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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

Lookup NU author(s): Dr Norah O'Farrell

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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 (PCMC). © 2006 Wiley Periodicals, Inc.

Publication metadata

Author(s): O'Farrell N, Kreiner M, Moore BD, Parker M-C

Publication type: Article

Publication status: Published

Journal: Biotechnology and Bioengineering

Year: 2006

Volume: 95

Issue: 4

Pages: 767-771

ISSN (print): 0006-3592

ISSN (electronic): 1097-0290

Publisher: John Wiley & Sons, Inc.

URL: http://dx.doi.org/10.1002/bit.20792

DOI: 10.1002/bit.20792

PubMed id: 16917861

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