A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations
✍ Scribed by Norah O'Farrell; Michaela Kreiner; Barry D. Moore; Marie-Claire Parker
- Publisher
- John Wiley and Sons
- Year
- 2006
- Tongue
- English
- Weight
- 117 KB
- Volume
- 95
- Category
- Article
- ISSN
- 0006-3592
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✦ Synopsis
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).