Liu X, Yang Q, Nakamura C, Miyake J
National Institute for Advanced Interdisciplinary Research, Agency of Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
J Chromatogr B Biomed Sci Appl. 2001 Jan 5;750(1):51-60. doi: 10.1016/s0378-4347(00)00427-8.
Unilamellar liposomes with entrapped fluorescent dye calcein were stably immobilized in gel beads by avidin-biotin-binding. The immobilized liposomes remained extremely stable upon storage and chromatographic runs. The immobilized calcein-entrapped liposomes were utilized for fluorescent analysis of solute-membrane interactions, which in some cases are too weak to be detected by chromatographic retardation. A liposome column was used as a sensitive probe to detect the interactions of membranes with pharmaceutical drugs, peptides and proteins. Retardation of the solutes was monitored using a UV detector. Perturbation of the membranes, reflected as leakage of the entrapped calcein by some of the solutes, can thus be detected on-line using a flow-fluorescent detector. For the amphiphilic drugs or synthetic peptides, perturbation of membranes became more pronounced when the retardation (hydrophobicity) of the molecules increased. On the other hand, in the case of positively-charged peptides, polylysine, or partially denatured bovine carbonic anhydrase, significant dye leakage from the liposomes was observed although the retardation was hardly to be measured. Weak protein-membrane interactions can thus be assumed from the large leakage of calcein from the liposomes. This provides additional useful information for solute-membrane interactions, as perturbation of the membranes was also indicated by avidin-biotin-immobilized liposome chromatography (ILC).
通过抗生物素蛋白-生物素结合，将包裹有荧光染料钙黄绿素的单层脂质体稳定地固定在凝胶珠中。固定化脂质体在储存和色谱运行过程中保持极其稳定。固定化的包裹钙黄绿素的脂质体用于溶质-膜相互作用的荧光分析，在某些情况下，这种相互作用太弱，无法通过色谱阻滞来检测。脂质体柱用作灵敏探针，以检测膜与药物、肽和蛋白质的相互作用。使用紫外检测器监测溶质的阻滞情况。一些溶质导致包裹的钙黄绿素泄漏，反映出膜的扰动，因此可以使用流动荧光检测器在线检测。对于两亲性药物或合成肽，当分子的阻滞（疏水性）增加时，膜的扰动变得更加明显。另一方面，对于带正电荷的肽、聚赖氨酸或部分变性的牛碳酸酐酶，尽管几乎无法测量阻滞情况，但观察到脂质体中有明显的染料泄漏。因此，可以从脂质体中大量钙黄绿素泄漏推测出蛋白质-膜之间存在微弱的相互作用。这为溶质-膜相互作用提供了额外的有用信息，因为抗生物素蛋白-生物素固定化脂质体色谱法（ILC）也表明了膜的扰动。
