Shai Y, Hadari Y R, Finkels A
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
J Biol Chem. 1991 Nov 25;266(33):22346-54.
The interaction of pardaxin, a shark-repellent neurotoxin, and its charge-modified analogues with vesicles and human erythrocytes is described. The following six analogues and derivatives were synthesized by a solid phase method: [Glu8, Glu16]pardaxin, [N1-succinamido,Glu8,Glu16]pardaxin, [N1,Lys8,Lys16-triacetyl]pardaxin, des-[1----9]pardaxin (Shai, Y., Bach, D., and Yanovsky, A. (1990) J. Biol. Chem. 265, 20202-20209), and des-[1----9] [Glu16]pardaxin. The relative hydrophobic characteristics of the analogues were examined using reverse-phase high performance liquid chromatography. The pH-dependent spectroscopic and functional characteristics of the analogues were also investigated at either neutral or acidic pH. Spectroscopic characterization was achieved by measuring circular dichroism both before and after binding to vesicles, at either neutral or acidic pH. The ability of the peptides to dissipate a diffusion potential, to cause calcein release or the pH-dependent release of 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt/p-xylene-bis[pyridinium bromide] from sonicated unilamellar liposomes, as well as measurements of cytolytic activity on human erythrocytes, served to functionally characterize the peptides. We show a direct correlation between alpha-helical content, the analogues' hydrophobicity, and their pore-forming properties at the different pH values tested. We also demonstrate that the charge of the N terminus and of the peptide backbone, but not of the C terminus, affects the secondary structure as well as the activities of the analogues. Finally, we show that the cytolytic activity of pardaxin at neutral pH is not retained by any of the analogues.
本文描述了一种驱鲨神经毒素——豹蟾鱼毒素及其电荷修饰类似物与囊泡和人红细胞的相互作用。通过固相法合成了以下六种类似物和衍生物：[Glu8, Glu16]豹蟾鱼毒素、[N1-琥珀酰氨基, Glu8, Glu16]豹蟾鱼毒素、[N1, Lys8, Lys16-三乙酰基]豹蟾鱼毒素、去-[1----9]豹蟾鱼毒素（Shai, Y., Bach, D., and Yanovsky, A. (1990) J. Biol. Chem. 265, 20202 - 20209）以及去-[1----9] [Glu16]豹蟾鱼毒素。使用反相高效液相色谱法检测了这些类似物的相对疏水特性。还在中性或酸性pH条件下研究了类似物的pH依赖性光谱和功能特性。通过在中性或酸性pH条件下测量与囊泡结合前后的圆二色性来实现光谱表征。肽消散扩散电位的能力、导致钙黄绿素释放或从超声处理的单层脂质体中pH依赖性释放8-氨基萘-1,3,6-三磺酸钠盐/对二甲苯双[溴化吡啶]的能力，以及对人红细胞的细胞溶解活性测量，用于对肽进行功能表征。我们展示了在测试的不同pH值下，α-螺旋含量、类似物的疏水性与其成孔特性之间的直接相关性。我们还证明，N端和肽主链的电荷而非C端的电荷会影响类似物的二级结构以及活性。最后，我们表明在中性pH条件下，豹蟾鱼毒素的细胞溶解活性没有被任何一种类似物保留。
