X-Ray Diffraction and Reflectivity Validation of the Depletion Attraction in the Competitive Adsorption of Lung Surfactant and Albumin

Abstract

Lung surfactant (LS) and albumin compete for the air-water interface when both are present in solution. Equilibrium favors LS because it has a lower equilibrium surface pressure, but the smaller albumin is kinetically favored by faster diffusion. Albumin at the interface creates an energy barrier to subsequent LS adsorption that can be overcome by the depletion attraction induced by polyethylene glycol (PEG) in solution. A combination of grazing incidence x-ray diffraction (GIXD), x-ray reflectivity (XR), and pressure-area isotherms provides molecular-resolution information on the location and configuration of LS, albumin, and polymer. XR shows an average electron density similar to that of albumin at low surface pressures, whereas GIXD shows a heterogeneous interface with coexisting LS and albumin domains at higher surface pressures. Albumin induces a slightly larger lattice spacing and greater molecular tilt, similar in effect to a small decrease in the surface pressure. XR shows that adding PEG to the LS-albumin subphase restores the characteristic LS electron density profile at the interface, and confirms that PEG is depleted near the interface. GIXD shows the same LS Bragg peaks and Bragg rods as on a pristine interface, but with a more compact lattice corresponding to a small increase in the surface pressure. These results confirm that albumin adsorption creates a physical barrier that inhibits LS adsorption, and that PEG in the subphase generates a depletion attraction between the LS aggregates and the interface that enhances LS adsorption without substantially altering the structure or properties of the LS monolayer.

Overcoming rapid inactivation of lung surfactant: Analogies between competitive adsorption and colloid stability

“…8,10) [79, 80, 82–84, 97] that shows albumin and LS coexist at an air-water interface. As surfactant adsorption increases, the serum proteins must be displaced from the interface; as the surface pressure increases above the serum Π eq , the serum proteins are displaced from the interface and return to the subphase [13, 79, 80, 82–84, 97]. At higher serum concentrations in the subphase, the interfacial density of serum is also higher, and surfactant must displace more protein to adsorb and raise the surface pressure.…”

“…8a) and Bragg rods (Fig. 8b) from [155] monolayers of the LS, Survanta, as a function of surface pressure [97]. At Π = 20 mN/m, two Bragg peaks are observed, with the integrated intensity of the q xy = 1.44 Å −1 or {1,0} peak, roughly twice that of the q xy = 1.48 Å −1 or {1,−1} peak, indicating a distorted hexagonal lattice similar to DPPC and DPPC/palmitic acid mixtures under similar conditions [124, 125, 138, 142].…”

“…9), ρ / ρ sub is significantly greater than 1 for 50 < Z < 100 Å. This suggests that a second layer of albumin [97] may form with the albumin long axis parallel to the interface. Both neutron reflectivity [164] and ellipsometry [165] also indicate a dense, closely packed albumin monolayer with a less dense second layer.…”

“…At all surface pressures, 10 kDa PEG in the subphase induces minimal changes in the Survanta Bragg peaks and Bragg rods (Table 1) [97], indicating that PEG, like albumin, does not significantly modify the molecular lattice of the ordered domains of the surfactant. Additionally, Table 1 shows that at a specific surface pressure (Π = 40 mN/m) and subphase condition (5% wt.…”

“…The equilibrium spreading pressure, collapse pressure, LS aggregate sizes and shapes in solution, the bilayer d-spacing and the monolayer lattice spacing and tilt are not affected by albumin or serum at concentrations that inactivate LS performance [97]. Hence, this mechanism of inactivation does not involve alterations to the surfactant bilayer or monolayer.…”