Probing perturbation of bovine lung surfactant extracts by albumin using DSC and 2H-NMR
Kaushik Nag 1*, Kevin M.W. Keough 1 and Michael Robert Morrow 1
1 Memorial University of Newfoundland
* To whom correspondence should be addressed. E-mail: kaushikn{at}mun.ca.
Submitted on November 8, 2005
Revised on February 1, 2006
Accepted on 3 February 2006
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Abstract |
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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. Multi-lamellar vesicular dispersions of a clinically-used replacement, bovine lipid extract surfactant (BLES), to which (2% by weight) chain-perdeuterated dipalmitoylphosphatidycholine (DPPC-d62) had been added, were studied using deuterium nuclear magnetic resonance spectroscopy (2H-NMR) and differential scanning calorimetry (DSC). DSC scans of BLES showed a broad gel to liquid crystalline phase transition between 10-35°C, with a temperature of maximum heat flow (Tmax) around 27°C. Incorporation of the DPPC-d62 into BLES reconstituted vesicles did not alter the Tmax or the transition range as observed by DSC, or the hydrocarbon stretching modes of the lipids observed using infrared spectroscopy. Transition enthalpy change and 2H-NMR order parameter profiles were not significantly altered by addition of calcium and cholesterol to BLES. 2H-NMR spectra of the DPPC-d62 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-d62 environment by rapid diffusion between small domains on a short timescale relative to that characteristic of the 2H-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 2H-NMR spectral features characteristic of coexisting fluid and ordered phases. This suggests the persistence of phase-separated domains throughout the transition regime (5-35°C) of BLES with albumin. The study suggests albumin can cause segregation of protein bound-lipid domains in surfactant at NMR timescales (10-5sec). Persistent phase separation at physiological temperature, may provide for a basis for loss of surface activity of surfactant in dysfunction and disease.
Key Words:
Phase transition, bilayer dispersions, domains, order parameter, perdeuterated lipid, soluble protein-lipid interactions
