(44) Assay Card

General Information
Summary Violacin A showed markedly decreased hemolytic activity relative to the prototypic cyclotide kalata B1
Condition Peptide samples were dissolved in water and diluted serially in phosphate-buffered saline (PBS) to give 20 ºl test solutions in a 96-well U-bottomed microtiter plate (Nunc). Human type A red blood cells were washed with PBS and centrifuged at 4000 rpm for 60 s in a microcentrifuge several times until a clear supernatant was obtained. A 0.25% (v/v) suspension of washed red blood cells in PBS was added (100 ºl) to the peptide solutions. The plate was incubated at 37 ¬C for 1 h and centrifuged at 150g for 5 min. Aliquots of 100 ºl were transferred to a 96-well flat-bottomed microtiter plate (Falcon) and the absorbance was measured at 405 nm with an automatic Multiskan Ascent plate reader (Labsystems). The amount of hemolysis was calculated as the percentage of maximum lysis (1% (v/v) Triton X-100 control) after adjusting for minimum lysis (PBS control). Synthetic melittin (Sigma) was used for comparison. The hemolytic dose necessary to lyse 50% of the red blood cells (HD50) was calculated using the regression constant from the linear portion of the hemolytic titration curve (Graphpad Prism software).
Result Violacin A showed markedly decreased hemolytic activity relative to the prototypic cyclotide kalata B1
AssayType Hemolytic

References
Ireland DC, Colgrave ML, Nguyencong P, Daly NL, Craik DJ (2006) Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins. J Mol Biol 357:1522-35

Cross-references
Proteins Assayed violacin A