Functional tests for the characterization of surfactant protein B (SP-B) and a fluorescent SP-B analog.

Surfactant protein B (SP-B) enhances lipid insertion into the alveolar air/liquid interface upon inhalation. The aim of this study was (i) to apply a palette of tests for a detailed biochemical and biophysical characterization of SP-B and (ii) to use these tests to compare native SP-B with a fluorescent (Bodipy) SP-B analog. The method of labeling was fast and resulted in a covalent fluorophore-protein bond. The ability of both proteins to spread a surfactant film on top of a buffer surface was determined in a spreading tray using the Wilhelmy plate technique to allow detection of alterations in surface tension and calculation of spreading velocities. In a captive bubble surfactometer surface tensions of spread films were measured. Similar biophysical properties were found for both native and Bodipy-labeled SP-B. It is concluded that the combination of tests used allows detection of small differences in structure and activity between the two proteins.

Surfactant-associated proteins: functions and structural variation.

Pulmonary surfactant is a barrier material of the lungs and has a dual role: firstly, as a true surfactant, lowering the surface tension; and secondly, participating in innate immune defence of the lung and possibly other mucosal surfaces. Surfactant is composed of approximately 90% lipids and 10% proteins. There are four surfactant-specific proteins, designated surfactant protein A (SP-A), SP-B, SP-C and SP-D. Although the sequences and post-translational modifications of SP-B and SP-C are quite conserved between mammalian species, variations exist. The hydrophilic surfactant proteins SP-A and SP-D are members of a family of collagenous carbohydrate binding proteins, known as collectins, consisting of oligomers of trimeric subunits. In view of the different roles of surfactant proteins, studies determining the structure-function relationships of surfactant proteins across the animal kingdom will be very interesting. Such studies may reveal structural elements of the proteins required for surface film dynamics as well as those required for innate immune defence. Since SP-A and SP-D are also present in extrapulmonary tissues, the hydrophobic surfactant proteins SP-B and SP-C may be the most appropriate indicators for the evolutionary origin of surfactant. SP-B is essential for air-breathing in mammals and is therefore largely conserved. Yet, because of its unique structure and its localization in the lung but not in extrapulmonary tissues, SP-C may be the most important indicator for the evolutionary origin of surfactant.

Effect of the hydrophobic surfactant proteins on the surface activity of spread films in the captive bubble surfactometer.

The main function of pulmonary surfactant, a mixture of lipids and proteins, is to reduce the surface tension at the air/liquid interface of the lung. The hydrophobic surfactant proteins SP-B and SP-C are required for this process. When testing their activity in spread films in a captive bubble surfactometer, both SP-B and SP-C showed concentration dependence for lipid insertion as well as for lipid film refinement. Higher activity in DPPC refinement of the monolayer was observed for SP-B compared with SP-C. Further differences between both proteins were found, when subphase phospholipid vesicles, able to create a monolayer-attached lipid reservoir, were omitted. SP-C containing monolayers showed gradually increasing minimum surface tensions upon cycling, indicating that a lipid reservoir is required to prevent loss of material from the monolayer. Despite reversible cycling dynamics, SP-B containing monolayers failed to reach near-zero minimum surface tensions, indicating that the reservoir is required for stable films.