Kevin M. W. Keough

Interfacial properties of surfactant proteins

Interfacial properties of surfactant proteins Jesüs Përez-Gil a, Kevin M.W. Keough b;* a Dept. Bioqu|̈mica, Fac. Biolog|̈a, Universidad Complutense, 28040 Madrid, Spain b Department of Biochemistry, Memorial University of Newfoundland, St. Johns, A1B 3X9, Canada Dedicated to John Clements. It is an honor and a pleasure to have contributed to this issue to celebrate the 75th birthday of John Clements. Seldom does one get a chance to know a founder of a ¢eld of scienti¢c endeavour. To know John Clements is a scienti¢c stimulation and a personal pleasure. His contributions to this ¢eld have been noteworthy not only for their relevance, but also in their breadth and insight. Much of what is discussed has been written about by John Clements over many years. Being the consummate scientist he is, he has led us to as many questions as answers throughout his work. Happy Birthday, and long may your big jib draw. Received 17 March 1998; received in revised form 4 June 1998; accepted 4 June 1998

Phase Transitions in Films of Lung Surfactant at the Air-Water Interface

Pulmonary surfactant maintains a putative surface-active film at the air-alveolar fluid interface and prevents lung collapse at low volumes. Porcine lung surfactant extracts (LSE) were studied in spread and adsorbed films at 23 +/- 1 degrees C using epifluorescence microscopy combined with surface balance techniques. By incorporating small amounts of fluorescent probe 1-palmitoyl-2-nitrobenzoxadiazole dodecanoyl phosphatidylcholine (NBD-PC) in LSE films the expanded (fluid) to condensed (gel-like) phase transition was studied under different compression rates and ionic conditions. Films spread from solvent and adsorbed from vesicles both showed condensed (probe-excluding) domains dispersed in a background of expanded (probe-including) phase, and the appearance of the films was similar at similar surface pressure. In quasistatically compressed LSE films the appearance of condensed domains occurred at a surface pressure (pi) of 13 mN/m. Such domains increased in size and amounts as pi was increased to 35 mN/m, and their amounts appeared to decrease to 4% upon further compression to 45 mN/m. Above pi of 45 mN/m the LSE films had the appearance of filamentous materials of finely divided dark and light regions, and such features persisted up to a pi near 68 mN/m. Some of the condensed domains had typical kidney bean shapes, and their distribution was similar to those seen previously in films of dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant. Rapid cyclic compression and expansion of LSE films resulted in features that indicated a possible small (5%) loss of fluid components from such films or an increase in condensation efficiency over 10 cycles. Calcium (5 mM) in the subphase of LSE films altered the domain distribution, decreasing the size and increasing the number and total amount of condensed phase domains. Calcium also caused an increase in the value of pi at which the maximum amount of independent condensed phase domains were observed to 45 mN/m. It also induced formation of large amounts of novel, nearly circular domains containing probe above pi of 50 mN/m, these domains being different in appearance than any seen at lower pressures with calcium or higher pressures in the absence of calcium. Surfactant protein-A (SP-A) adsorbed from the subphase onto solvent-spread LSE films, and aggregated condensed domains in presence of calcium. This study indicates that spread or adsorbed lung surfactant films can undergo expanded to condensed, and possibly other, phase transitions at the air-water interface as lateral packing density increases. These phase transitions are affected by divalent cations and SP-A in the subphase, and possibly by loss of material from the surface upon cyclic compression and expansion.

Pulmonary surfactant protein SP-C causes packing rearrangements of dipalmitoylphosphatidylcholine in spread monolayers

The hydrophobic pulmonary surfactant protein SP-C has been isolated from porcine lung surfactant, and it has been incorporated into monolayers of dipalmitoylphosphatidylcholine (DPPC). The monolayers, which contained 1 mol% of a fluorescently-labeled phosphatidylcholine, were observed under various states of compression in an epifluorescence surface balance. SP-C altered the packing arrangements of DPPC in the monolayer, causing the production of many more, smaller condensed lipid domains in its presence than in its absence.

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: II. Monolayers of pulmonary surfactant protein SP-C and phospholipids.

