(45) Assay Card

General Information
Summary violacin A was impervious to digestion by the endopeptidases trypsin and thermolysin following incubation for 6 h
Condition Endoprotease enzymatic digestion assays were performed at a 50:1 substrate (peptide) to enzyme ratio for trypsin and thermolysin. Trypsin and thermolysin stocks were prepared in 100 mM NH4HCO3 buffer (pH 8) (containing 10 mM CaCl2 for thermolysin digests) and their incubation with the peptide was halted upon a 5 ºl injection into 95 ºl of 0.5% (v/v) formic acid. Exoprotease enzymatic digestion assay was performed at a 50:1 substrate (peptide) to enzyme ratio for aminopeptidase M. Aminopeptidase M was prepared in 0.5 M TrisÄHCl (pH 8.5), 0.025 M MnCl2, 0.125 M MgCl2 with incubation with the peptide halted upon addition of 5 ºl of 0.5% formic acid. For all assays, peptides were incubated with enzyme at 37 ¬C for up to 6 h with the exception of the thermolysin digest, which was conducted at 65 ¬C. All digestion assay data wee analyzed by LC/MS chromatography on an Agilent 1100 series HPLC coupled to a QStar mass spectrometer (Applied Biosystems) equipped with an electrospray ionization source.
Result violacin A was impervious to digestion by the endopeptidases trypsin and thermolysin following incubation for 6 h
AssayType Enzymatic Digest

References
Ireland DC, Colgrave ML, Nguyencong P, Daly NL, Craik DJ (2006) Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins. J Mol Biol 357:1522-35

Cross-references
Proteins Assayed violacin A