Surfactant protein B: effects on lipid domain formation and intermembrane lipid flow

Pulmonary surfactant is a mixture of (phospho)lipids and surfactant specific proteins, lining the alveolar space. During each respiration cycle phospholipids are transferred between the phospholipid monolayer at the air/water interface and a variety of underlying membranes. Surfactant proteins may play a role in facilitating the insertion and removal of phospholipids by affecting the lipid organization of the bilayer and monolayer. The experiments described in this article were carried out in order to investigate the influence of surfactant protein B (SP-B) on the distribution of phospholipids in membranes and on the mixing of lipids between membranes. To determine the distribution of the non-labeled phospholipids in small unilamellar vesicles (SUV), the relative clustering of pyrene-labeled phospholipids was used, by measuring the ratio of excimer-to-monomer (E/M) pyrene fluorescence. In the absence of SP-B it was found that the clustering of the pyrenePC molecules was dependent on the proportion of saturated acyl chains and not on the proportion of negative charges. Addition of the positively charged SP-B to a mixture of DPPC and PG, led to an increase of approximately 20% in E/M ratio, indicating a clustering of the negatively charged PG molecules. This effect was intensified by addition of calcium ions. If pyrenePC-containing SUV were mixed with excess non-labeled SUV in the presence of SP-B and calcium ions, the E/M ratio decreased, corresponding with a flow of the pyrenePC molecules into the acceptor membranes. It is concluded that presence of domains of phospholipids can be detected with the use of pyrene-labeled phospholipids. Furthermore, SP-B showed a concentrating effect on the distribution of the negatively charged phospholipids, a process that could be important in regulating the phospholipid composition of the monolayer.