(5) Assay Card

General Information
Summary Resistance to enzymatic digest shown for kalata B1 and acyclic permutants. Reduced protein was susceptible.
Condition Peptide solutions were dissolved in appropriate buffers at a concentration of 1 mg/mL. Trypsin, endoprotease Glu-C (endoGlu-C), pepsin, and thermolysin (Sigma-Aldrich) were prepared as 1 mg/mL stock solutions in MilliQ H2O. These were then diluted in appropriate buffers to give a final working solution with a concentration of 20 g/mL. Trypsin and endoGlu-C were prepared in 100 mM ammonium bicarbonate (pH 8.1). Pepsin digestions were undertaken in a 100 mM acetic/formic acid solution (pH 2.1). Thermolysin was prepared in 100 mM ammonium bicarbonate (pH 8.1) containing 10 mM CaCl2 as an enzymatic cofactor. Digestions using trypsin, endoGlu-C, and pepsin were conducted at 37 C, while thermolysin was incubated at 65 C. Equal volumes of peptide and enzyme working solutions were mixed to yield a final substrate/enzyme ratio of 50/1 in all cases. Aliquots (1uL) were taken at appropriate time intervals and diluted 100-fold in H2O for immediate analysis by LC-MS or acidified and run at a later time.
Result Kalata B1 was impervious to trypsin, endo Glu-C, pepsin, and thermolysin, as were acyclic permutants des(24-28)kB1and des(19-20)kB1. Reduced and alkylated kalata B1 was degraded rapidly by trypsin, approx 50% was degraded after one and a half hours with endo Glu-C and was degraded after 90 min with thermolysin. It was resistant to pepsin cleavage. Reduced and alkylated acyclic permutants were degraded by trypsin (<15min), endo Glu-C (<4h) and thermolysin (<5h) and were stable to pepsin.
AssayType Enzymatic Digest

Colgrave ML, Craik DJ (2004) Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry 43:5965-75

Proteins Assayed des(24-28)kB1
kalata b1-5
kalata B1