A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations
| Autor: | Barry D. Moore, Marie-Claire Parker, Norah O'Farrell, Michaela Kreiner |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Electrospray Electrospray ionization Bioengineering Applied Microbiology and Biotechnology chemistry.chemical_compound chemistry.chemical_classification Serine protease Binding Sites Chromatography biology Serine Endopeptidases fungi Titrimetry Proteins Active site Serine hydrolase Enzymes Phenylmethylsulfonyl Fluoride Enzyme chemistry Biochemistry biology.protein PMSF Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 95:767-771 |
| ISSN: | 1097-0290 0006-3592 |
| DOI: | 10.1002/bit.20792 |
| Popis: | 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). |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text