Understanding the role of lung surfactant specific proteins in lipid monolayers is essential for improved treatments for Respiratory Distress Syndrome, which is a leading cause of death in premature infants. Fluorescence (FM) and Atomic Force (AFM) microscopies reveal that the amino-terminal peptide of lung surfactant protein SP-B alters the behavior of palmitic acid (PA) monolayers, enhancing their in vivo performance. The combination of these techniques provides an excellent correlation between the protein-lipid interactions on the molecular level with the macroscopic properties of the monolayer.SP-B protein incorporates into monolayers of PA, an important component of natural and synthetic lung surfactants monolayers. The effect of the protein on the monolayer is evidenced in the isotherm data shown in Fig 1, in which the area per PA molecule, compressibility, and surface pressure at collapse all increase as a function of increasing protein concentration. The protein accomplishes this by inhibiting the formation of ordered phases of PA. This is seen via FM as a transition from a homogeneous, dark ordered phase without protein (Fig 2a) to a network of a disordered, bright phase (the fluorescent lipid probes used in this study prefer to partition into disordered phases, making them appear as bright regions in FM images) that separates ordered phase domains at coexistence (Fig. 2b). The network is stabilized by the low line tension between the bright phase and other lipid phases as confirmed by the formation of extended linear domains of bright phase in a dark background, or “stripe” phases (Fig. 3a) under certain subphase conditions. Similar stripe phases also occur in single component fluorescein-labeled SP-B monolayers (Fig 3b), implying that the protein is responsible for the reduction in line tension. The formation of the fluid phase network is responsible for the increased collapse resistance of these mixed monolayers. The mechanism of collapse shifts from a heterogeneous process of nucleation and growth of large rigid crystalline collapse phases (Fig. 4a) to a more homogeneous process with nucleation and growth of smaller domains distributed uniformly across the film (Fig. 4b). This is due to the protein-induced network breaking up and isolating the domains of ordered phase, effectively lowering the probability of finding a heterogeneous nucleation site within each domain (analagous to the classic experiments of Turnbull on supercooled microemulsions of metallic liquids). The partitioning of the protein into the bright phase network was confirmed through the use of a dual-probe system. Fluorescein-labeled SP-B was added to a PA monolayer incorporating a fluorescent lipid analogue that emits at a higher wavelength. Upon imaging the same region of a monolayer at the different wavelengths (Fig. 5a and 5b), the protein is seen to be located in the bright phase network regions as expected.
Fluorescence, polarized fluorescence, and Brewster angle microscopy of palmitic acid and lung surfactant protein B monolayers