We have shown previously that lateral compression of pulmonary surfactant monolayers initially induces separation of two phases but that these remix when the films become more dense (1). In the studies reported here, we used fluorescence microscopy to examine the role of the different surfactant constituents in the remixing of the separated phases. Subfractions containing only the purified phospholipids (PPL), the surfactant proteins and phospholipids (SP and PL), and the neutral and phospholipids (N and PL) were obtained by chromatographic separation of the components in extracted calf surfactant (calf lung surfactant extract, CLSE). Compression of the different monolayers produced nonfluorescent domains that emerged for temperatures between 20 and 41 °C at similar surface pressures 6-8 mN/m higher than values observed for dipalmitoyl phosphatidylcholine (DPPC), the most prevalent component of pulmonary surfactant. Comparison of the different preparations showed that the neutral lipid increased the total nonfluorescent area at surface pressures up to 25 mN/m but dispersed that total area among a larger number of smaller domains. The surfactant proteins also produced smaller domains, but they had the opposite effect of decreasing the total nonfluorescent area. Only the neutral lipids caused remixing. In images from static monolayers, the domains for N and PL dropped from a maximum of 26 ± 3% of the interface at 25 mN/m to 4 ± 2% at 30 mN/m, similar to the previously reported behavior for CLSE. During continuous compression through a narrow range of pressure and molecular area, in N and PL, CLSE, and mixtures of PPL with 10% cholesterol, domains became highly distorted immediately prior to remixing. The characteristic transition in shape and abrupt termination of phase coexistence indicate that the remixing caused by the neutral lipids occurs at or close to a critical point.
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