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A rapid and direct method for the determination of active site accessibility in proteins based on ESI‐MS and active site titrations
Author(s) -
O'Farrell Norah,
Kreiner Michaela,
Moore Barry D.,
Parker MarieClaire
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20792
Subject(s) - phenylmethylsulfonyl fluoride , active site , pmsf , chemistry , subtilisin , serine protease , electrospray ionization , chromatography , enzyme , serine , mass spectrometry , titration , biochemistry , protease , organic chemistry
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). © 2006 Wiley Periodicals, Inc.