The rate of change of surface pressure, pi, in a Langmuir trough following the deposition of surfactant suspensions on subphases containing serum, with or without polymers, is used to model a likely cause of surfactant inactivation in vivo: inhibition of surfactant adsorption due to competitive adsorption of surface active serum proteins. Aqueous suspensions of native porcine surfactant, organic extracts of native surfactant, and the clinical surfactants Curosurf, Infasurf, and Survanta spread on buffered subphases increase the surface pressure, pi, to approximately 40 mN/m within 2 min. The variation with concentration, temperature, and mode of spreading confirmed Brewster angle microscopy observations that subphase to surface adsorption of surfactant is the dominant form of surfactant transport to the interface. However (with the exception of native porcine surfactant), similar rapid increases in pi did not occur when surfactants were applied to subphases containing serum. Components of serum are surface active and adsorb reversibly to the interface increasing pi up to a concentration-dependent saturation value, pi(max). When surfactants were applied to subphases containing serum, the increase in pi was significantly slowed or eliminated. Therefore, serum at the interface presents a barrier to surfactant adsorption. Addition of either hyaluronan (normally found in alveolar fluid) or polyethylene glycol to subphases containing serum reversed inhibition by restoring the rate of surfactant adsorption to that of the clean interface, thereby allowing surfactant to overcome the serum-induced barrier to adsorption.

A theory based on the Smolukowski analysis of colloid stability shows that the presence of charged, surface-active serum proteins at the alveolar air-liquid interface can severely reduce or eliminate the adsorption of lung surfactant from the subphase to the interface, consistent with the observations reported in the companion article (pages 1769-1779). Adding nonadsorbing, hydrophilic polymers to the subphase provides a depletion attraction between the surfactant aggregates and the interface, which can overcome the steric and electrostatic resistance to adsorption induced by serum. The depletion force increases with polymer concentration as well as with polymer molecular weight. Increasing the surfactant concentration has a much smaller effect than adding polymer, as is observed. Natural hydrophilic polymers, like the SP-A present in native surfactant, or hyaluronan, normally present in the alveolar fluids, can enhance adsorption in the presence of serum to eliminate inactivation.

Although the effects of surfactant protein B (SP-B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure-activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N-terminal SP-B peptides in lipids and structure-promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP-B1_25 (residues 1-25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP-B,-25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP-B,_,, sequence. Residues Cys-8-Gly-25 are largely a-helix in methanol, whereas the N-terminal segment Phe-1-Cys-8 had turn and helical propensities. Addition of SP-Bl-25 spin-labeled at the N-terminal Phe (i.e., SP-BT_25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP-B1-25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP-BI-25 argues that the reporter on Phe-1 resides in the headgroup of the lipid dispersions. The blue-shift in the endogenous fluorescence of Trp-9 near the N-terminus of SP-B1-25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP-BI-2S interacting with lipids.

The mechanisms by which pulmonary surfactant protein B (SP-B) affects the surface activity of surfactant lipids are unclear. We have studied the peptide/lipid interactions of the amino-terminal amphipathic domain of SP-B by comparing the secondary conformations and surface activities of a family of synthetic peptides based on the native human SP-B sequence, modified by site-specific amino acid substitutions. Circular dichroism measurements show an ahelical structure correlating with the ability of the peptides to interact with lipids and with the surface activity of peptide/lipid dispersions. Amino acid substitutions altering either the charge or the hydrophobicity of the residues lowered the helical content and reduced the association of the aminoterminal segment with lipid dispersions. Surface activity of peptide/lipid mixtures was maximally altered by reversal of charge in synthetic peptides. These observations indicate that electrostatic interactions and hydrophobicity are important factors in determining optimal structure and function of surfactant peptides in lipid dispersions.Surfactant protein B (SP-B, Mr 9000; refs. 1 and 2) is encoded on chromosome 2 and synthesized by type 2 pneumocytes (3-6). The primary translation product for SP-B is a 381-residue glycosylated preprotein (Mr 42,000) (7). Amino-and carboxyl-terminal proteolytic cleavage produces a 79-residue peptide, with amino terminus located at residue 201 of the preprotein. Mature SP-B lacks glycosylation sites, is highly hydrophobic, and has a basic pl. In contrast to surfactant protein C (SP-C), SP-B has no covalently linked lipids, although it coisolates with SP-C and surfactant lipids (8). Four of 10 positively charged residues (two arginines, two lysines) appear at the amino terminus. Negatively and positively charged residues are found at the carboxyl terminus (one aspartate, one glutamate, four arginines). Intra-and intermolecular sulfhydryl bonds are important structural features of the mature protein, which contains seven cysteines and is oligomeric under oxidizing conditions (9, 10).Together with surfactant protein A (SP-A) and SP-C, SP-B interacts with lipids to form mammalian pulmonary surfactant. SP-B is present in lamellar bodies in type II pneumocytes and appears in bronchoalveolar lavage. SP-B from bovine and porcine sources contributes to the function of surfactant preparations used in replacement therapies (11)(12)(13)(14)(15). Synthetic SP-B (16) and synthetic peptides from predicted amphipathic sequences of human SP-B (17) emulate native SP-B in surfactant lipid dispersions (13,(16)(17)(18)(19).Quantitative measurements of the amphipathicity of the human amino acid sequence of SP-B using the Eisenberg hydrophobic algorithm (20) predict that the amino-terminal sequence has a high a hydrophobic moment (9, 21). Therefore, we investigated the amino-terminal sequence of SP-B to identify secondary structures and specific conformations associated with surface activity in peptide/lipid dispersions.Based on the native amino-te...

Substances (for example, serum proteins or meconium) that interfere with the activity of pulmonary surfactant in vitro may also be important in the pathogenesis or progression of acute lung injury. Addition of polymers such as dextran or polyethylene glycol (PEG) to surfactants prevents and reverses surfactant inactivation. The purpose of this study was to find out whether surfactant/polymer mixtures are more effective for treating one form of acute lung injury than is surfactant alone. Acute lung injury in adult rats was created by tracheal instillation of human meconium. Injured animals, which were anesthetized, paralyzed, and ventilated with 100% oxygen and not treated with surfactant mixtures, remained hypoxic and required high ventilator pressures to maintain Pa(CO(2)) in the normal range over the 3 h of the experiment. Uninjured animals maintained normal values for oxygen and compliance of the respiratory system. The greatest improvement in both oxygenation (178%) and compliance (42%) occurred in animals with lung injury that were treated with Survanta and PEG (versus untreated control animals; p < 0.01), whereas little improvement was found after treatment with Survanta alone. Similar results were found when postmortem pulmonary pressure-volume curves and histology were examined. We conclude that adding PEG to Survanta improves gas exchange, pulmonary mechanics, and histologic appearance of the lungs in a rat model of acute lung injury caused by meconium.