Sargent D F, Bean J W, Schwyzer R
Department of Molecular Biology and Biophysics, Swiss Federal Institute of Technology, Zürich.
Biophys Chem. 1989 Oct;34(2):103-14. doi: 10.1016/0301-4622(89)80048-1.
Interaction of substance P with electrically neutral, planar lipid bilayers prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and with anionic bilayers prepared from mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and brain phosphatidylserine was measured using the capacitance minimization method for monitoring the membrane surface potential caused by the positive charges and electric dipole moment of adsorbed peptide. Substance P bound to the electrically neutral bilayers from 9 mM KCl (buffered to pH 5.5 with 2.0 mM 2-(N-morpholino)ethanesulfonate) with a maximal binding density of about 1 x 10(-2) molecules per nm2 and a dissociation constant of about 2 x 10(-4) M. Measurement of the surface potential at different ionic strengths (shielding of surface charges) allowed distinction between the fixed-charge surface potential and a dipole potential. Ascribing this dipole potential to membrane-bound substance P would imply an effective dipole moment normal to the bilayer surface of about 20 Debye per molecule. Magnitude and polarity are consistent with an alpha-helical domain at the C-terminal end of substance P which is oriented normal to the surface of the membrane, and inserted so as to be inaccessible to the aqueous phase. Consistent measurements were obtained with anionic membranes at low substance P concentrations (10(-7)-10(-6) M; pH 7.2). They indicated electrostatic accumulation of the triply charged peptide on the surface of the membrane followed by hydrophobic interaction with the same parameters as for neutral membranes. The results agree with the membrane structure of substance P determined with infrared attenuated total reflection spectroscopy, circular dichroism measurements, and thermodynamic estimations.
使用电容最小化方法测量了P物质与由1-棕榈酰-2-油酰基-sn-甘油-3-磷酸胆碱制备的电中性平面脂质双层以及由1,2-二油酰基-sn-甘油-3-磷酸胆碱和脑磷脂酰丝氨酸混合物制备的阴离子双层之间的相互作用，以监测由吸附肽的正电荷和电偶极矩引起的膜表面电位。P物质从9 mM KCl（用2.0 mM 2-（N-吗啉代）乙磺酸盐缓冲至pH 5.5）中与电中性双层结合，最大结合密度约为每nm2 1×10(-2)个分子，解离常数约为2×10(-4) M。在不同离子强度下测量表面电位（表面电荷的屏蔽）允许区分固定电荷表面电位和偶极电位。将该偶极电位归因于膜结合的P物质意味着每个分子垂直于双层表面的有效偶极矩约为20德拜。大小和极性与P物质C末端的α-螺旋结构域一致，该结构域垂直于膜表面定向，并插入使得水相无法接触。在低P物质浓度（10(-7)-10(-6) M；pH 7.2）下用阴离子膜获得了一致的测量结果。它们表明三电荷肽在膜表面的静电积累，随后与中性膜具有相同参数的疏水相互作用。结果与用红外衰减全反射光谱、圆二色性测量和热力学估计确定的P物质的膜结构一致。
