Surface Dilatational Behavior of Pulmonary Surfactant Components

Surface dilatational behavior of spread monolayers of the surfactant protein B (SP-B) and its mixture ... surfactant proteins, SP-A and SP-D, the hydr...
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Langmuir 2002, 18, 1125-1130

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Surface Dilatational Behavior of Pulmonary Surfactant Components Spread on the Surface of a Pendant Drop. 2. Dipalmitoyl Phosphatidylcholine and Surfactant Protein B R. Wu¨stneck,† N. Wu¨stneck,‡ B. Moser,† and U. Pison*,‡ Universita¨ t Potsdam, Institut fu¨ r Physik, Am Neuen Palais 10, D-14415 Potsdam, Germany, and Humboldt-Universita¨ t Berlin, Charite´ Campus Virchow-Klinikum, Anaesthesiologie, Augustenburger Platz 1, D-13353 Berlin, Germany Received July 31, 2001. In Final Form: November 2, 2001 Surface dilatational behavior of spread monolayers of the surfactant protein B (SP-B) and its mixture with dipalmitoyl phosphatidylcholine (DPPC + 0.5 mol % SP-B) were investigated on the surface of a pendant phosphate buffered saline drop. Surface pressure/area isotherms of these pulmonary surfactant components were recorded in a captive bubble device. We found that the dilatational viscosity of SP-B and DPPC/SP-B films strongly decreases in the frequency range of respiration, creating an elastic film. Temperature (20 versus 30 °C) influences surface rheology quantitatively but not qualitatively. DPPC films containing either of the two hydrophobic pulmonary surfactant proteins, SP-B or SP-C, are predominantly elastic at breathing frequencies (0.04-0.2 Hz). We conclude that the hydrophobic surfactant proteins balance the surface rheological behavior of the thin film that covers the alveolar lining layer, by forming an interface of low compressibility at surface pressures below the protein squeeze-out. In the region of the protein squeeze-out, the film compressibility, however, increases. This behavior reduces the mechanical work required for respiration.

Introduction One of the main deformation processes during respiration is compression and expansion of the pulmonary layer that covers the lung surface. Therefore, surface dilatational rheological behavior of pulmonary surfactant determines the dynamic properties of the thin film that floats on the alveolar surface. The surface dilatational elasticities and viscosities are the parameters that have to be used to evaluate the capability of pulmonary surfactant to reduce the mechanical work required for respiration. Pulmonary surfactant, consisting of about 90% lipids (mostly phospholipids) and 8-10% surfactant-associated proteins, is synthesized in alveolar type II cells and secreted in lamellar bodies, where the various surfactant components are assembled in a series of densely packed bilayer aggregates in the bulk phase.1 The formation of the interfacial layer that covers alveoli requires the opening of these surfactant bilayer aggregates for the transfer of lipids and proteins into the interface by adsorption. Cyclic compression and expansion causes a continuous loss of material from the interface that leads to the cellular uptake and reprocessing of surfactant lipids and proteins.2 In contrast to the hydrophilic pulmonary surfactant proteins, SP-A and SP-D, the hydrophobic surfactant proteins, SP-B and SP-C, are assumed to influence the properties of the alveolar lining layer decisively, contributing to its surface dilatational rheological behavior.