The interaction of the hydrophobic pulmonary surfactant protein SP-C with dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and DPPC:DPPG (7:3, mol:mol) in spread monolayers at the air-water interface has been studied. At low concentrations of SP-C (about 0.5 mol% or 3 weight%protein) the protein-lipid films collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At initial protein concentrations higher than 0.8 mol%, or 4 weight%, the isotherms displayed kinks at surface pressures of about 50 mN.m-1 in addition to the collapse plateaux at the higher pressures. The presence of less than 6 mol%, or 27 weight%, of SP-C in the protein-lipid monolayers gave a positive deviation from ideal behavior of the mean areas in the films. Analyses of the mean areas in the protein-lipid films as functions of the monolayer composition and surface pressure showed that SP-C, associated with some phospholipid (about 8-10 lipid molecules per molecule of SP-C), was squeezed out from the monolayers at surface pressures of about 55 mN.m-1. The results suggest a potential role for SP-C to modify the composition of the monolayer at the air-water interface in the alveoli.

Cholesterol in condensed and fluid phosphatidylcholine monolayers studied by epifluorescence microscopy

Epifluorescence microscopy was used to investigate the effect of cholesterol on monolayers of dipalmitoylphosphatidylcholine (DPPC) and 1 -palmitoyl-2-oleoyl phosphatidylcholine (POPC) at 21 +/- 2 degrees C using 1 mol% 1-palmitoyl-2-[12-[(7-nitro-2-1, 3-benzoxadizole-4-yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) as a fluorophore. Up to 30 mol% cholesterol in DPPC monolayers decreased the amounts of probe-excluded liquid-condensed (LC) phase at all surface pressures (pi), but did not effect the monolayers of POPC, which remained in the liquid-expanded (LE) phase at all pi. At low pi (2-5 mN/m), 10 mol% or more cholesterol in DPPC induced a lateral phase separation into dark probe-excluded and light probe-rich regions. In POPC monolayers, phase separation was observed at low pi when > or =40 mol% or more cholesterol was present. The lateral phase separation observed with increased cholesterol concentrations in these lipid monolayers may be a result of the segregation of cholesterol-rich domains in ordered fluid phases that preferentially exclude the fluorescent probe. With increasing pi, monolayers could be transformed from a heterogeneous dark and light appearance into a homogeneous fluorescent phase, in a manner that was dependent on pi and cholesterol content. The packing density of the acyl chains may be a determinant in the interaction of cholesterol with phosphatidylcholine (PC), because the transformations in monolayer surface texture were observed in phospholipid (PL)/sterol mixtures having similar molecular areas. At high pi (41 mN/m), elongated crystal-like structures were observed in monolayers containing 80-100 mol% cholesterol, and these structures grew in size when the monolayers were compressed after collapse. This observation could be associated with the segregation and crystallization of cholesterol after monolayer collapse.

Differential scanning calorimetric studies of aqueous dispersions of phosphatidylcholines containing two polyenoic chains

The thermotropic behavior of aqueous dispersions of phosphatidylcholines (PC) that contain two identical unsaturated acyl chains, either linoleoyl, alpha-linolenoyl, or arachidonoyl chains, has been investigated by differential scanning calorimetry. These dipolyenoic PC displayed very broad thermal transitions, which occurred over a range of approximately 40 C deg, that started between -77 degrees C (20:4-20:4 PC) and -71 degrees C (18:2-18:2 PC). The enthalpy changes associated with the transitions were small (1 to 2 kcal/mol, 4 to 8 kJ/mol). The broad, low-enthalpy transitions suggest that these lipids might have a very disordered gel phase, or that the transitions from the gel to the liquid crystal might proceed through some intermediate states. The temperatures over which the transitions occurred were influenced only slightly by the number of double bonds between two and four per chain.

Fluorescently labeled pulmonary surfactant protein C in spread phospholipid monolayers

Pulmonary surfactant, a lipid-protein complex, secreted into the fluid lining of lungs prevents alveolar collapse at low lung volumes. Pulmonary surfactant protein C (SP-C), an acylated, hydrophobic, alpha-helical peptide, enhances the surface activity of pulmonary surfactant lipids. Fluorescein-labeled SP-C (F-SP-C) (3, 6, 12 wt%) in dipalmitoylphosphatidylcholine (DPPC), and DPPC:dipalmitoylphosphatidylglycerol (DPPG) [DPPC:DPPG 7:3 mol/mol] in spread monolayers was studied by epifluorescence microscopy. Mass spectometry of F-SP-C indicated that the protein is partially deacylated and labeled with 1 mol fluorescein/1 mol protein. The protein partitioned into the fluid, or liquid expanded, phase. Increasing amounts of F-SP-C in DPPC or DPPC:DPPG monolayers decreased the size and total amounts of the condensed phase at all surface pressures. Calcium (1.6 mM) increased the amount of the condensed phase in monolayers of DPPC:DPPG but not of DPPC alone, and such monolayers were also perturbed by F-SP-C. The study indicates that SP-C perturbs the packing of neutral and anionic phospholipid monolayers even when the latter systems are condensed by calcium, indicating that interactions between SP-C and the lipids are predominantly hydrophobic in nature.

