正电性多肽copoly(Lys/Tyr)与磷脂膜的相互作用研究

Interaction of cationic copoly (Lys/Tyr) with phospholipid membranes

目的 研究正电性多肽copoly(Lys/Tyr) (CPLT)在模拟生物膜上的透过性。方法 ①配制由卵磷脂(EPC)、2-油酰基磷脂酰乙醇胺(DOPE)和大豆磷脂(SBPL)组成的二分子磷脂膜。②Zeta电位测定法(zetapotential method,ZP)测定由上述磷脂组成的脂质体在加入CPLT后的膜表面Zeta电位。③园二色谱法(circulardichroism spectroscopy,CD)检测CPLT分子与磷脂膜作用时的构象情况。④电生理学方法(electrophysioloy tech—nique,ET)测量CPLT分子在磷脂膜上引起的跨膜电流。⑤荧光光度分析法(fluorescence spectroscop,FS)检测CPLT分子与磷脂膜的作用过程中的荧光强度变化。⑥共焦点激光扫描显微分析法(confocal laser scanning mi—croscopy,CLSM)研究CPLT在磷脂膜上透过及其相对透过效率。结果 ①随着CPLT的加入和其浓度的增加,磷脂膜Zeta电位逐渐增加并趋向饱和。②CPLT分子在水相取β—sheet构象,当与磷脂膜结合后,其β-sheet构象的波峰发生红移,但构象基本不变。③CPLT分子能在一定浓度和一定外加电压条件下,透过二分子磷脂膜引起膜电流。④CPLT与磷脂膜的作用可分为三步:第一步,CPLT分子吸附于磷脂膜上;第二步,CPLT分子通过磷脂膜的疏水区;第三步,CPLT分子从二分子膜的内膜解吸,进入膜内水相。⑤CPLT分子在磷脂膜上?

OBJECTIVE To investigate the transference of copoly (Lys/Tyr) across lipid membranes and estimate the relative transfer efficiency. METHODS The interaction of positively charged copoly (Lys/Tyr) with lipid membranes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), as well as soybean phospholipid (SBPL) was examined using zeta potential method (ZP), circular dichroism spectroscopy (CD), electrophysiology technique (ET), fluorescence spectroscopy (FS) and confocal laser scanning microscopy (CLSM). 4-Fluoro-7-nitrobenzofu-razan ( NBD - F) labeled copoly (Lys/Tyr) was used in CLSM experiment to follow the process of peptide-membrane interaction. RESULTS (1)Copoly (Lys/Tyr) was associated with lipid membranes and gradually saturated on the membranes either electrostatically or hydrophobically or both. (2)β-sheet conformation of copoly (Lys/Tyr) was red-shifted upon binding to SPBL and EPC/DOPE liposome membrane but with almost no change in conformation type. (3)Positively charged copoly (Lys/Tyr) could cause transmembrane currents under an appropriate polypeptide concentration and applied voltage, suggesting its transferrence across lipid membranes. (4)The transference of polypeptide across lipid membranes could be separated into three steps, adsorption, transportation and desorption, which was afterward reaffirmed by confocal laser scanning microscopy. CONCLUSIONS The transference of copoly (Lys/Tyr) across SBPL, EPC/DOPE lipid bilayer membranes was proved. The transfer efficiency of copoly (Lys/Tyr) across lipid membranes largely depended on the composition of the lipid membrane. Addition of anionic lipid in membrane composition would have a negative effect on the transfer efficiency. In addition, electrostatic attraction was the main factor for the initial state of the peptide-membrane interaction when polypeptide is ptxsitively charged while lipid membrane is negatively charged.