title: A fluorescence study of the binding of cytochrome C to mixed-phospholipid microvesicles : evidence for a preferred orientation of the bound protein.
abstract: Kinetic and equilibrium experiments are reported on the binding of the fluorescent probe 1,8-anilino-naphtalene sulfonate (ANS) to microvesicles of natural lecithin containing 10 per cent of an anionic phospholipip (90 : 10 mixtures). Kinetics discriminated between fast binding to the outer leaflet of the bilayer and apparently slow binding to the inner leaflet controlled by the diffusion of the probe across the bilayer. The equilibrium distribution of ANS between the two leaflets was not dependent on the nature of the anionic species and the spectral properties of bound ANS were identical in all cases investigated. A hyperbolic saturation was observed allowing to propose an affinity scale for the binding of ANS to mixtures of lecithin with phosphatidic acid, phosphatidylinositol, and cardiolipin. The effects on binding of ionic strength and sodium dodecylsulfate were also considered. The binding of horse heart ferricytochrome c to ANS-labelled microvesicles was studied quantitatively making use of the quenching of the probes fluorescence by the heme. Perrin-Förster energy transfer could be analysed on the basis of a simple model of the physical arrangement of the system which was elaborated from published data referring to ANS and cytochrome c binding to phospholipids. Experimental and theoretical computed values of the quenching efficiency were compared and led to conclude in favor of a preferred orientation of the heme crevice fully accessible from the external space at the lipid interface.
mesh_terms: D000817:Anilino Naphthalenesulfonates; D003574:Cytochrome c Group; D007700:Kinetics; D008567:Membranes, Artificial; D008968:Molecular Conformation; D010743:Phospholipids; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D000817:Anilino Naphthalenesulfonates; D003574:Cytochrome c Group; D008567:Membranes, Artificial; D010743:Phospholipids