A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations

Moore, B.D. and O'Farrell, Norah and Kreiner, M. and Parker, Marie-Claire (2006) A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations. Biotechnology and Bioengineering, 95 (4). pp. 767-771. ISSN 0006-3592

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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