The collapse of monolayers containing pulmonary surfactant phospholipids is kinetically determined

Prior studies have shown that during and after slow compressions of monomolecular films containing the complete set of purified phospholipids (PPL) from calf surfactant at an air/water interface, surface pressures (pi) reach and sustain values that are remarkably high relative to expectations from simple systems with model lipids. Microscopy shows that the liquid-expanded, tilted-condensed, and collapsed phases are present together in the PPL films between 45 and 65 mN/m. The Gibbs phase rule restricts equilibrium coexistence of three phases to a single pi for films with two components but not for more constituents. We therefore determined if the surprising stability of PPL reflects release from the thermodynamic restrictions of simple model systems by the presence of multiple components. Experiments with binary films containing dioleoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine first tested the predictions of the phase rule. The onset of three-phase coexistence, determined by fluorescence microscopy, and its termination, established by relaxation of collapsing films on a captive bubble, occurred at similar pi. Experiments for PPL using the same methods suggested that the three phases might coexist over a range of pi, but limited to approximately 2 mN/m, and extending below rather than above the coexistence pi for the binary films. Our results show that the PPL films at high pi must deviate from equilibrium and that they must then be metastable.     

Surface respreading after collapse of monolayers containing major lipids of pulmonary surfactant

Isotherms have been obtained near 37 degrees C for a series of repetitive compressions and expansions of monolayers that contain major components of lung surfactant. The minimum surface tension or maximum surface pressure which could be achieved under conditions of dynamic compression, and the rate of return of lipid from excluded phase to the monolayers were measured. Monolayers of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or of DPPC plus 10 or 30 mol% of the calcium salt of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG) (POPG-Ca) achieved very high surface pressures or low surface tensions (near 0 mN m-1), but they showed no return of material from the collapse phases under the test conditions. Monolayers of POPG-Ca alone collapsed at relatively low surface pressures (high surface tensions), but showed good return of material from the collapse phase into the monolayer. Monolayers containing more complex mixtures of lipids (DPPC, phosphatidylglycerol (PG), unsaturated phosphatidylcholine (PC), cholesterol (chol] in ratios similar to those found in surfactant achieved minimum surface tensions intermediate between those of monolayers with less complex compositions. These more complex mixtures showed a better rate of return of lipids from the collapse phases to the monolayer than did simple DPPC-POPG mixtures. 31P-NMR and differential scanning calorimetric investigations of the mixture DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POP G/DPPG/chol (10:4:2:1:3) showed that in the bulk phase at 37 degrees C, it was in bilayers in the liquid-crystalline state.     

The melting of pulmonary surfactant monolayers.

Monomolecular films of phospholipids in the liquid-expanded (LE) phase after supercompression to high surface pressures (pi), well above the equilibrium surface pressure (pi(e)) at which fluid films collapse from the interface to form a three-dimensional bulk phase, and in the tilted-condensed (TC) phase both replicate the resistance to collapse that is characteristic of alveolar films in the lungs. To provide the basis for determining which film is present in the alveolus, we measured the melting characteristics of monolayers containing TC dipalmitoyl phosphatidylcholine (DPPC), as well as supercompressed 1-palmitoyl-2-oleoyl phosphatidylcholine and calf lung surfactant extract (CLSE). Films generated by appropriate manipulations on a captive bubble were heated from < or =27 degrees C to > or =60 degrees C at different constant pi above pi(e). DPPC showed the abrupt expansion expected for the TC-LE phase transition, followed by the contraction produced by collapse. Supercompressed CLSE showed no evidence of the TC-LE expansion, arguing that supercompression did not simply convert the mixed lipid film to TC DPPC. For both DPPC and CLSE, the melting point, taken as the temperature at which collapse began, increased at higher pi, in contrast to 1-palmitoyl-2-oleoyl phosphatidylcholine, for which higher pi produced collapse at lower temperatures. For pi between 50 and 65 mN/m, DPPC melted at 48-55 degrees C, well above the main transition for bilayers at 41 degrees C. At each pi, CLSE melted at temperatures >10 degrees C lower. The distinct melting points for TC DPPC and supercompressed CLSE provide the basis by which the nature of the alveolar film might be determined from the temperature-dependence of pulmonary mechanics.     

