SEPARATION OF SUBFRACTIONS OF THE HYDROPHOBIC COMPONENTS OF CALF LUNGSURFACTANT

This study reports the biochemical separation of the hydrophobic const ituents of calf lung surfactant into separate fractions from which spe cific components are excluded. Gel permeation chromatography on LH-20 with acidified chloroform-methanol separated the constituents of calf lung surfactant extract (CLSE) into fractions containing purified phos pholipids (PPL), the neutral lipids and phospholipids (N&PL), or the h ydrophobic surfactant proteins (SP)-B and -C together with the phospho lipids (SP&PL). Extraction of acid to prevent phospholipid degradation after separation reduced recovery of the apoproteins in SP&PL. This f raction was therefore supplemented with protein purified separately to attain the initial levels present in CLSE. Biochemical analyses confi rmed that the resulting preparations had the expected composition not only of protein, neutral lipids and phospholipids, but also of the pho spholipid head groups. In addition to these fractions obtained with ac idified solvent, elution of CLSE with chloroform-methanol without acid yielded the zwitterionic phospholipids substantially depleted of anio nic phosphatidylglycerol and phosphatidylinositol. Limited interfacial measurements also demonstrated that the process of separation did not alter the fundamental surface characteristics of the surfactant const ituents. Recombined CLSE (rCLSE) reconstituted from all of the separat ed components had surface activity indistinguishable from the original CLSE. The individual fractions of surfactant components also had aver age molecular areas at the air-liquid interface which agreed with pred ictions based on their biochemical composition. jlr These well defined preparations of the hydrophobic constituents of pulmonary surfactant provide the basis for future studies to establish the role of individu al components in the function of this complex surface active material.