There are three important peculiarities, which have to be considered when investigating surface rheological properties of pulmonary surfactant. The first one is the requirement of a sufficiently high surface pressure (π ) σ0 - σ; σ0 is the surface tension of the solvent, and σ is the actual surface tension). Respiration stretches almost saturated interfacial layers, with surface pressure varying between 30 and 60 mN/m.3 Proteins are squeezed out of the interfacial layer during compression in this range to build up a surface film associated reservoir.4 The second peculiarity is wetting. Because compressed pulmonary surfactant films have the ability to reduce surface tension at the air/fluid interface to extremely low values (≈0 mN/ m),5 wetting occurs that disfavors the use of many classical methods of surface chemistry to characterize pulmonary surface film behavior. A captive bubble surfactometer can be used to characterize pulmonary surfactant’s film behavior as was first described by Schu¨rch et al.5 A pendant drop technique in combination with axisymmetric drop shape analysis was also successfully used to determine surface behavior of pulmonary surfactant,6 although the application of this device is limited by the surface pressures even when using highly hydrophobic capillaries for drop creation.7,8 The third peculiarity is a general problem of investigating rheological film properties. The surface dilatational parameters are not constants of matter but depend on the surface concentration of the pulmonary components, which is associated with a defined film pressure, and the velocity of film deformation. Due to these

* Corresponding author. Ulrich Pison, MD, Medizinische Fakulta¨t Charite´, Humboldt-Universita¨t Berlin, Campus VirchowKlinikum, Klinik fu¨r Anaesthesiologie und operative Intensivmedizin, Augustenburger Platz 1, D-13353 Berlin, Germany. Phone: +49 (0)30 450-559678. Fax: +49 (0)30 450-551900. E-mail: [email protected]. † Universita ¨ t Potsdam. ‡ Humboldt-Universita ¨ t Berlin.

(3) Keough, K. M. W. Physical Chemistry of Pulmonary Surfactant in the Terminal Air Space. In Pulmonary Surfactant, from Molecular Biology to Clinic Practice; Robertson, B., van Golde, L. M. G., Batenburg, J. J., Eds.; Elsevier: Amsterdam, 1992; pp 109-163. (4) Rodriguez-Capote, K.; Nag, K.; Schu¨rch, S.; Possmayer, F. Am. J. Physiol. 2001, 281, L231-L242. (5) Schu¨rch, S.; Lee, M.; Gehr, P. Pure Appl. Chem. 1992, 64, 1745. (6) Jyoti, A.; Prokop, R. M.; Neumann, A. W. Colloids Surf. 1997, 8, 115-124. (7) Wu¨stneck, R.; Wu¨stneck, N.; Grigoriev, D. O.; Pison, U.; Miller, R. Colloids Surf., B 1999, 15, 275-288. (8) Wu¨stneck, R.; Wu¨stneck, N.; Moser, B.; Karageorgieva, V.; Pison, U. Langmuir 2002, 18, 1119-1124.

(1) Ikegami, M.; Jobe, A. H. Biochim. Biophys. Acta 1998, 1408, 218225. (2) Wright, J. R.; Clements, J. A. Am. Rev. Respir. Dis. 1987, 135, 426-444.

10.1021/la011216x CCC: $22.00 © 2002 American Chemical Society Published on Web 01/26/2002

