(14) Assay Card

General Information
Summary Hemolytic activity for established for kalata B1 but not acyclic permutants
Condition Human erythrocytes (blood group O) were washed several times with phosphate-buffered saline and centrifuged at 3500¬rpm until a clear supernatant was obtained. A 1% solution of the red blood cells in phosphate-buffered saline was used in the assays, which were performed by adding 20¬¬µl of test compound to 80¬¬µl of the 1% suspension of red blood cells. A 1% solution of SDS was used as a control for 100% hemolysis. The mixtures were left at room temperature for 1¬h and then centrifuged at 3500¬rpm for 1¬min. The supernatant (20¬¬µl) was diluted with 580¬¬µl of deionized (Milli-Q) water, and the absorbance was measured at 415¬nm.
Result Kalata B1 has previously been shown to have hemolytic activity, and this was confirmed in the current study, where 50% hemolysis occurs on incubation of erythrocytes with 50¬¬µM kalata B1. Recent studies have shown that factors such as high amphipathicity, high hydrophobicity, and high helicity correlate with high hemolytic activity. Thus, it is not surprising that the level of hemolytic activity observed for the non-helical kalata B1 is rather mild and less than that for highly helical peptides such as melittin (<1 ¬µM for 50% hemolysis). However, despite the ready detection of activity for the parent kalata molecule, no lysis was observed for the acyclic permutants at concentrations of up to ~60 ¬µM.
AssayType Hemolytic

Daly NL, Craik DJ (2000) Acyclic permutants of naturally occurring cyclic proteins. Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1. J Biol Chem 275:19068-75

Proteins Assayed kalata b1-2
kalata b1-3
kalata b1-5
kalata b1-6a
kalata b1-6b
kalata b1-1
kalata b1-4
kalata B1