Rapid compression transforms interfacial monolayers of pulmonary surfactant

Films of pulmonary surfactant in the lung are metastable at surface pressures well above the equilibrium spreading pressure of 45 mN/m but commonly collapse at that pressure when compressed in vitro. The studies reported here determined the effect of compression rate on the ability of monolayers containing extracted calf surfactant at 37 degrees C to maintain very high surface pressures on the continuous interface of a captive bubble. Increasing the rate from 2 A(2)/phospholipid/min (i.e., 3% of (initial area at 40 mN/m)/min) to 23%/s produced only transient increases to 48 mN/m. Above a threshold rate of 32%/s, however, surface pressures reached > 68 mN/m. After the rapid compression, static films maintained surface pressures within +/- 1 mN/m both at these maximum values and at lower pressures following expansion at < 5%/min to > or = 45 mN/m. Experiments with dimyristoyl phosphatidylcholine at 37 degrees C produced similar results. These findings indicate that compression at rates comparable to values in the lungs can transform at least some phospholipid monolayers from a form that collapses readily at the equilibrium spreading pressure to one that is metastable for prolonged periods at higher pressures. Our results also suggest that transformation of surfactant films can occur without refinement of their composition.     

Influence of liquid-layer thickness on pulmonary surfactant spreading and collapse

Pulmonary surfactant spreads on the thin ( approximately 0.1 microm) liquid layer that lines the alveoli, forming a film that reduces surface tension and allows normal respiration. Pulmonary surfactant deposited in vitro on liquid layers that are several orders of magnitude thicker, however, does not reach the low surface tensions ( approximately 0.001 N/m) achieved in the lungs during exhalation when the surfactant film compresses. This is due to collapse, a surface phase transition during which the surfactant film, rather than decreasing surface tension by increasing its surface density, becomes thicker at constant surface tension ( approximately 0.024 N/m). Formation of the collapse phase requires transport of surfactant to collapse sites, and this transport can be hindered in thinner liquid layers by viscous resistance to motion. Our objective is to determine the effect of the liquid-layer thickness on surfactant transport, which might affect surfactant collapse. To this end, we developed a mathematical model that accounts for the effect of the liquid-layer thickness on surfactant transport, and focused on surfactant spreading and collapse. Model simulations showed a marked decrease in collapse rates for thinner liquid layers, but this decrease was not enough to completely explain differences in surfactant film behavior between in vitro and in situ experiments.     

Lipid compositional analysis of pulmonary surfactant monolayers and monolayer-associated reservoirs

Pulmonary surfactant is a lipid:protein complex containing dipalmitoyl-phosphatidylcholine (DPPC) as the major component. Recent studies indicate adsorbed surfactant films consist of a surface monolayer and a monolayer-associated reservoir. It has been hypothesized that the monolayer and its functionally contiguous reservoir may be enriched in DPPC relative to bulk phase surfactant. We investigated the compositional relationship between the monolayer and its reservoir using paper-supported wet bridges to transfer films from adsorbing dishes to clean surfaces on spreading dishes. Spreading films appear to form monolayers in the spreading dishes. We employed bovine lipid extract surfactant [BLES(chol)] containing [3H]DPPC and either [14C]palmitoyl, oleoyl-phosphatidylcholine (POPC), [14C]dipalmitoyl-phosphatidylglycerol (DPPG), [14C]palmitoyl, oleoyl-phosphatidylglycerol (POPG), or [14C]cholesterol. Radiolabeled phosphatidylglycerols were prepared using phospholipase D. The studies demonstrated that the [3H]DPPC-[14C] POPC ratios were the same in the prepared BLES dispersions as in Langmuir-Blodgett films, indicating a lack of DPPC selectivity during film formation. Furthermore, identical 3H-14C isotopic ratios were observed with DPPC and either 14C-labeled POPC, DPPG, POPG, or cholesterol in the original dispersions, the bulk phases in adsorption dish D1, and monolayers recovered from spreading dish D2. These relationships remained unperturbed with 2-fold increases in bulk concentrations in D1 and 10-fold variations in D1-D2 surface area. These results indicate adsorbed surfactant monolayers and their associated reservoirs possess similar lipid compositions and argue against selective adsorption of DPPC.     

Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 microg/ml SP-A and 0, 1.64, or 5 mM CaCl(2). In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca(2+). Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures (pi approximately 5 mN/m) and the domains grew in size with increasing pi. Above 25 mN/m, the domain size decreased with increasing pi. The amount of observable dark phase was maximal at 18% of the total film area at pi approximately 25 mN/m, then decreased to approximately 3% at pi approximately 40 mN/m. The addition of 0.16 microg/ml SP-A with 0 or 1.64 mM Ca(2+) in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to approximately 25% between pi of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca(2+) and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca(2+). PSLE films were spread on a subphase containing 0.16 microg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca(2+), TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca(2+) resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.     

Structural models and surface equation of state for pulmonary surfactant monolayers

A simple surface equation of state is proposed to describe pi-A isotherms of pulmonary surfactant monolayers. The monolayer is considered as undergoing three characteristic states during the compression: the disordered liquid-expanded (LE) state, the ordered liquid-condensed (LC) state and the collapse state. Structural models of pure protein (SP-B and SP-C) monolayer are proposed to interpret the behavior characteristics of monolayer in the states. The area, ALC, is defined as an instantaneous LC-state area when the monolayer is under the complete LC state. The area, At, is defined as a transition area from the ordered LC state to the collapse state. And the collapse pressure, pi(max), is defined as the maximum surface pressure that the monolayer can bear before collapse. The ideal equation of state is revised by ALC, At and pi(max), and a new equation of state is obtained, which is applicable for pure components of pulmonary surfactant. The theoretical pi-A isotherms described by the equation of state are compared with the experimental ones for SP-B, SP-C, DPPC and DPPG, and good agreements are obtained. The equation of state is generalized to protein-lipid binary mixtures by introducing mixing rules. The predicted pi-A isotherms agree with the experimental ones for various pulmonary surfactant components and the average deviation is about 9.2%.     

Fluorescently labeled pulmonary surfactant protein C in spread phospholipid monolayers.

Pulmonary surfactant, a lipid-protein complex, secreted into the fluid lining of lungs prevents alveolar collapse at low lung volumes. Pulmonary surfactant protein C (SP-C), an acylated, hydrophobic, alpha-helical peptide, enhances the surface activity of pulmonary surfactant lipids. Fluorescein-labeled SP-C (F-SP-C) (3, 6, 12 wt%) in dipalmitoylphosphatidylcholine (DPPC), and DPPC:dipalmitoylphosphatidylglycerol (DPPG) [DPPC:DPPG 7:3 mol/mol] in spread monolayers was studied by epifluorescence microscopy. Mass spectometry of F-SP-C indicated that the protein is partially deacylated and labeled with 1 mol fluorescein/1 mol protein. The protein partitioned into the fluid, or liquid expanded, phase. Increasing amounts of F-SP-C in DPPC or DPPC:DPPG monolayers decreased the size and total amounts of the condensed phase at all surface pressures. Calcium (1.6 mM) increased the amount of the condensed phase in monolayers of DPPC:DPPG but not of DPPC alone, and such monolayers were also perturbed by F-SP-C. The study indicates that SP-C perturbs the packing of neutral and anionic phospholipid monolayers even when the latter systems are condensed by calcium, indicating that interactions between SP-C and the lipids are predominantly hydrophobic in nature.     

Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant

Pulmonary surfactant forms a surface film that consists of a monolayer and a monolayer-associated reservoir. The extent to which surfactant components including the main component, dipalmitoylphosphatidylcholine (DPPC), are adsorbed into the monolayer, and how surfactant protein SP-A affects their adsorptions, is not clear. Transport of cholesterol to the surface region from dispersions of bovine lipid extract surfactant [BLES(chol)] with or without SP-A at 37 degrees C was studied by measuring surface radioactivities of [4-(14)C]cholesterol-labeled BLES(chol), and the Wilhelmy plate technique was used to monitor adsorption of monolayers. Results showed that transport of cholesterol was lipid concentration dependent. SP-A accelerated lipid adsorption but suppressed the final level of cholesterol in the surface. Surfactant adsorbed from a dispersion with or without SP-A was transferred via a wet filter paper to a clean surface, where the surface radioactivity and surface tension were recorded simultaneously. It was observed that 1) surface radioactivity was constant over a range of dispersion concentrations; 2) cholesterol and DPPC were transferred simultaneously; and 3) SP-A limited transfer of cholesterol.These results indicate that non-DPPC components of pulmonary surfactant can be adsorbed into the monolayer. Studies in the transfer of [1-(14)C]DPPC-labeled BLES(chol) to an equal or larger clean surface area revealed that SP-A did not increase selective adsorption of DPPC into the monolayer. Evaluation of transferred surfactant with a surface balance indicated that it equilibrated as a monolayer. Furthermore, examination of transferred surfactants from dispersions with and without prespread BLES(chol) monolayers revealed a functional contiguous association between adsorbed monolayers and reservoirs.     

Interactions of pulmonary surfactant protein A with phospholipid monolayers change with pH.

The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the pi-A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at the edges of condensed-expanded phase domains, and distributed evenly at lower concentration into the fluid phase with increasing pressure. At pH 4.5, TR-SP-A expanded DPPC monolayers to a slightly lower extent than at pH 7.4. It interacted primarily at the phase boundaries but it did not distribute into the fluid phase with increasing pressure. Films of DPPC/dipalmitoylphosphatidylglycerol (DPPG) 7:3 mol/mol were somewhat expanded by TR-SP-A at pH 7.4. The protein was distributed in aggregates only at the condensed-expanded phase boundaries at all surface pressures. At pH 4.5 TR-SP-A caused no expansion of the pi-A isotherm of DPPC/DPPG, but its fluorescence was relatively homogeneously distributed throughout the expanded phase at all pressures studied. These observations can be explained by a combination of factors including the preference for SP-A aggregates to enter monolayers at packing dislocations and their disaggregation in the presence of lipid under increasing pressure, together with the influence of pH on the aggregation state of SP-A and the interaction of SP-A with zwitterionic and acidic lipid.     

Molecular dynamics simulations of lung surfactant lipid monolayers

Pulmonary surfactant provides for a lipid rich film at the lung air-water interface, which prevents alveolar collapse at the end of expiration. The films are likely enriched in the major surfactant component dipalmitoylphosphatidylcholine (DPPC), which, due to its saturated fatty acid chains, can withstand high surface pressures up to 70 mN/m, thereby reducing surface tension in that interface to very low values (close to 1 mN/m). Despite many experimental measurements in situ, as well as in vitro for native lung surfactant films, the exact mechanism by which other fluid lipid components of surfactant, in combination with surfactant proteins, allow for such low surface tension values to be reached is not well understood. We have performed molecular dynamics simulation of films composed of DPPC alone and in mixtures with other fluid and acidic lipid components of surfactant at the high densities relevant to the low surface tension regime. 10-50 ns simulations were performed with the software GROMACS, with 40-64 lipids molecules plus water, using 5 different lipid compositions and 7 different areas per lipid. The primary focus was to learn how differences in lipid composition affect the response of the monolayer to compression, such as the development of curvature or the loss of lipids to the exterior of the monolayer. The systems studied exhibit features of two of the major schools of thought of lung surfactant mechanisms, in that although unsaturated lipids did not appear to prevent the monolayers from achieving high surface pressure, POPG did appear to be selectively squeezed out of the DPPC/POPG monolayers at high lipid densities.     