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pecularities, understanding of surface rheological behavior of pulmonary surfactant and its components requires a methodological design that allows one to reach sufficiently high surface pressures in a breathing-relevant frequency range. Reliable experimental data for this range are not published. In previous works, we used harmonic oscillation of drop and bubble surfaces to characterize the surface mechanical behavior of pulmonary surfactant components.8-11 This is a convenient way to characterize dilatational behavior in a defined range of deformation frequencies. In the time scale of some 10-100 s/oscillation, this technique covers reorientation processes of surface clusters and domains, formed by lipids and proteins. Film properties in this time scale are viscoelastic. In the range of respiration frequencies of many mammals, that is, 0.04-0.2 Hz, we found that the dilatational viscosity of dipalmitoyl phosphatidylcholine (DPPC) and DPPC/SP-C films strongly decreases, thus leading to highly elastic films in the range of sufficiently high surface coverage.8 Here, we report the surface dilatational rheological behavior of SP-B and a DPPC/SP-B mixture and compare these results with those found for SP-C and mixtures of DPPC/SP-C.8,11 We used the pendant drop microfilm balance12 to determine surface dilatational parameters. Surface pressure/area (π/A) isotherms, which are necessary to classify surface rheological film properties, were determined by using the captive bubble method. Material and Experimental Details Chloroform and methanol were p.a. grade and purchased from Baker (J.T. Baker B.V. Deventer-Holland). L-Dipalmitoyl phosphatidylcholine (DPPC) was purchased from Sigma and used without further purification (99% purity). SP-B was isolated from chloroform/methanol lipid extracts of sheep lung washings using a semipreparative HPLC column with Vydac C4, a butyl silica gel with 5% 0.1 N trifluoroacetic acid acidified chloroform/ methanol (1:1, v/v) solvent as the mobile phase.13 The SP-B obtained was lipid free. DPPC, SP-B, and their mixtures were dissolved in chloroform/methanol (1:1, v/v). The water used was double distilled. The mixtures of DPPC and SP-B were prepared by mixing stock solutions of each component. The films were spread on 10 mM phosphate buffered saline (150 mM NaCl, pH ) 7). The chosen concentration relation between DPPC and SP-B is based on the assumption that the alveolar film contains 8-10 wt % proteins.15-16 The relation of lipid to the whole protein is 10:1 in lung surfactant. Experiments were performed with a DPPC/SP-B mixture containing 11.8 wt % of SP-B dimer, which corresponds approximately to 0.5 mol % relative to DPPC.17 This seems to be a good approximation to compare the mixtures of DPPC/SP-B (0.5 mol %) with mixtures of DPPC/SP-C (2 mol %) investigated before8 when taking into account the molecular weight ratio of native SP-B/SP-C (17380/4200). The pendant drop technique in combination with the axisymmetric drop shape analysis (ADSA) was used as a microfilm (9) Wu¨stneck, R.; Enders, P.; Wu¨stneck, N.; Pison, U.; Miller, R. PhysChemComm 1999, 11. (10) Wu¨stneck, N.; Wu¨stneck, R.; Fainerman, V. B.; Miller, R.; Pison, U. Colloids Surf., A 2000, 164, 267-278. (11) Wu¨stneck, N.; Wu¨stneck, R.; Fainerman, V. B.; Miller, R.; Pison, U. Colloids Surf., B 2001, 21, 191-205. (12) Kwok, D. Y.; Vollhardt, D.; Miller, R.; Li, D.; Neumann, A. W. Colloids Surf., A 1994, 88, 15-58. (13) Bu¨nger, H.; Kaufner, L.; Pison, U. J. Chromatogr., A 2000, 870, 363-369. (14) Haagsman, H. P.; van Golde, L. M. G. Annu. Rev. Physiol. 1991, 53, 441-464. (15) Taneva, S. G.; Keough, K. M. W. Biochemistry 2000, 39, 60836093. (16) Discher, B. M.; Maloney, K. M.; Grainger, D. W.; Sousa, C. A.; Hall, S. B. Biochemistry 1999, 38, 374-383. (17) Perez-Gil, J.; Keough, K. M. W. Biochim. Biophys. Acta 1998, 1408, 203-217.

