Structural and Functional Characterization of Native Complexes of Pulmonary Surfactant Proteins Purified with Detergents

Abstract

Pulmonary surfactant is a specialized lipid-protein complex, which essential function is to stabilize the gas exchange surface of the respiratory epithelium against physical forces tending to its collapse. Hydrophobic surfactant proteins SP-B and SP-C are critical to promote very rapid adsorption of phospholipids into the interface and to stabilize the surface active film along the compression-expansion respiratory cycles. Mature SP-B is a homodimeric protein of approximately 18kDa (two 79 amino-acid polypeptides), while SP-C is an apparently monomeric lipopeptide of 35 residues (3.7kDa).To date, the classical method to extract and purify these proteins from surfactant membranes involves the use of organic solvents. This method can disrupt native supramolecular protein complexes. We have optimized the purification of surfactant membrane proteins upon solubilization of surfactant membranes by the zwitterionic detergent CHAPS. Different fractions have been purified from the solubilised material by gradient centrifugation and exclusion-size chromatography, showing the presence of two types of protein complexes in terms of size and density, heterogeneously composed of different oligomeric states of surfactant proteins.An ionic-exchange chromatography of solubilized surfactant yielded a fraction of cationic protein complexes consisting mainly of surfactant protein SP-B. Their structural characterization by circular dichroism and fluorescence emission revealed a structure entirely analogous to that of SP-B purified in organic solvents. However, detergent-purified SP-B showed a much more oligomeric structure, as analyzed by blue-native electrophoresis, analytical centrifugation or electron microscopy. This SP-B complex has been reconstituted into surfactant phospholipids and the resulting lipid/protein complexes showed in the captive bubble surfactometer (CBS) a similar surface active behaviour than provided by SP-B purified using the classic chloroform/methanol-based method.