Am J Physiol Lung Cell Mol Physiol 283: L897–L906, 2002;
10.1152/ajplung.00431.2001.
Content-dependent activity of lung surfactant protein B
in mixtures with lipids
Z. WANG,1 J. E. BAATZ,2 B. A. HOLM,3,4 AND R. H. NOTTER1,5,6
Departments of Pediatrics, 5Environmental Medicine, and 6Chemical Engineering, University of
Rochester, Rochester 14642; 3Departments of Pediatrics and 4Obstetrics and Gynecology, State
University of New York at Buffalo, Buffalo, New York 14214; and 2Department of Pediatrics,
Medical University of South Carolina, Charleston, South Carolina 29425
1
Received 2 November 2001; accepted in final form 18 March 2002
surfactant proteins; lung surfactant; exogenous surfactants;
calf lung surfactant extract
is a complex mixture of lipids and three biophysically active proteins:
surfactant protein (SP)-A, SP-B, and SP-C. The physiological necessity of surfactant in respiratory function
has led to extensive research on the biophysics, cell and
molecular biology, and physiology of this complex
material (11, 20, 34, 38). Although lipids (primarily
phospholipids) comprise the great majority of lung surfactant in a quantitative sense, the apoprotein constituents of the surfactant system are highly active and
ENDOGENOUS PULMONARY SURFACTANT
Address for reprint requests and other correspondence: R. H.
Notter, Dept. of Pediatrics (Neonatology, Box 850), Univ. of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642.
http://www.ajplung.org
crucial in a functional sense. This is also the case in
most of the exogenous surfactant preparations used in
the therapy of the neonatal and acute respiratory distress syndromes. The hydrophobic surfactant proteins
SP-B and SP-C both have strong molecular interactions with lipids. Because of its combination of polar
and nonpolar residues and overall amphipathic structure, SP-B can interact with both phospholipid head
groups and fatty chains and is particularly active in
enhancing surface active behavior in endogenous and
exogenous lung surfactants (13, 25, 34, 44, 46, 48, 56,
58). However, the content-dependent effects of SP-B on
the surface and physiological activity of lipids have not
been fully quantitated.
A better understanding of SP-B’s content-dependent
actions in mixtures with lipids is directly relevant for
the function of animal-derived clinical exogenous surfactants, which vary significantly in their compositional levels of this apoprotein (4, 19, 39, 47). The
present study examines the effects of ascending
amounts of bovine SP-B in increasing the adsorption
and dynamic surface activity of 1,2 dipalmitoyl-sn-3phosphocholine (DPPC), mixed synthetic lipids (SL),
and purified surfactant phospholipids (PPL) isolated
by gel permeation chromatography from calf lung surfactant extract (CLSE). Adsorption is measured in an
apparatus with a stirred subphase to remove diffusion
resistance, and dynamic surface tension lowering is
assessed during cycling at rapid physiological rates in
a pulsating bubble surfactometer. Surface pressurearea (␲-A) isotherms and dynamic respreading are also
defined for solvent-spread phospholipid-SP-B films at
the air-water interface in a Wilhelmy surface balance.
In addition to surfactant biophysical studies, the content-dependent activity of SP-B combined with synthetic lipids is examined in a well-characterized excised rat lung model used in the quality control
manufacture of several Food and Drug Administrationapproved clinical exogenous surfactants (Survanta, Infasurf) (3). Biophysical and excised lung experiments
are used to study the activity of SP-B contents increThe costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society
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Wang, Z., J. E. Baatz, B. A. Holm, and R. H. Notter.
