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

B.D. Moore, Norah O'Farrell, M. Kreiner, Marie-Claire Parker

Research output: Contribution to journalArticle

4 Citations (Scopus)

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
Original languageEnglish
Pages (from-to)767-771
Number of pages5
JournalBiotechnology and Bioengineering
Volume95
Issue number4
DOIs
Publication statusPublished - 2006

Keywords

  • enzyme
  • inhibition
  • organic solvent
  • active-site titration
  • mass spectrometry

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