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Figure 1. Surface pressure/area isotherms of SP-B spread at a bubble surface at 20 °C (red diamonds, compression; blue diamonds, expansion). balance for harmonic oscillation experiments. This method was described in detail earlier.18 The captive bubble device in combination with ADSA was used to obtain π/A isotherms, as described in detail elsewhere.19 In our harmonic oscillation experiments with the pendant drop microfilm balance, we changed the drop volume periodically, which causes compression and expansion of the spread surface layer at the drop surface. Resulting changes of the surface pressure were recorded by taking images of the drop profile with 2 images/s using ADSA. The ratio between the amplitudes of the surface area and the surface pressure yields the dilatational elasticity; the shift of frequency, the dilatational viscosity. A spline-fit was used to create planes of elasticity and viscosity via frequency and surface pressure, which are based on a symmetric 9 × 9 frequency/surface pressure matrix. The confidence intervals (95% confidence level) of the elasticities were about 1-4%; those of the corresponding viscosities were in the order of 10-15%. Other experimental details can be found in our preceding study.8 Rheological investigation of surface structures requires small deformation, to avoid destruction of the surface layer. In the preceding part of this report, we showed that for high surface pressure the harmonic deformation of the drop surface may cause an overoscillation of the resulting surface pressure, which became evident for spread DPPC/SP-C layers at π ≈ 55 mN/m.8 The measuring range of the pendant drop device is limited by possible spreading of the surface-active material over the outer surface of the capillary.7 By using FEP (fluoroethylene-propylene) instead of poly(tetrafluoroethylene) (PTFE) capillaries, the measuring range of the pendant drop device could be increased up to about π ≈ 67 mN/m for the present study. For spreading on pendant drop surfaces, a microdroplet injection system (microdrop-Gesellschaft fu¨r Mikrodosiertechnik, Norderstedt, Germany) was used. The reliability of this technique was tested earlier.8 Using this injection system, accurate spreading of extremely small amounts of liquid onto the surface of a pendant drop is possible in the range of some picoliters, without contacting the drop surface. For spreading at the captive bubble surface, a Hamilton microsyringe was used that allows injection of 0.05-0.15 µL of spreading solution through contacting the bubble. π/A isotherms were determined by continuous compression of the spread monolayer at the bubble surface taking images for bubble shape analysis every 5 s using ADSA. The compression rate was 7.4 × 10-4 nm2 s-1.

Experimental Results π/A Isotherms. With pure SP-B spread on the captive bubble’s surface, we obtained π/A isotherms during compression and expansion as shown in Figure 1. Surface pressures in the captive bubble nearly reached monolayer collapse. Our graph gives surface pressure versus area (18) Wu¨stneck, R.; Moser, B.; Muschiolik, G. Colloids Surf., B 1999, 15, 263-273. (19) Wu¨stneck, R.; Wu¨stneck, N.; Vollhardt, D.; Miller, R.; Pison, U. Mater. Sci. Eng., C 1999, 8-9, 57-64.

Pulmonary Surfactant Components on a Pendant Drop

Figure 2. Surface pressure/area isotherms of mixtures of DPPC + 0.5 mol % SP-B for 20 °C (red diamonds, compression; blue diamonds, expansion) and 30 °C (yellow circles, compression).

per molecule. The minimum area demand for a single SP-B dimer is nearly 1 order of magnitude higher than for a single phospholipid molecule. The principle shape of the isotherms resembles that of most synthetic surfactants or polymers. After a surface pressure lift-off, the isotherm passes a more or less pronounced plateau that can be explained by the formation of surface aggregates and/or changing of the molecular structure of the monolayer components (LE/LC). In the present case, we also found some kind of plateau, which occurs during compression between 30 and 40 mN/m. After this plateau, the surface pressure further increases up to comparable high surface pressures for proteins or polymers. At about 65 mN/m, the isotherm starts to level off, indicating layer collapse. The shape of the isotherm during expansion shows a rapid surface pressure decay, which supports the assumption

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of monolayer collapse. Corresponding to the rapid surface pressure decay, there is also a well-pronounced hysteresis, which does not appear if the film is not compressed to maximum. The plateau observed during compression is repeated during expansion but is shifted to lower surface pressure. This behavior differs from that of pure DPPC and its mixtures with proteins.7,11 With the DPPC/SP-B mixture spread on the captive bubble’s surface, we obtained π/A isotherms as shown in Figure 2. The isotherms obtained at 20 and 30 °C both show a well-pronounced LC/LE plateau that is shifted to higher surface pressures at the higher temperature. The slope of the isotherm during compression starts to change at 0.5-0.45 nm2/molecule, indicating protein squeeze-out. Due to the relatively small amount of protein in the spreading mixtures, there is no pronounced squeeze-out plateau at 20 °C and only a slightly outlined plateau at 30 °C. Above the protein squeeze-out region, the π/A isotherms are similar for both temperatures. In contrast to the π/A isotherms of pure SP-B, there is no rapid breakdown of surface pressure during expansion even after monolayer overcompression, that is, after compressing the layer to a surface coverage lower than the cross section of a vertically orientated DPPC molecule. Nevertheless, a small hysteresis is observed, but actually the π/A slope during expansion repeats that found during compression. Drop Oscillation Experiments. During harmonic oscillation of the pendant drop volume with the DPPC/ SP-B mixture spread, the drop surface and the surface pressure changed as shown in Figure 3. Even with a starting surface pressure of 53 mN/m at 20 °C, π oscillated harmonically. This improvement of the measuring range of the pendant drop microfilm balance, as compared to

