The antimicrobial peptide Ltc1-K and its own derivates without one, two, then three N-terminal amino acid residues were studied predicated on the hypothesis (backed by some experimental data) which the hydrophobic N-terminal moiety of linear cationic antimicrobial peptides defines their haemolytic activity. peptides, predicated on the functional and structural analysis of a lot of -helix AMPs. Using the exemplory case of latarcins Ltc5 and Ltc2a, it was showed that the experience of AMPs regarding eukaryotic cells could be decreased via removing their N-terminal fragment or via stage amino acid substitutes that lower their hydrophobic potential [5]. We follow this path and present the outcomes of a study in to the properties of AMP Ltc1-K ( ), which corresponds to the non-processed form of latarcin Ltc1 with an additional lysine residue within the C-terminus. In terms of haemolytic activity and bactericide effect with respect to and , Ltc1-K does not differ from mature Ltc1 [6]. It was shown by NMR that when Ltc1-K binds to sodium dodecylsulfate micelles (the simplest membrane-imitating model), the N-terminal fragment of the -helix peptide is definitely embedded into the hydrophobic region of a micelle [7]. Taking into account the NMR data [7] and structural and practical analysis [5], we assumed the N-terminal fragment enhances the binding of Ltc1-K to the membranes consisting of zwitterionic lipids and determines the cytotoxic activity of a peptide. Three Ltc1-K analogues shortened from your N-terminus by one, two, and then three 1533426-72-0 GNASXL amino acid residues ( ) were synthesized in order to verify this hypothesis. The comparative analysis of their constructions, activity, and features of haemolytic action was performed. Table 1 Amino acid sequences of Ltc1-K and its derivatives -glycero-3-phosphocholine (DOPC) and 1-myristoyl-2-hydroxy- -glycero-3-phosphocholine (LMPC) (Avanti Polar Lipids, United States); Mueller C Hinton broth, carboxyfluorescein (CF), fluorescein-labelled dextrans with molecular excess weight 70 and 500?kDa (FD70 and FD500), fluorescent dyes FM1-43, propidium iodide, and Hoechst 33342 (Sigma Aldrich, United States); culture medium Roswell Park Memorial Institute 1640 (RPMI-1640) purchased from PanEco (Russia); and heparin (Sintez, Russia). The remaining reagents were of unique purity grade. The peptides ( ) were synthesized by solid phase synthesis in the Peptide Synthesis Laboratory of the Institute of Bioorganic Chemistry of the Russian Academy of Sciences (Moscow) (team head N.S. Egorova) 1533426-72-0 and seen as a HPLC and mass spectrometry (MALDI-MS) as defined previously [4, 6]. Peptide concentrations in aqueous solutions had been determined based on UV absorption utilizing a SF-256 spectrophotometer (LOMO, Russia) as well as the 1533426-72-0 molar extinction coefficients 5500?M -1 cm -1 for (-3)Ltc1-K and 11000?M -1 cm -1 for Ltc1-K, (-1)Ltc1-K, and (-2)Ltc1-K at a wavelength of 280?nm. Documenting and evaluation from the round dichroism (Compact disc) spectra The Compact disc spectra from the peptides had been recorded within a phosphate buffered saline (110?mM NaCl, 50?mM NaH 2 PO 4 ,pH 7.4) and in LMPC 1533426-72-0 micelles (30?mM regarding lipid) within a phosphate-buffered saline in a peptide focus of 40?M, utilizing a Jasco J-810 spectropolarimeter (Jasco, Japan). To be able to attain an equilibrium distribution from the peptide within the micelles, LMPC-containing examples had been pre-incubated for 30?min in a heat range of 25. The spectra had been recorded within a variety from 190 to 250?nm using a 0.2?nm increment (the slit spectral width getting 1 nm). An example was placed right into a cell using a detachable screen (Hellma, Germany) with an optical route amount of 0.01?cm. The supplementary structures from the peptides had been calculated based on the Compact disc spectra using CONTILL software program [8]. The computation results had been averaged based on two independent tests. Planning of erythrocyte suspension system The donors capillary bloodstream (100C150?l) was blended with a RPMI-1640 moderate containing heparin (10?u/ml). The cells had been precipitated by 1533426-72-0 centrifugation for 2?min in 300?g; the precipitate was re-suspended within a heparin-free moderate until your final erythrocyte focus of 2??10 7 ?CFU/ml was attained. All tests had been carried out.