Probing perturbation of bovine lung surfactant extracts by albumin using DSC and 2H-NMR

Abstract

Lung surfactant (LS), a lipid-protein mixture, forms films at the lung air-water interface and prevents alveolar collapse at end expiration. In lung disease and injury, the surface activity of LS is inhibited by leakage of serum proteins such as albumin into the alveolar hypophase. Multilamellar vesicular dispersions of a clinically used replacement, bovine lipid extract surfactant (BLES), to which (2% by weight) chain-perdeuterated dipalmitoylphosphatidycholine (DPPG mixtures-d(62)) had been added, were studied using deuterium-NMR spectroscopy ((2)H-NMR) and differential scanning calorimetry (DSC). DSC scans of BLES showed a broad gel to liquid-crystalline phase transition between 10-35 degrees C, with a temperature of maximum heat flow (T(max)) around 27 degrees C. Incorporation of the DPPC-d(62) into BLES-reconstituted vesicles did not alter the T(max) or the transition range as observed by DSC or the hydrocarbon stretching modes of the lipids observed using infrared spectroscopy. Transition enthalpy change and (2)H-NMR order parameter profiles were not significantly altered by addition of calcium and cholesterol to BLES. (2)H-NMR spectra of the DPPC-d(62) probes in these samples were characteristic of a single average lipid environment at all temperatures. This suggested either continuous ordering of the bilayer through the transition during cooling or averaging of the DPPC-d(62) environment by rapid diffusion between small domains on a short timescale relative to that characteristic of the (2)H-NMR experiment. Addition of 10% by weight of soluble bovine serum albumin (1:0.1, BLES/albumin, dry wt/wt) broadened the transition slightly and resulted in the superposition of (2)H-NMR spectral features characteristic of coexisting fluid and ordered phases. This suggests the persistence of phase-separated domains throughout the transition regime (5-35 degrees C) of BLES with albumin. The study suggests albumin can cause segregation of protein bound-lipid domains in surfactant at NMR timescales (10(-5) s). Persistent phase separation at physiological temperature may provide for a basis for loss of surface activity of surfactant in dysfunction and disease.

FIGURE 1

ESI-MS (a) and MALDI-TOF (b) spectra of BLES in organic solvent obtained in the positive ion mode. The major phosphatidylcholine species in panel a are detected as a singly charged species and represent the major PC present in the lipid extract, and the 734 peak represents the 1,2, dipalmitoylphosphatidylcholine (16:0/16:0 PC). The other species detected with different acyl chain distribution of PC species at m/z are 706 (14:0/16:0); 721 (16:0-sphigomyelin); 732 (16:0/16:1); 747-18:1-sphinogomyelin; 761- (16:0/18:1); 786- (18:2/18:2) phospholipids. In panel b the MALDI-TOF spectra of BLES in organic solvent, the singly charged monomeric species of bovine SP-C at 4041 Da, and SP-B monomers at 8.6 kDa and dimers at 17.3 kDa are detected.

FIGURE 2

(a) DSC endotherms of i), BLES, ii), BLES with cholesterol, iii), BLES with cholesterol and calcium, and iv), BLES with cholesterol, calcium, and albumin (1:0.1, wt/wt). (b) DSC endotherm for BLES-DPPC-d₆₂. (c) The FTIR spectra of BLES (top) and BLES-DPPC-d₆₂ (bottom). In panel c the C-D₂ symmetric and asymmetric stretching modes at 2195 cm⁻¹ and 2090 cm⁻¹ appear as a separate pair of peaks due to the DPPC-d₆₂ present in BLES; however, the relative distribution of the CH₂ vibrations (2922 and 2852 cm⁻¹) are not altered in either sample.

FIGURE 3

²H-NMR spectra at selected temperatures for BLES-DPPC-d₆₂ dispersions. Samples were cooled from 45 to 0°C.

FIGURE 4

²H-NMR spectra at selected temperatures for BLES-DPPC-d₆₂ dispersions with (a) cholesterol, (b) cholesterol plus calcium, and (c) cholesterol plus calcium plus albumin. Samples were cooled from 45 to 0°C.

FIGURE 5

Temperature dependence of ²H-NMR first spectral moments (M₁) for (○) BLES, (□) BLES-cholesterol, (▵) BLES-cholesterol-calcium, and (•) BLES-cholesterol-calcium-albumin samples containing DPPC-d₆₂.