title: Critical assessment of the spectroscopic activity assay for monitoring trypsin activity in organic-aqueous solvent.
abstract: Solvent-assisted protein digestion involves enzymatic hydrolysis in mixed aqueous-organic solvents. With trypsin, acetonitrile is the modifying solvent of choice, recommended at concentrations from 10 to 80% to improve protein sequence coverage in mass spectrometry. Spectroscopic activity assays employing substrate mimics such as N-benzoyl arginine ethyl ester (BAEE) appear to show a relative enhancement of trypsin activity in mixed solvent systems. However, as reported here, the changing solvent polarity induces bias in the absorbance measurement, lending upward of 35% error in the apparent trypsin activity as the acetonitrile is raised to 70%. Furthermore, time-dependent spectroscopic and mass spectrometric measurements reveal a progressive deactivation of trypsin over a 5- to 10-min period in as little as 30% acetonitrile.
authors: Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
authors: Andrew M J
authors: AM
authors: Crowell
authors: Emma J
authors: EJ
authors: Stewart
authors: Zahra S
authors: ZS
authors: Take
authors: Alan A
authors: AA
authors: Doucette
mesh_terms: D000097:Acetonitriles / Q000737:chemistry*; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry; D014106:Tosylarginine Methyl Ester / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*; D014867:Water / Q000737:chemistry*
chemical_list: D000097:Acetonitriles; D011506:Proteins; D012997:Solvents; D014867:Water; D014106:Tosylarginine Methyl Ester; D001120:Arginine; C009727:benzoylarginine ethyl ester; D014357:Trypsin; C032159:acetonitrile