(10) Assay Card

General Information
Summary Resistance to enzymatic digest shown for kalata B1 and acyclic permutants. Reduced protein was susceptible.
Condition Peptides were dissolved in DMSO and serially diluted in phosphate-buffered saline (PBS) to give 20 uL 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 30 s in a microcentrifuge several times until a clear supernatant was obtained. A 0.25% suspension of washed RBCs in PBS was added (100 uL) to the peptide solutions. The plate was incubated at room temperature for 1 h and centrifuged at 150g for 5 min. Aliquots of 100 uL 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 [buffer (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 Under a standard set of assay conditions, our results show that native kalata B1 induces 50% hemolysis (HD50) at approximately 300 uM
AssayType Hemolytic

References
Barry DG, Daly NL, Clark RJ, Sando L, Craik DJ (2003) Linearization of a naturally occurring circular protein maintains structure but eliminates hemolytic activity. Biochemistry 42:6688-95

Cross-references
Proteins Assayed des(24-28)kB1