Content-dependent activity of lung surfactant protein B in
mixtures with lipids. Am J Physiol Lung Cell Mol Physiol
283: L897–L906, 2002; 10.1152/ajplung.00431.2001.—The
content-dependent activity of surfactant protein (SP)-B was
studied in mixtures with dipalmitoyl phosphatidylcholine
(DPPC), synthetic lipids (SL), and purified phospholipids
(PPL) from calf lung surfactant extract (CLSE). At fixed SP-B
content, adsorption and dynamic surface tension lowering
were ordered as PPL/SP-B ⬇ SL/SP-B ⬎ DPPC/SP-B. All
mixtures were similar in having increased surface activity as
SP-B content was incrementally raised from 0.05 to 0.75% by
weight. SP-B had small but measurable effects on interfacial
properties even at very low levels ⱕ0.1% by weight. PPL/
SP-B (0.75%) had the highest adsorption and dynamic surface activity, approaching the behavior of CLSE. All mixtures
containing 0.75% SP-B reached minimum surface tensions
⬍1 mN/m in pulsating bubble studies at low phospholipid
concentration (1 mg/ml). Mixtures of PPL or SL with SP-B
(0.5%) also had minimum surface tensions ⬍1 mN/m at 1
mg/ml, whereas DPPC/SP-B (0.5%) reached ⬍1 mN/m at 2.5
mg/ml. Physiological activity also was strongly dependent on
SP-B content. The ability of instilled SL/SP-B mixtures to
improve surfactant-deficient pressure-volume mechanics in
excised lavaged rat lungs increased as SP-B content was
raised from 0.1 to 0.75% by weight. This study emphasizes
the crucial functional activity of SP-B in lung surfactants.
Significant differences in SP-B content between exogenous
surfactants used to treat respiratory disease could be associated with substantial activity variations.
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CONTENT-DEPENDENT SP-B ACTIVITY
mentally varying from 0.05 to 0.75% by weight in
mixtures with lipids.
MATERIALS AND METHODS
AJP-Lung Cell Mol Physiol • VOL
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Synthetic phospholipids and lipids. Synthetic DPPC, egg
phosphatidylcholine (egg PC), egg phosphatidylglycerol (egg
PG), egg phosphatidylinositol (egg PI), egg phosphatidylethanolamine (egg PE), and bovine brain sphingomyelin (Sph)
for activity studies were purchased from Avanti Polar Lipids
(Alabaster, AL). Cholesterol was reagent grade from Sigma
(St. Louis, MO). DPPC was ⬎99% pure as supplied and gave
a single well-defined spot on thin-layer chromatography with
solvent system C of Touchstone et al. (51). Egg PC, egg PG,
egg PI, egg PE, and Sph gave broader single spots on thinlayer analysis, consistent with their content of multiple fatty
chain derivatives. Activity experiments utilized either pure
DPPC or SL, the latter roughly based on the lipid composition of lavaged lung surfactant (SL ⫽ 40:40:10:5:2.5:2.5
molar ratio DPPC/egg PC/egg PG/egg PI/egg PE/Sph ⫹ 5%
cholesterol).
CLSE and PPL. Mixed phospholipids were isolated by gel
permeation chromatography from solvent-extracted, lavaged
calf lung surfactant (18). Intact lungs from freshly killed
calves (Gold Medal Packing, Rome, NY) were lavaged with
2–3 l of cold 0.15 M NaCl. Lavage fluid was immediately
centrifuged at 12,000 g to obtain whole surfactant, which was
then extracted with chloroform-methanol (5) to obtain CLSE.
The composition of CLSE by weight was 93% phospholipid,
4.5% cholesterol, 1.5% protein (a mixture of SP-B and SP-C),
and 1% other. The phospholipid class distribution was ⬃85%
PC, 5% PG, 4% PI (including phosphatidylserine), 3% PE, 1%
Sph, and 2% other (51). ELISA testing using a monoclonal
antibody to bovine SP-B (rabbit derived) has reported a level
of 0.9% SP-B by weight relative to phospholipid in CLSE
material similar to that used here (39). Mixed lung surfactant phospholipids (PPL) were isolated from CLSE by column
chromatography with an elution solvent of 1:1 (vol/vol) chloroform-methanol plus 5% 0.1 N HCl (18). Two passes through
a Sephadex LH-20 column (Pharmacia-LKB Biotechnology,
Piscataway, NJ) separated phospholipids away from hydrophobic surfactant apoproteins and neutral lipids. Acid remaining in PPL fractions was removed by a second chloroform-methanol extraction. Final PPL fractions had a protein
content below the limits of detection of both the Folin-phenol
reagent assay of Lowry et al. (27) and the amido black assay
of Kaplin and Pedersen (26) modified by the addition of
5–15% sodium dodecyl sulfate (SDS).
