Ringstad Lovisa, Protopapa Elisabeth, Lindholm-Sethson Britta, Schmidtchen Artur, Nelson Andrew, Malmsten Martin
Department of Pharmacy, Uppsala University, Uppsala, Sweden.
Langmuir. 2008 Jan 1;24(1):208-16. doi: 10.1021/la702538k. Epub 2007 Dec 5.
Electrochemical methods employing the hanging mercury drop electrode were used to study the interaction between variants of the complement-derived antimicrobial peptide CNY21 (CNYITELRRQH ARASHLGLAR) and dioleoyl phosphatidylcholine (DOPC) monolayers. Capacitance potential and impedance measurements showed that the CNY21 analogues investigated interact with DOPC monolayers coating the mercury drop. Increasing the peptide hydrophobicity by substituting the two histidine residues with leucine resulted in a deeper peptide penetration into the hydrophobic region of the DOPC monolayer, indicated by an increase in the dielectric constant of the lipid monolayer (Deltaepsilon = 2.0 after 15 min interaction). Increasing the peptide net charge from +3 to +5 by replacing the histidines by lysines, on the other hand, arrests the peptide in the lipid head group region. Reduction of electroactive ions (Tl+, Pb2+, Cd2+, and Eu3+) at the monolayer-coated electrode was employed to further characterize the types of defects induced by the peptides. All peptides studied permeabilize the monolayer to Tl+ to an appreciable extent, but this effect is more pronounced for the more hydrophobic peptide (CNY21L), which also allows penetration of larger ions and ions of higher valency. The results for the various ions indicate that charge repulsion rather than ion size is the determining factor for cation penetration through peptide-induced defects in the DOPC monolayer. The effects obtained for monolayers were compared to results obtained with bilayers from liposome leakage and circular dichroism studies for unilamellar DOPC vesicles, and in situ ellipsometry for supported DOPC bilayers. Trends in peptide-induced liposome leakage were similar to peptide effects on electrochemical impedance and permeability of electroactive ions for the monolayer system, demonstrating that formation of transmembrane pores alone does not constitute the mechanism of action for the peptides investigated. Instead, our results point to the importance of local packing defects in the lipid membrane in close proximity to the adsorbed peptide molecules.
采用悬汞滴电极的电化学方法研究了补体衍生抗菌肽CNY21（CNYITELRRQH ARASHLGLAR）变体与二油酰磷脂酰胆碱（DOPC）单层膜之间的相互作用。电容电位和阻抗测量表明，所研究的CNY21类似物与覆盖汞滴的DOPC单层膜相互作用。用亮氨酸取代两个组氨酸残基增加了肽的疏水性，导致肽更深入地渗透到DOPC单层膜的疏水区域，脂质单层膜的介电常数增加表明了这一点（相互作用15分钟后Δε = 2.0）。另一方面，用赖氨酸取代组氨酸将肽的净电荷从 +3增加到 +5，使肽滞留在脂质头部基团区域。利用单层膜覆盖电极上电活性离子（Tl +、Pb2 +、Cd2 +和Eu3 +）的还原进一步表征肽诱导的缺陷类型。所有研究的肽都能使单层膜对Tl +有相当程度的通透，但这种效应在疏水性更强的肽（CNY21L）中更明显，它还能使更大的离子和更高价态的离子渗透。各种离子的结果表明，电荷排斥而非离子大小是阳离子通过肽诱导的DOPC单层膜缺陷渗透的决定因素。将单层膜的结果与脂质体泄漏和单层DOPC囊泡的圆二色性研究以及支撑DOPC双层膜的原位椭偏测量的双层膜结果进行了比较。肽诱导的脂质体泄漏趋势与肽对单层膜系统的电化学阻抗和电活性离子渗透性的影响相似，表明仅形成跨膜孔并不构成所研究肽的作用机制。相反，我们的结果表明脂质膜中紧邻吸附肽分子的局部堆积缺陷很重要。
