Retzinger G S, Meredith S C, Lau S H, Kaiser E T, Kézdy F J
Anal Biochem. 1985 Oct;150(1):131-40. doi: 10.1016/0003-2697(85)90451-8.
We have developed a rapid method for probing the affinity of peptides toward an amphiphilic surface. Hydrophobic polystyrene-divinylbenzene beads of 5.7 +/- 1.5 micron diameter are coated with a monomolecular film of egg lecithin to achieve the equilibrium spreading density of the phospholipid, 6 X 10(-3) molecule/A2. The coated beads are ideally suited for assessing the affinity of peptides for phospholipid surfaces: Large quantities of lipid-coated beads of known surface area can be prepared easily and rapidly. Within the pH range 2.0 to 9.0, the adsorbed phospholipids are relatively resistant to hydrolysis and remain bound indefinitely. Following incubation with peptide ligands, beads can be separated from the reaction mixture by centrifugation. Peptides, such as melittin, which destroy or cause fusion of single bilayer phospholipid vesicles, cannot disrupt lecithin-coated beads in a comparable way, and do not displace lecithin from the surface of beads. After incubating these beads in solutions of peptides and proteins, we have determined the parameters for the binding of several ligands to the phospholipid surface. The binding of many amphiphilic peptides obeys a Langmuir adsorption isotherm, i.e., saturable reversible binding to independent and equivalent sites on the bead. That the binding is a true reversible equilibrium is shown by desorption of the ligand upon dilution. From the isotherm, the surface areas occupied by the ligand molecules were calculated, and were observed to be similar to those observed in monolayers at the air-water interface. In comparing the binding of amphiphilic peptides to that of completely hydrophilic peptides, we observed that only the former bind at levels measurable by our techniques. Thus, this method can serve as a rapid assay for detecting amphiphilicity in peptides of putative amphiphilic character.
我们开发了一种快速方法来探测肽与两亲性表面的亲和力。将直径为5.7±1.5微米的疏水性聚苯乙烯 - 二乙烯基苯珠粒用卵磷脂单分子膜包被，以达到磷脂的平衡铺展密度，即6×10⁻³分子/Ų。包被的珠粒非常适合评估肽对磷脂表面的亲和力：可以轻松快速地制备大量已知表面积的脂质包被珠粒。在pH值2.0至9.0范围内，吸附的磷脂相对抗水解，并且能无限期保持结合状态。与肽配体孵育后，可通过离心将珠粒与反应混合物分离。诸如蜂毒肽之类的肽，能破坏或导致单层磷脂囊泡融合，但无法以类似方式破坏卵磷脂包被的珠粒，也不会从珠粒表面取代卵磷脂。在将这些珠粒在肽和蛋白质溶液中孵育后，我们确定了几种配体与磷脂表面结合的参数。许多两亲性肽的结合遵循朗缪尔吸附等温线，即在珠粒上独立且等效的位点上进行可饱和的可逆结合。稀释时配体的解吸表明这种结合是真正的可逆平衡。根据等温线计算出配体分子占据的表面积，观察到其与在气 - 水界面单分子层中观察到的相似。在比较两亲性肽与完全亲水性肽的结合时，我们发现只有前者能以我们的技术可测量的水平结合。因此，该方法可作为一种快速检测方法，用于检测具有假定两亲性特征的肽的两亲性。
