Determination of active enzyme concentration using activity-based probes and direct mass spectrometric readout.
Activity-based probes (ABPs) are specific covalent inhibitors developed for different classes of enzymes. We have titrated a serine protease and a lipase with their specific ABPs and measured the extent of inhibition using nanoelectrospray mass spectrometry (nanoESI-MS). Because ABPs only interact with the active enzyme form, the approach allows to accurately measure the active enzyme concentration in solution. This is even possible in the presence of contaminants. The concentrations of the two enzymes were also investigated by UV spectroscopy, which appears to give higher concentrations than those measured with the active site titration method.
Bovet
Cédric
C
Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland.
Zenobi
Renato
R
D005656:Fungal Proteins; D008049:Lipase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D036103:Nanotechnology; D010261:Paraoxon / Q000494:pharmacology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014105:Tosyl Compounds / Q000494:pharmacology*
D005656:Fungal Proteins; D015842:Serine Proteinase Inhibitors; D014105:Tosyl Compounds; C003614:4-toluenesulfonyl fluoride; D008049:Lipase; C431349:lipase B, Candida antarctica; D013381:Subtilisins; D010261:Paraoxon