Purified SP-B. SP-B was purified from CLSE on a 450-ml
Silica C8 column with 7:1:0.4 methanol-chloroform-0.1 N HCl
or 7:1:0.4 methanol-chloroform-H2O ⫹ 0.1% trifluoroacetic
acid as elution solvents. Eluted fractions from the first and
second column passes were screened by an ultraviolet detector (Pharmacia Biotech, Uppsala, Sweden) at 254 nm, and
final pooled SP-B isolates were analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and NH2-terminal
amino acid analysis. Fractions for SDS-PAGE were dried,
resuspended in NuPage sample buffer (Novex, San Diego,
CA), and applied to 4–10% gradient acrylamide NuPage gels.
Electrophoresis was at 200 V for 40 min with a 2-(N-morpholino)ethanesulfonic acid buffer (MES; Novex) containing (in
mM) 50 MES, 50 Tris base, 3.5 SDS, and 1 EDTA, pH 7.7.
Silver staining for detection of protein bands was according
to Morrissey (29). NH2-terminal sequence analysis (7 cycles)
was done with an Applied Biosystems Procise amino acid
analyzer (pure SP-B yielded a single sequence of Phe-Pro-IlePro-Ile-Pro-Tyr). SP-B fractions were also analyzed by West-
ern blots using rabbit anti-bovine SP-B antibody. Proteins for
Western blots were transferred from gels to 0.22-␮m nitrocellulose using a Novex electrotransfer unit with current
preset at 3 mA/cm2 and a 60-min transfer time. After transfer, the nitrocellulose was blocked in 25 mM Tris base buffer,
pH 7.7, containing 0.05% vol/vol Tween 20 and 5% wt/vol
nonfat dry milk (Blotto), followed by three 15-min washes
with 25 mM Tris base and 0.05% Tween 20, pH 7.7. The
nitrocellulose was then incubated in primary polyclonal antibody (rabbit anti-bovine SP-B antibody) in Blotto (1:10,000
dilution) for 60 min, washed several times with Tris-Tween
buffer, incubated for 30 min with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:2,500 dilution; Santa Cruz
Biotechnology, Santa Cruz, CA), washed with 25 mM Tris
base (pH 7.7), and incubated with Luminol reagent (Santa
Cruz Biotechnology) for 60 s followed by removal of excess
reagent and film development.
Lipid-SP-B mixture formulation. DPPC, PPL, or SL were
combined with SP-B as follows. Appropriate amounts of lipids dissolved in chloroform were initially mixed with SP-B in
chloroform-methanol and evaporated to dryness under nitrogen. Amounts of phospholipid were determined by phosphate
assay (7), and levels of SP-B protein were based on the
Folin-phenol reagent assay of Lowry et al. (27) modified by
the addition of 15% SDS. Dried lipid-SP-B mixtures were
redissolved in 9:1 vol/vol hexane-ethanol (HPLC grade) for
Wilhelmy balance film studies or were dispersed at desired
concentrations in buffered saline (10 mM HEPES, 1.5 mM
CaCl2, 150 mM NaCl, pH 7.0) by probe sonication for adsorption, pulsating bubble, or excised lung studies. Dispersion
was with a Heat Systems sonicator (model W-220F) at 40 W
of power applied for three to five 15-s bursts on ice. Highly
pure distilled and deionized water (Milli-Q UV Plus system;
Millipore, Bedford, MA) was used in all dispersions.