Figure 3. Harmonic drop oscillation experiment with DPPC + 0.5 mol % SP-B spread on phosphate buffered saline (pH 7) at 20 °C. The DPPC/SP-B film was compressed before oscillation to a surface pressure of 55.5 mN/m. The amplitude of the drop volume oscillation was (0.25 µL. Surface area (S, blue circles), surface pressure (π, red diamonds), drop volume (V, green triangles).

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Figure 4. (a) Surface dilatational elasticity of SP-B spread on phosphate buffered saline (pH 7) at 20 °C: surface dilatational elasticity, ; surface pressure, π; oscillation frequency, f. (b) Surface dilatational viscosity of SP-B spread on phosphate buffered saline (pH 7) at 20 °C: surface dilatational viscosity, η; surface pressure, π; oscillation frequency, f.

the results given in our preceding report,8 was achieved by using FEP instead of PTFE capillaries. Taking into account the considerable high amplitudes of the surface pressure in this range of π, it became possible to increase the starting surface pressure to about 55 mN/m, where the peaks of the surface pressure definitely exceed the surface pressure of the protein squeeze-out. Therefore, we were able to characterize the pulmonary layers up to surface pressures slightly exceeding the protein squeezeout. The dependencies of the surface dilatational elasticities and viscosities on frequency and surface pressure for pure SP-B films are presented in Figure 4a,b. The surface pressures used for these graphs are the starting values. During drop oscillation, π oscillates around this value. The surface dilatational elasticity of SP-B (Figure 4a) increases to a surface pressure in the range of 20 mN/m where a plateau starts that continues up to 35 mN/m over the whole range of frequencies. From 35 mN/m on, the elasticity continuously increases up to the collapse range. This, however, is in the range of methodical limitations. Because of the quite high surface pressure amplitudes, no further oscillations were possible without destruction of the surface structure. The surface dilatational viscosity of SP-B (Figure 4b) has a maximum in the range of low frequencies, with comparable high viscosities even at low surface pressures. Nevertheless, for higher frequencies the viscosity rapidly drops. In the range of breathing frequencies (0.04-0.2 Hz), the dilatational viscosity

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Figure 5. (a) Surface dilatational elasticity of DPPC + 0.5 mol % SP-B spread on phosphate buffered saline (pH 7) at 20 °C: surface dilatational elasticity, ; surface pressure, π; oscillation frequency, f. (b) Surface dilatational viscosity of DPPC + 0.5 mol % SP-B spread on phosphate buffered saline (pH 7) at 20 °C: surface dilatational viscosity, η; surface pressure, π; oscillation frequency, f.

