| Summary |
Cycloviolacin O13, O14, O16 and O24 were tested for their stability against proteolytic degradation by pepsin, trypsin and thermolysin.No degradation by proteases was observed for any of the cyclotides tested following 6 hours of incubation with enzymes, while the linear control peptide was completely degraded in less than 5 minutes. |
| Condition |
Enzymatic digestion assays were performed at a 50:1 substrate (peptide):enzyme ratio (w/v) for trypsin, pepsin and thermolysin. A linear 34-amino acid peptide corresponding to a truncated form of the bacterial chaperone Escherichia coli DnaK 577-610 (DnaK-f) was utilized as a control peptide. Kalata B1 was also included for comparison. For all assays, peptides were incubated with enzyme at 37¬C for up to 6 hours. Peptide solutions were prepared in the appropriate buffer as follows. 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 0.5% CH2O2 solution. Pepsin stock solution was prepared in 100 mM CH- 3COOH:CH2O2 (1:1 v/v) at pH 2 and the reaction was halted by diluting into 100 mM NH4HCO3 buffer (pH 8). All digestion assay data was 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 |
Cycloviolacin O13, O14, O16 and O24 were tested for their stability against proteolytic degradation by pepsin, trypsin and thermolysin.No degradation by proteases was observed for any of the cyclotides tested following 6 hours of incubation with enzymes, while the linear control peptide was completely degraded in less than 5 minutes. |
| AssayType |
Enzymatic Digest |
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