(105) Assay Card

General Information
Summary G6A and E7A mutants of kalata B1 were completely impervious to enzymatic digestion by the three endoproteinases trypsin, chymotrypsin, and proteinase K, as was the prototypic cyclotide kalata B1.
Condition 1 mg/ml solutions of WT-kB1, G6A, or E7A were prepared in 100 mM ammonium bicarbonate buffer (pH 8.1). The enzymes used in these studies were trypsin, chymotrypsin, and proteinase K. Enzymatic digestions (50:1 substrate-to-enzyme ratio) were carried out at 37 C with aliquots taken at set times up to 24 h.
Result G6A and E7A mutants of kalata B1 were completely impervious to enzymatic digestion by the three endoproteinases trypsin, chymotrypsin, and proteinase K, as was the prototypic cyclotide kalata B1.
AssayType Enzymatic Digest

References
Simonsen SM, Sando L, Rosengren KJ, Wang CK, Colgrave ML, Daly NL, Craik DJ (2008) Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity J Biol Chem 283:9805-9813

Cross-references
Proteins Assayed kalata B1
[G6A]kalata B1
[E7A]kalata B1