(107) Assay Card

General Information
Summary Substitution of certain residues of kalata B1 to alanine result in loss of hemolytic activity. Residues in loops 1 and 3 along with a few other isolated residues strongly affect activity.
Condition Stock solutions of each peptide were prepared and standardized using UV spectroscopy ({epsilon} = 6410 cm-1-1 at 215 nm) at 350 µM ± 3% then serially diluted in triplicate. Human erythrocytes were washed with repeated centrifugation in phosphate-buffered saline (pH 7.4) to remove serum contaminants. A 0.25% v/v stock solution was prepared from the cell pellet, and 100 µl of this stock was added to each diluted peptide solution aliquot and then incubated at 37 °C for 1 h. After centrifugation of intact cells the percentage of hemolysis of the supernatant was measured by visual absorption spectroscopy ({lambda} = 405 nm)
Result Relative to 100% lysis control standard of 10 uL of 1% w/v Triton X-100. kB1 (69%), G1A (68%), L2A (12%), P3A (37%), V4A (34%), G6A (1%), E7A (1%), T8A (8%), V10A (49%), G11A (61%), G12A (11%), T13A (46%), N15A (4%), T16A (5%), P17A (49%), G18A (62%), T20A (62%), S22A (59%), V25A (9%), T27A (69%), R28A (15%), N29A (54%)
AssayType Hemolytic

Simonsen SM, Sando L, Rosengren KJ, Wang CK, Colgrave ML, Daly NL, Craik DJ (2008) Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity J Biol Chem 283:9805-9813

Proteins Assayed [G1A]kalata B1
[L2A]kalata B1
[V4A]kalata B1
[G6A]kalata B1
[E7A]kalata B1
[T8A]kalata B1
[V10A]kalata B1
[G11A]kalata B1
[G12A]kalata B1
[T13A]kalata B1
[N15A]kalata B1
[T16A]kalata B1
[P17A]kalata B1
[G18A]kalata B1
[T20A]kalata B1
[S22A]kalata B1
[V25A]kalata B1
[T27A]kalata B1
[R28A]kalata B1
[N29A]kalata B1
kalata B1
[P3A]kalata B1