(100) Assay Card

General Information
Summary Cycloviolacins Y4 and Y5 are the most hemolytic of the new cyclotides, both being more potent than the prototypic cyclotide kalata B1. By contrast, cycloviolacin Y1 is substantially less active than all of the other cyclotides tested.
Condition Peptides were dissolved in water and serially diluted in phosphate-buffered saline (PBS) to give 20 mL test solutions in a 96-well U-bottomed microtiter plate (Nunc). Human type A red blood cells (RBCs) 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% suspension of washed RBCs in PBS was added (100 µL) to the peptide solutions. The plate was incubated at room temperature for 1 h and centrifuged at 150 g for 5 min. Aliquots of 100 µL 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 level of hemolysis was calculated as the percentage of maximum lysis (1% Triton X-100 control) after adjusting for minimum lysis (PBS control). Synthetic melittin (Sigma) was used for comparison.
Result HD50 for kalata B1 (11.7 uM), cycloviolacin Y4 (9.3 uM), cycloviolacin Y5 (8.7 uM). cycloviolacin Y1 had a very low hemolytic result.
AssayType Hemolytic

Wang CK, Colgrave ML, Gustafson KR, Ireland DC, Göransson U, Craik DJ (2007) Anti-HIV Cyclotides from the CHinese Medicinal Herb Viola yedoensis J Nat Prod 71:47-52

Proteins Assayed kalata B1
kalata S
cycloviolacin Y1
cycloviolacin Y4
cycloviolacin Y5