Microstructure and dynamic surface properties of surfactant protein SP-B/dipalmitoylphosphatidylcholine interfacial films spread from lipid-protein bilayers

Abstract Suspensions of dipalmitoylphosphatidylcholine (DPPC) bilayers containing 5, 10 or 20% (w/w) surfactant protein SP-B have been reconstituted and spread at air-liquid interfaces. Compression isotherms of DPPC/SP-B monolayers spread from these preparations were qualitatively comparable to the isotherms of the corresponding DPPC/SP-B monolayers spread from solvents. SP-B was squeezed-out at higher pressures from vesicle-spread films than from solvent-spread monolayers. SP-B caused a marked decrease on the rate of relaxation of DPPC collapse phases to equilibrium pressures in all the lipid/protein films assayed. This stabilizing effect was higher in vesicle-spread than in solvent-spread monolayers. Inclusion in the films of traces of the fluorescent probe NBD-PC (1 mol%) and use of a fluorescent derivative of SP-B labeled with a rhodamine derivative, Texas Red, allowed for direct observation of protein and lipid domains at the interface by epifluorescence microscopy. Upon compression, SP-B altered the packing of phospholipids in the bilayer-spread films, observed as a SP-B-induced reduction of the area of liquid-condensed domains, in a way similar to its effect in solvent-spread monolayers. SP-B was not associated with condensed regions of the films. Fluorescence images from vesicle-spread films showed discrete fluorescent aggregates that could be consistent with the existence of lipid-protein vesicles in close association with the monolayer. Both the retention of SP-B at higher surface pressures and the greater stability of collapse phases of DPPC/SP-B films prepared by spreading from liposomes in comparison to those spread from solvents can be interpreted as a consequence of formation of complex bilayer-monolayer interacting systems.

Cholesterol does not remove the gel-liquid crystalline phase transition of phosphatidylcholines containing two polyenoic acyl chains

Homoacid (single-acid) phosphatidylcholines containing two linoleate (18:2), arachidonate (20:4), or docosahexaenoate (22:6) chains were dispersed in water to form multilamellar vesicles. The influence of cholesterol on the gel to liquid phase transitions was studied by differential scanning calorimetry (DSC). The pure dipolyenoic phosphatidylcholines exhibited very broad endothermic transitions (widths of 28 to 38 C degrees at scanning rates of 5 C degrees/min) in the temperature range of about -80 degrees C to -30 degrees C. The mixing of cholesterol into the phospholipids in proportions up to 50 mol% had little effect on the temperatures, widths or enthalpy changes associated with the phase transitions. The data suggest that cholesterol does not interfere with the packing of these lipids in the gel state, possibly because the packing is already irregular or because the solubility of cholesterol in these lipids is low, or both.

The influence of unsaturation on the phase transition temperatures of a series of heteroacid phosphatidylcholines containing twenty-carbon chains

A series of heteroacid sn-1,2 diacyl phosphatidylcholines (PC) with twenty-carbon fatty acyl chains has been synthesized. Each PC contained eicosanoate (20:0) in the sn-1 position and one of a group of eicosaenoic acids with increasing numbers of cis double bonds in the sn-2 position. The double bonds were at positions delta 11 (20:1), delta 11,14 (20:2), delta 11,14,17 (20:3), or delta 5,8,11,14 (20:4). The disaturated PC containing two eicosanoate chains was also studied. Aqueous dispersions of these PC were analyzed by differential scanning calorimetry, and data for the gel to liquid-crystalline transitions (given as PC: Tc (degrees C), Tmax (degrees C), delta H (kcal/mol)) were as follows - 20:0-20:0 PC: 66.8, 68.4, 15; 20:0-20:1 PC: 19.8, 22.2, 8; 20:0-20:2 PC: -4.3, 1.8, 5; 20:0-20:3 PC: 1.2, 4.4, 7; 20:0-20:4 PC: -10.7, -6.8, 3. Double bonds in excess of two per chain did not substantially change the transition temperatures of these heteroacid PC. There was a small effect of the location of the multiple double bonds on the transition temperature. The data is consistent with the model that the transition temperatures are determined by a balance between a decrease in the packing density in the gel and a decrease in the rotational freedom of the chains in the liquid crystal, both caused by the double bonds ((1983) Biochemistry 22, 1466-1473).