Surfactant proteins B and C (SP-B and SP-C), together with phospholipids, are important constituents of pulmonary surfactant and of preparations used for treatment of respiratory distress syndrome (RDS). SP-B belongs to the saposin family of homologous proteins, which include other lipid-interacting proteins, like the membranolytic NK-lysin. SP-B, in contrast to other saposins, is hydrophobic and a disulfide-linked dimer, and its mechanism of action is not known. A model of the three-dimensional structure of one SP-B subunit was generated from the structure of monomeric NK-lysin determined by nuclear magnetic resonance, and the SP-B dimer was formed by joining two subunits via the intersubunit disulfide bond Cys48-Cys48'. After energy minimization, intersubunit hydrogen bonds/ion pairs were formed between the strictly conserved residues Glu51 and Arg52, which creates a central non-polar region located in between two clusters of positively charged residues. The structural features support a function of SP-B in cross-linking of lipid membranes. Mixtures of phospholipids, an SP-C analogue and polymyxin B (which cross-links lipid vesicles but is structurally unrelated to SP-B) exhibit in vitro surface activity which is indistinguishable from that of analogous mixtures containing SP-B instead of polymyxin B. This suggests an avenue for identification of SP-B analogues that can be used in synthetic surfactants for treatment of RDS.

Surfactant protein C (SP-C) is a lipopeptide that contains two thioester-linked palmitoyl groups and is considered to be important for formation of the alveolar surface active lipid film. Here, a non- or dipalmitoylated SP-C analogue (SP-C(Leu)), in which all helical Val residues were replaced with Leu and Cys-5 and Cys-6 were replaced with Ser, was tested for surface activity in a captive bubble system (CBS). SP-C(Leu), either palmitoylated at Ser-5 and Ser-6 or non-palmitoylated, was added to mixtures of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidyl glycerol (PG)/palmitic acid (PA), 68:22:9, (by mass) at a concentration of 2 and 5%. With 2% peptide, surface film formation was rapid, reaching a surface tension below 25 mN/m within 5 s, but the samples with 5% SP-C(Leu) required more than 20 s to reach values below 25 mN/m. Minimum surface tension for the samples with dipalmitoylated SP-C(Leu) was below 1.5 mN/m and very stable, as the surface tension increased by less than 0.5 mN/m within 10 min at constant bubble volume. Minimum surface tension for the non-palmitoylated SP-C(Leu) was approximately 2 and 5 mN/m for 2 and 5% peptide, respectively, but the films were less stable as seen by frequent bubble clicking at low surface tensions. Films with dipalmitoylated SP-C(Leu) that were dynamically cycled at 20-30 cycles/min were substantially less compressible at a surface tension of 20 mN/m (0.007 m/mN) than those that contained the non-palmitoylated peptide (0.02 m/mN). After subphase depletion, the incorporation of lipids into the surface active film during initial bubble expansion occurred at a relatively low surface tension (about 35 mN/m) for the samples with dipalmitoylated SP-C(Leu) compared to approximately 45 mN/m for those containing the non-palmitoylated peptide. Furthermore, for samples that contained non-palmitoylated SP-C(Leu), the ability to reach near zero stable surface tension was lost after a few adsorption steps, whereas with the dipalmitoylated peptide the film quality did not deteriorate even after more than 10 expansion steps and the incorporation of reservoir material equivalent to more than two monolayers. It appears that the covalently linked palmitoyl groups of the SP-C analogue studied are important for the mechanical stability of the lipid film, for the capacity to incorporate material from the reservoir into the surface active film upon area expansion, and for the low film compressibility of dynamically cycled films.

Surfactant preparations for the treatment of respiratory distress syndrome (RDS) that contain phospholipids and small amounts of the two hydrophobic proteins, SP-B and SP-C, are presently obtained from animal lungs. Since structural information about SP-B and SP-C is available, it appears possible to design analogues that can replace the native proteins in synthetic surfactants. SP-C contains a single helix, but analogues with the poly-Val sequence of the native molecule do not fold into a native-like α-helical conformation. However, replacement of all Val with Leu yields efficient folding into a helical structure and Leu-based SP-C analogues effectively accelerate spreading of surfactant lipids and exhibit some physiological activity in animal models of RDS. The inferior in vivo activity of synthetic surfactants containing SP-C only compared to that of surfactant preparations derived from natural sources may be caused by a lack of covalently linked palmitoyl groups in the analogues and/or absence of SP-B. SP-B is significantly larger than SP-C and has a tertiary fold of several amphipathic helices in a dimeric structure. A single simplified amphipathic helical peptide containing only Leu and Lys does not mimic the surface properties of SP-B in vitro. These circumstances make the design of SP-B analogues from solely structural considerations less likely to be successful than in the case of SP-C.

