemistry, WestChem, University of Glasgow, University Avenue, Glasgow G12 8QQ, United Kingdom. norah.ofarrell@ncl.ac.uk
authors.forename: Norah
authors.initials: N
authors.lastname: O'Farrell
authors.forename: Michaela
authors.initials: M
authors.lastname: Kreiner
authors.forename: Barry D
authors.initials: BD
authors.lastname: Moore
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Parker
mesh_terms: D001665:Binding Sites / Q000502:physiology; D004798:Enzymes / Q000737:chemistry; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017186:Titrimetry
chemical_list: D004798:Enzymes; D011506:Proteins; D010664:Phenylmethylsulfonyl Fluoride; D012697:Serine Endopeptidases