Interactions of pulmonary surfactant protein SP-A with monolayers of dipalmitoylphosphatidylcholine and cholesterol: roles of SP-A domains

Pulmonary surfactant protein A (SP-A) is an oligomeric glycoprotein that binds dipalmitoylphosphatidylcholine (DPPC). Interactions of rat SP-A and recombinant SP-As with pure and binary monolayers of DPPC and cholesterol were studied using a rhomboid surface balance at 37 degrees C. A marked inflection at equilibrium surface tension (23 mN/m) in surface tension-area isotherm of a pure DPPC film was abolished by rat SP-A. The inflection was decreased and shifted to 18 mN/m with wild-type recombinant SP-A (SP-Ahyp). Both rat SP-A and SP-Ahyp decreased surface area reduction required for pure DPPC films to reach near zero surface tension from 30 to 25%. SP-Ahyp, E195Q,R197D, mutated in carbohydrate recognition domain (CRD) known to be essential for SP-A-vesicle interactions, conveyed a detrimental effect on DPPC surface activity. SP-ADeltaG8-P80, with deletion of collagen-like domain, had little effect. Both SP-Ahyp, C6S (Ser substitution for Cys6) and SP-Ahyp,DeltaN1-A7 (N-terminal segment deletion) which appear mainly as monomers on non-reducing SDS-PAGE analysis, increased required surface area reduction for minimal surface tension. All SP-As reduced collapse surface tension of a pure cholesterol film from 27 to 23 mN/m in the presence of Ca2+. When mixed films were formed by successive spreading of DPPC/SP-A/cholesterol, rat SP-A, SP-Ahyp, or SP-ADeltaG8-P80 blocked the interaction of cholesterol with DPPC; SP-Ahyp,E195Q,R197D could not impede the interaction; SP-Ahyp,C6S or SP-Ahyp,DeltaN1-A7 only partially blocked the interaction, and cholesterol appeared to stabilize SP-Ahyp,C6S-DPPC association. These results demonstrate the importance of CRD and N-terminal dependent oligomerization in SP-A-phospholipid associations. The findings further indicate that SP-A-cholesterol interactions differ from SP-A-DPPC interactions and may be nonspecific.     

Electrostatic barrier to recovery of dipalmitoylphosphatidylglycerol monolayers after collapse

The reincorporation of lipids into monolayers at the air-water interface after collapse is important to the maintenance of low surface tensions on subsequent expansion and compression cycles. For single component, anionic dipalmitoylphosphatidylglycerol monolayers, the fraction of recovered lipid is proportional to the subphase ionic strength. The collapse mechanism and structure of the collapsed materials appear unchanged with ionic strength. A simple electrostatic barrier model shows that the fractional recovery depends exponentially on the Debye length; this is verified by experiment. This simple model suggests possible catalytic roles for the cationic lung surfactant specific proteins SP-B and SP-C that induce structural changes in the monolayer that may act as charge-neutralizing docking sites for surfactant in the subphase, leading to faster and more efficient recovery.     

Compositional and structural characterization of monolayers and bilayers composed of native pulmonary surfactant from wild type mice

This work comprises a structural and dynamical study of monolayers and bilayers composed of native pulmonary surfactant from mice. Spatially resolved information was obtained using fluorescence (confocal, wide field and two photon excitation) and atomic force microscopy methods. Lipid mass spectrometry experiments were also performed in order to obtain relevant information on the lipid composition of this material. Bilayers composed of mice pulmonary surfactant showed coexistence of distinct domains at room temperature, with morphologies and lateral packing resembling the coexistence of liquid ordered (lo)/liquid disordered (ld)-like phases reported previously in porcine lung surfactant. Interestingly, the molar ratio of saturated (mostly DPPC)/non-saturated phospholipid species and cholesterol measured in the innate material corresponds with that of a DOPC/DPPC/cholesterol mixture showing lo/ld phase coexistence at a similar temperature. This suggests that at quasi-equilibrium conditions, key lipid classes in this complex biological material are still able to produce the same scaffold observed in relevant but simpler model lipid mixtures. Also, robust structural and dynamical similarities between mono- and bi-layers composed of mice pulmonary surfactant were observed when the monolayers reach a surface pressure of 30 mN/m. This value is in line with theoretically predicted and recently measured surface pressures, where the monolayer–bilayer equivalence occurs in samples composed of single phospholipids. Finally, squeezed out material attached to pulmonary surfactant monolayers was observed at surface pressures near the beginning of the monolayer reversible exclusion plateau (~ 40 mN/m). Under these conditions this material adopts elongated tubular shapes and displays ordered lateral packing as indicated by spatially resolved LAURDAN GP measurements.     