Natural surfactant preparations containing phospholipids and the hydrophobic surfactant proteins B and C (SP-B and SP-C) are effective in the treatment of respiratory distress syndrome in premature infants. The limited supply, and the risk of infectious agents and immunological reactions have promoted the evaluation of synthetic peptides in surfactant preparations. However, the folding of synthetic SP-C into an alpha-helix is inefficient and alpha-helical SP-C analogues with Val-->Leu substitutions form oligomers. In order to circumvent these problems we have synthesized an SP-C analogue, named SP-C(LKS), which differs from SP-C mainly by the exchange of most of the Val residues in positions 16-28 with Leu residues to promote an alpha-helical conformation, and by the introduction of Lys residues at positions 17, 22 and 27 in order to locate positive charges around the helical circumference and thereby avoid self polymerization. CD spectroscopy showed a spectrum typical for alpha-helical peptides and SDS/PAGE disclosed a single band. The biophysical activity of artificial surfactant preparations containing SP-C(LKS) and phospholipids, with and without native SP-B, was measured using a Wilhelmy balance and a pulsating bubble surfactometer. SP-C(LKS) (3%, w/w) in a mixture of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidylglycerol/palmitic acid (68:22:9, by wt.) suspended in 150 mM NaCl, showed rapid spreading at the air-liquid interface and produced a surface tension of <1 mN/m at minimum bubble size (gammamin) and 42 mN/m at maximum bubble size (gammamax) in the pulsating bubble surfactometer. The addition of 2% (w/w) SP-B to the preparation reduced the maximum surface tension to 33-35 mN/m, i.e. both gammamin and gammamax values were similar to those of natural surfactant preparations. Optimal in vitro characteristics were also obtained from a preparation containing SP-C(LKS), SP-B, DPPC and phosphatidylglycerol, i.e. when palmitic acid was omitted from the lipid mixture. SP-B containing surfactant preparations made up in Hepes buffer at pH 6.9, instead of in 150 mM NaCl, had similar biophysical activity provided that palmitic acid was omitted, but decreased activity in the presence of palmitic acid.

Surfactant proteins B and C (SP-B and SP-C) are present in natural derived surfactant preparations used for treatment of respiratory distress syndrome. Herein the surface activity of an SP-C analogue (SP-C(LKS)), a hybrid peptide between SP-C and bacteriorhodopsin (SP-C/BR) and a model peptide (KL(4)) was studied with a captive bubble surfactometer (CBS). The peptides were mixed with either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidylglycerol (PG) (7:3, by weight) or DPPC/PG/palmitic acid (68:22:9, by weight) at a concentration of 1 mg/ml in HEPES buffer, pH 6.9 and a polypeptide/lipid weight ratio of 0.02--0.03. In some lipid/peptide preparations also 2% of SP-B was included. Adsorption, monitored as surface tension vs. time for 10 min after bubble formation did not show discernible differences for the whole set of preparations. Equilibrium surface tensions of approximately 25 mN/m were reached after 5--10 min for all preparations, although those with SP-C/BR appeared not to reach end point of adsorption within 10 min. Area compression needed to reach minimum surface tension of 0.5--2.0 mN/m was least for the KL(4) preparation, about 13% in the first cycle. 3% SP-C(LKS) in DPPC:PG (7:3, by weight) reached minimum surface tension upon 27% compression in the first cycle. If DPPC:PG:PA (68:22:9, by weight) was used instead only 16% area compression was needed and 14% if also 2% SP-B was included. 3% SP-C(LKS) in DPPC:PG (7:3, by weight)+2% SP-B needed 34% compression to reach minimum surface tension. The replenishment of material from a surface associated surfactant reservoir was estimated with subphase depletion experiments. With the 2% KL(4) preparation incorporation of excess material took place at a surface tension of 25--35 mN/m during stepwise bubble expansion and excess material equivalent to 4.3 monolayers was found. When 2% SP-B was added to 3% SP-C(LKS) in DPPC:PG (7:3, by weight) the number of excess monolayers increased from 1.5 to 3.6 and the incorporation took place at 30--40 mN/m. When SP-B was added to 3% SP-C(LKS) in DPPC:PG:PA (68:22:9, by weight) the number of excess monolayers increased from 0.5 to 3.4 and incorporation took place at 40--50 mN/m. With 2% SP-C/BR incorporation took place at 40--45 mN/m, frequent instability clicks were observed and excess material of approximately 1.1 monolayer was estimated.