becomes nearly zero or is at least so small that the resulting surface rheological behavior becomes mainly elastic in the whole range of surface pressures. The dependencies of the surface dilatational elasticities and viscosities on frequency and surface pressure for the DPPC/SP-B mixture are given for 20 and 30 °C in Figures 5 and 6. Temperature influenced elasticity and viscosity quite similarly. The surface dilatational elasticity of DPPC/ SP-B increases up to a surface pressure of about 50-52 mN/m nearly independently of frequency. At surface pressures higher than 52 mN/m, the elasticity starts to decrease again. The increase of elasticity up to the protein squeeze-out, however, is not monotonic. Both the elasticities at 20 and 30 °C pass some kind of plateau where the elasticity remains nearly constant, that is, for 20° in the range of 35-40 mN/m and for 30 °C between 20 and 40 mN/m. The maximum elasticities and the level of the elasticity plateau are much smaller at 30 °C compared to those at 20 °C, suggesting a more flexible and compressible structure at higher temperature. That means the compressibility of the films generally increases with raising temperature. In contrast to the elasticities, the surface dilatational viscosities of DPPC/SP-B strongly depend on frequency with a well-pronounced maximum for low frequencies and very low viscosities at high frequencies for all surface pressures. The maximum values at low frequencies occur in a surface pressure range of about 50 mN/m at 30 °C and at 40-45 mN/m at 20 °C. This range is in good agreement with that of the maximum elasticities for 30

Pulmonary Surfactant Components on a Pendant Drop

Figure 6. (a) Surface dilatational elasticity of DPPC + 0.5 mol % SP-B spread on phosphate buffered saline (pH 7) at 30 °C: surface dilatational elasticity, ; surface pressure, π; oscillation frequency, f. (b) Surface dilatational viscosity of DPPC + 0.5 mol % SP-B spread on phosphate buffered saline (pH 7) at 30 °C: surface dilatational viscosity, η; surface pressure, π; oscillation frequency, f.

°C. At higher surface pressures, the viscosity starts to decrease for both temperatures, as the corresponding elasticities did. The main finding is that independently of temperature and surface pressure the surface dilatational viscosities become almost zero at high frequencies, that is, in the range of the respiration frequencies. That means the deformation of the films is elastic in this particular range. Discussion As an attempt to understand in more detail the relationship between stress and strain of the alveolar lining surface layer during breathing, we characterized the rheological behavior of single pulmonary surfactant components, DPPC,7 DPPG,9 SP-C/DPPC,8,11 and SP-B and SP-B/DPPC. Our previous data determined by stress relaxation experiments show that the main relaxation times of DPPC7 and DPPG9 monolayers are outside the range relevant for respiration. There are, however, data from Panaiotov et al.20 showing relaxation times for pulmonary surfactant in the range of some seconds that may be important for respiration. Therefore, a more detailed investigation was carried out by harmonic oscillation in a captive bubble device for DPPC/SP-C in the range of the main relaxation (20) Panaiotov, I.; Ivanova, T.; Proust, J.; Boury, F.; Denizot, B.; Keough, K.; Taneva, S. Colloids Surf., B 1996, 6, 243-260.

