(47) Assay Card

General Information
Summary At a concentration of 25 ºM, a more than 6-fold difference exists between the most hemolyticcyclotide, cycloviolacin O24 (~75% hemolysis), and the least hemolytic cyclotide cycloviolacin O14 (~11% hemolysis). The sensitivity of hemolytic activity to variations in the peptide sequence is evident when comparing cycloviolacin O2 and O13. Here, the only sequence deviation is a single residue substitution of a serine in loop 3 of O2 (HD50 ~36 ºM) to an alanine in the homologous position in O13 (HD50 ~11 ºM). The loss of this single hydroxy group changes the HD50 more than three-fold.
Condition Peptides were dissolved in water and serially diluted 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 (RBCs) were washed with PBS and centrifuged at 4000 rpm for 60 sec in a microcentrifuge several times until a clear supernatant was obtained. A 0.25% suspension of washed RBCs in PBS (100 ºL) was added to the peptide solutions. The plate was incubated at 37¬C for one hour and centrifuged at 150 g for five minutes. 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% 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 RBCs (HD50) was calculated using the regression constant from the linear portion of the hemolytic titration curve (Graphpad Prism software).
Result At a concentration of 25 ºM, a more than 6-fold difference exists between the most hemolyticcyclotide, cycloviolacin O24 (~75% hemolysis), and the least hemolytic cyclotide cycloviolacin O14 (~11% hemolysis). The sensitivity of hemolytic activity to variations in the peptide sequence is evident when comparing cycloviolacin O2 and O13. Here, the only sequence deviation is a single residue substitution of a serine in loop 3 of O2 (HD50 ~36 ºM) to an alanine in the homologous position in O13 (HD50 ~11 ºM). The loss of this single hydroxy group changes the HD50 more than three-fold.
AssayType Hemolytic

References
Ireland DC, Colgrave ML, Craik DJ (2006) A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability. Biochem J. 400:1-12

Cross-references
Proteins Assayed cycloviolacin O15
kalata S
cycloviolacin O13
cycloviolacin O14
cycloviolacin O24
kalata B1
cycloviolacin O2