(8) Assay Card

General Information
Summary Kalata B1 was observed to be resistant to acid hydrolysis.
Condition Solutions containing 1 mg/mL kB1 in either 0.5, 1, or 2 M HCl were incubated at 80 and/or 25 C to examine the stability of each peptide in acid. At specified times, 1 uL aliquots were removed and diluted 100-fold in water containing sufficient NaOH to neutralize the acid and hence stop the hydrolysis. The resulting solutions were analyzed using LC-MS as described below. DnaK-f and reduced, alkylated kalata B1 (kB1-RA) were also incubated at 80 C in 0.5 M HCl for comparison.
Result For the control DnaK-f peptide, cleavage of the backbone to yield multiple different peptide species was observed. More than 95% of the control peptide was degraded after 2 h compared to ~25% degradation of kB1. Approximately 65% of kB1-RA had been hydrolyzed after the same time, and complete hydrolysis occurred in <6 h. All of the reduced and oxidized kalata peptides were also examined after incubation in acid at 25 C, but degradation did not occur at this temperature. Under more acidic conditions (1 and 2 M HCl), the rate of hydrolysis of kB1 was increased and the point at which 95% of the peptide had been hydrolyzed was observed to be at 5 and 2 h, respectively.
AssayType Acid Hydrolysis

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 kalata B1