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times of DPPC (6 × 10-3 to 2.5 × 10-2 Hz).11 This is at the present time the only frequency range available for this method.8 Using this experimental design, some brittle surface structures in the range of surface pressure between 20 and 40 mN/m were found for spread DPPC layers. Both dilatational elasticities and viscosities have two maxima, one before and the other above the protein squeeze-out plateau. Furthermore, in the preceding part of the present report we have shown for the first time that the surface dilatational viscosity drastically decreases in the frequency range of 0.05-0.5 Hz for spread DPPC/SP-C monolayers.8 Our isotherms for SP-B show slopes similar to those from Taneva and Keough,15 although it has to be considered that surface pressures higher than 45 mN/m are hardly realized in a Langmuir trough. A direct comparison of our isotherm with those found in the literature is not easy. The shape of the SP-B isotherm depends on how the protein was purified, as pointed out by Taneva and coworkers.15 The plateaus recorded in our SP-B isotherm may be caused by aggregation and/or molecular rearrangement processes and will be a topic of future investigation. There is rapid breakdown of surface pressure during expansion of SP-B surface films, when ruptures of surface aggregates and collapsed structures occur.21 We found such breakdown of surface pressure after layer overcompression only with pure SP-B but not with the DPPC, DPPC/SP-B, and DPPC/SP-C. We interpreted these differences of film behavior by different monolayer folding during overcompression10 for DPPC and DPPC/SP-C, which is in agreement with findings of Lee et al.22 A second explanation is that liposome formation occurs, which is only possible if DPPC or DPPC with the hydrophobic proteins is present at the interface, but not for SP-B alone. The π/A isotherms of the DPPC/SP-B mixture show at which surface pressures the LE/LC transition occurs and at which surface pressure the protein squeeze-out becomes evident. The π/A isotherms of the DPPC/SP-B mixture are qualitatively similar to those of DPPC and DPPC/ SP-C mixtures reported before.8,11 The small hysteresis between the compression and expansion curves and the absence of a rapid breakdown of film pressure after overcompression support our earlier data and the interpretation that monolayer folding and liposome formation may occur. It also favors re-entering of proteins in the case of monolayer expansion.8 The temperature dependence corresponds to that known for lipid and lipid/protein systems reported elsewhere.8,23 The LE/LC transition starts at π of about 10 mN/m at 20 °C, which corresponds to that known for DPPC monolayers, and is shifted to 30 mN/m at 30 °C. The protein squeeze-out becomes evident at surface pressures exceeding 52 mN/m, which is very similar to those reported for DPPC/SP-C.11 For our oscillation experiments with the pendant drop microfilm balance, we tested how strongly the spread layer may be compressed without destroying the layer structure8 and without wetting the outer capillary wall.7 Using a FEP capillary, monolayers spread on the pendant drop surface could be compressed and oscillated up to surface pressures that exceed the pressures necessary for protein squeeze-out. This is not high enough to investigate DPPC/ (21) Vollhardt, D. Adv. Colloid Interface Sci. 1996, 64, 143-169. (22) Lee, K. Y. C.; von Nahmen, A.; Lipp, M.; Gopal, A.; Takamoto, D. Y.; Ding, J.; Waring, A. J. Proceedings of “Amphiphiles at Interfaces, From Structure Control to Properties”, May 24-28, 1999, Beijing, China. (23) Albrecht, O.; Gruler, H; Sackmann, E. J. Phys. (Paris) 1978, 39, 301-313.

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SP-B layers in the overcompression state but is sufficient to compare our results with those of DPPC/SP-C.11 The surface rheological behavior reported for pulmonary surfactant components is interpreted with the π/A isotherms in mind. Harmonic oscillation experiments with DPPC alone and DPPC/SP-C show two well-pronounced steps of maximum elasticities at 20 °C, that is, one at about 120 mN/m for low frequencies and the other at about 200 mN/m for higher frequencies.8 Harmonic oscillation experiments with DPPC/SP-B compared with those for DPPC/SP-C revealed similarities but also particularities, which are caused by the two different hydrophobic surfactant proteins. The surface dilatational behavior of spread SP-B layers fits well into the interpretation given in the preceding part of our paper. The maximum surface dilatational elasticities of SP-C alone seem to be lower than that of SP-B, but it should be taken into account that the maximum surface pressure used for SP-C was 35 mN/m and no plateau region was observed for elasticity up to this surface pressure.11 The maximum dilatational viscosities for both SP-B and SP-C are in the range of 200 mNs/m even at relatively low surface pressures.11 The dilatational elasticity increases with surface pressure and passes a maximum just before the range of protein squeeze-out. The maximum elasticities of the present system at 20 °C nearly agree with those of DPPC/SP-C. At 30 °C, however, the elasticity is about 200 mN/m in comparison to 120 mN/m for DPPC/SP-C. The influence of SP-C on surface film elasticity facilitates a more balanced surface mechanical behavior, which is distinguished by higher elasticity at low frequencies corresponding to lower film compressibility for the mixture.8 The effect of SP-B on the surface behavior of DPPC layers seems to be similar. It also increases the elasticity at low frequencies. This influence is also reflected by the occurrence of a well-established plateau of the elasticity at about 200 mN/m at 20 °C and 80 mN/m at 30 °C (Figure 5a and 6a), where a DPPC layer without protein is distinguished by very brittle film structures with irregular changes of the elasticity in a comparable range of surface pressure (see first part of this report). In the presence of one of the hydrophobic surfactant proteins in the monolayer, such irregularities nearly disappear over the whole range of frequencies investigated at 20 °C and vanish completely at 30 °C. Such changes of surface elasticity support the concept of strong molecular interactions between the hydrophobic surfactant proteins with the phospholipid surface film. However, what part of the change is caused simply by the mixing and what may be caused by specific interactions could not be learned from our data. The concept of very strong molecular interactions between the hydrophobic surfactant proteins and the lipid surface film is also supported by our surface dilatational viscosity data. In addition, the different surface dilatational viscosity of DPPC/SP-B films compared to those of all other surfactant components analyzed so far points to structural features that are specific for SP-B. With SPB/DPPC films, the viscosity becomes about 500 mNs/m at

