(138) Assay Card

General Information
Summary MCo-AT1-7 showed high stability in human serum, which was more stable than AT1-7 and linear MCo-AT1-7. MCo-AT1-7 was able to increase the intracellular concentration of NO as AT1-7 but not MCOTI-II.
Condition Human serum was spun down at 15,000 rpm for 10 min to separate the lipid components. Peptides (≈15 µg dissolved in 15 µL PBS (phosphate buffer saline)) were mixed with 150 µL human serum and incubated in a 37 °C water bath. Aliquot samples (15 µL) were taken at different time points and serum protein were precipitated with ≈70% MeCN at 4 °C for 10 min. The supernatant was separated, lyophilized,dissolved in 5% MeCN in water containing 0.1% formic acid, and analyzed by HPLC-MS/MS.
Result MCo-AT1-7 was only slightly less stable (τ1/2 = 39 ± 5 h) than the parent cyclotide MCoTI-I (τ1/2 = 55 ± 7 h). In contrast, peptide AT1-7 was degraded considerably faster under the same conditions (τ1/2 = 44 ± 3 min). A linearized, reduced and alkylated version of MCo-AT1-7 was also rapidly degraded (τ1/2 = 57 ± 5 min) indicating the importance of the circular Cys-knot topology for proteolytic stability.
AssayType Enzymatic Digest

References
Aboye, T., Meeks, C.J., Majumder, S., Shekhtman, A., Rodgers, K. and Camarero, J.A (2016) Design of a MCoTI-based cyclotide with angiotensin (1-7)-like activity Molecules 21:152-0

Cross-references
Proteins Assayed MCo-AT1-7