Phase separation in monolayers of pulmonary surfactant phospholipids at the air-water interface: composition and structure.

The phase behavior of monolayers containing the complete set of purified phospholipids (PPL) obtained from calf surfactant was investigated as a model for understanding the phase transitions that precede compression of pulmonary surfactant to high surface pressure. During compression, both fluorescence microscopy and Brewster angle microscopy (BAM) distinguished domains that separated from the surrounding film. Quantitative analysis of BAM grayscales indicated optical thicknesses for the PPL domains that were similar to the liquid condensed phase for dipalmitoyl phosphatidylcholine (DPPC), the most abundant component of pulmonary surfactant, and higher and less variable with surface pressure than for the surrounding film. BAM also showed the optical anisotropy that indicates long-range orientational order of tilted lipid chains for the domains, but not for the surrounding film. Fluorescence microscopy shows that addition of DPPC to the PPL increased the area of the domains. At fixed surface pressures from 20-40 mN/m, the total area of each phase grew in proportion with the mol fraction of DPPC. This constant variation allowed analysis of the DPPC mol fraction in each phase, construction of a simple phase diagram, and calculation of the molecular area for each phase. Our results indicate that the phase surrounding the domains is more expanded and compressible, and contains reduced amounts of DPPC in addition to the other phospholipids. The domains contain a mol fraction for DPPC of at least 96%.     

Differential partitioning of pulmonary surfactant protein SP-A into regions of monolayers of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol.

The interaction of the pulmonary surfactant protein SP-A fluorescently labeled with Texas Red (TR-SP-A) with monolayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC/dipalmitoylphosphatidylglycerol 7:3 w/w has been investigated. The monolayers were spread on aqueous subphases containing TR-SP-A. TR-SP-A interacted with the monolayers of DPPC to accumulate at the boundary regions between liquid condensed (LC) and liquid expanded (LE) phases. Some TR-SP-A appeared in the LE phase but not in the LC phase. At intermediate surface pressures (10-20 mN/m), the protein caused the occurrence of more, smaller condensed domains, and it appeared to be excluded from the monolayers at surface pressure in the range of 30-40 mN/m. TR-SP-A interaction with DPPC/dipalmitoylphosphatidylglycerol monolayers was different. The protein did not appear in either LE or LC but only in large aggregates at the LC-LE boundary regions, a distribution visually similar to that of fluorescently labeled concanavalin A adsorbed onto monolayers of DPPC. The observations are consistent with a selectivity of interaction of SP-A with DPPC and for its accumulation in boundaries between LC and LE phase.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: I. Monolayers of pulmonary surfactant protein SP-B and phospholipids.

The effects of pulmonary surfactant protein SP-B on the properties of monolayers of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), and a mixture of DPPC:DPPG (7:3, mol:mol) were studied using spread films at the air-water interface. The addition of SP-B to the phospholipid monolayers gave positive deviations from additivity of the mean areas in the films. At low protein concentrations (less than 45% amino acid residues which corresponds to 0.5 mol% or 10 weight% SP-B) monolayers of SP-B/DPPC, SP-B/DPPG and SP-B/(DPPC:DPPG) collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At higher concentrations of SP-B in the protein-lipid monolayers, kink points appeared in the isotherms at about 40-45 mN.m-1, implying possible exclusion of material from the films, hence, changes in the original monolayer compositions. Calculated analyses of the monolayer compositions as a function of surface pressure indicated that nearly pure SP-B, associated with small amounts of phospholipid (2-3 lipid molecules per SP-B dimer), was lost from SP-B/DPPC, SP-B/DPPG, and SP-B/(DPPC:DPPG) films at surface pressures higher than 40-45 mN.m-1. The results are consistent with a low effectiveness of SP-B in removing saturated phospholipids, DPPC or DPPG, from the spread SP-B/phospholipid films.