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20 °C and 400 mNs/m at 30 °C in the range below 0.01 Hz. These values are about twice as much as those found for DPPC or DPPC/SP-C layers.7,8,11 Such high viscosities found for SP-B/DPPC films at low frequencies correspond to deformation of mechanically very stable monolayers. Such a behavior could not be explained by electrostatic interaction, since the PI values of SP-B and SP-C are quite similar for both proteins, ranging between 8 and 9.24 The dominating difference therefore seems to be caused by different structural features of the two proteins. In contrast to SP-C, native SP-B is a dimer, which is formed by covalent cysteine bonds. This might have a stronger impact on the mechanical layer stability than the monomeric and smaller SP-C. Nag et al.25 considered that SP-C may decrease the size of different surfactant structural assemblies and promote the transfer of tensoactive molecules between the bulk and the interface. We assume that the possible binding of squeezed-out aggregates (liposomes) to the interface by SP-B14 may lead to a considerable impact on the dilatational surface viscosity of the mixed lipid/ protein layers. Perez-Gil et al. brought forward the view that compressed SP-C-containing monolayers are solid enough to stabilize the respiratory surface and, simultaneously, flexible enough to elastically recuperate when expanded.17 Obviously, this view also holds for DPPC/SP-B films, especially as the dilatational surface viscosity drastically reduces at higher frequencies. This is our main finding: the surface rheological behavior of pulmonary lining surfaces is strongly frequency dependent and becomes mainly elastic at frequencies that resemble mammalian respiration. Both mixtures of DPPC, the main phospholipid, and SP-C or SP-B, the hydrophobic surfactant proteins, cause high surface dilatational elasticities but very low dilatational viscosities in the range of frequencies higher 0.04 Hz. Up to the starting protein squeeze-out, the elasticities become very high, which corresponds to low compressibility of films. In the range of protein squeeze-out, the elasticity decreases again, and the surface film becomes more compressible. This rheological behavior of the pulmonary surfactant film is the prerequisite for reducing the mechanical work of breathing. We conclude that films formed by pulmonary surfactant components are mainly elastic at breathing frequencies. Both surfactant proteins, SP-B and SP-C, balance the surface rheological behavior forming films of low compressibility below protein squeezeout and highly compressible films in the range of the protein squeeze-out. Acknowledgment. The financial support by the Deutsche Forschungsgemeinschaft (Grant Pi 165/7, Wu¨ 187/6, Wu¨ 187/8) is gratefully acknowledged. Many thanks to Klaus Dannenberg from Virchow-Klinikum, Werkstatt, for helping us build the experimental setups. LA011216X (24) Perez-Gil, J.; Keough, K. M. W. Biochim. Biophys. Acta 1998, 1408, 203-217. (25) Nag, K.; Perez-Gil, J.; Cruz, A.; Keough, K. M. W. Biophys. J. 1996, 71, 246-256.