Adsorption methods. Adsorption was measured at 37 ⫾
0.5°C for surfactant dispersions in a Teflon dish containing a
35-ml buffered subphase (10 mM HEPES, 1.5 mM CaCl2, 150
mM NaCl, pH 7.0) (33, 35). The subphase was agitated
continuously by a Teflon-coated bar and magnetic stirrer to
minimize diffusion resistance. At time zero, a bolus of surfactant dispersion (2.5 mg phospholipid/5 ml buffer) was injected into the stirred subphase, and surface pressure (surface tension lowering below that of the pure subphase) was
measured as a function of time by a sandblasted platinum
slide connected to a force transducer. Final subphase concentration for adsorption studies was uniform at 0.063 mg phospholipid/ml (2.5 mg/40 ml of total subphase).
Pulsating bubble surfactometer methods. The ability of
surfactant mixtures to lower surface tension during rapid
cycling at 37°C was measured using a pulsating bubble
surfactometer (General Transco, Largo, FL; formerly Electronetics, Amherst, NY) (15). An air bubble, communicating
with ambient air, was formed in a buffered suspension of
surfactant held in a 40-␮l plastic sample chamber. The sample chamber was mounted on the instrument pulsator unit,
and the bubble was repetitively oscillated between maximum
and minimum radii of 0.55 and 0.4 mm at a rate of 20
complete cycles of compression-expansion per minute. The
pressure drop across the air-water interface was measured
by a precision pressure transducer. Surface tension at minimum bubble radius (minimum surface tension) was calculated as a function of time from the spherical form of the
Laplace equation (16). Surfactant dispersions were studied in
the pulsating bubble apparatus at concentrations of both 1
and 2.5 mg phospholipid/ml to increase the sensitivity of
measurements to discern the relative activity of the different
mixtures studied.
CONTENT-DEPENDENT SP-B ACTIVITY
AJP-Lung Cell Mol Physiol • VOL
RESULTS
The adsorption of mixtures of DPPC/SP-B, SL/SP-B,
and PPL/SP-B at 37°C is shown in Fig. 1, A–C, respectively. At fixed SP-B content, the rate of adsorption was
generally most rapid for mixtures containing PPL and
SL, followed by DPPC. However, the variation of adsorption as a function of SP-B content was very similar
in all three sets of mixtures. Very low amounts of SP-B
(ⱕ0.1% by weight) had a measurable effect in improving adsorption relative to lipids alone. However, the
rate and magnitude of adsorption increased continuously as the weight percentage of SP-B was incrementally raised up to 0.75%. PPL/SP-B containing 0.75%
apoprotein by weight reached equilibrium surface
pressures of 48.5 ⫾ 0.5 mN/m (surface tensions of just
under 22 mN/m) after 20 min of adsorption (Fig. 1C).
CLSE, which contains all the components in PPL/SP-B
plus cholesterol and SP-C, reached the same final equilibrium adsorption surface pressure but at a faster rate
(Fig. 1C). Mixtures of SL/SP-B (0.75%) and DPPC/SP-B
(0.75%) reached slightly lower final adsorption surface
pressures than PPL/SP-B (0.75%). Mixtures of lipids
with 0.5% SP-B by weight adsorbed less rapidly than
corresponding mixtures containing 0.75% SP-B. However, final surface pressures for mixtures containing
0.5% SP-B were only slightly lower than for those
containing 0.75% SP-B (Fig. 1, A–C).
The surface tension lowering of mixtures of DPPC/
SP-B, SL/SP-B, and PPL/SP-B during dynamic compression in pulsating bubble experiments is shown in
Figs. 2–4. The pattern of activity in these dynamic
studies also showed successive improvements as the
content of SP-B was raised from 0.05 to 0.75% by
weight relative to phospholipid. As in the adsorption
results above, overall dynamic surface activity was
generally ordered as PPL/SP-B ⬇ SL/SP-B ⬎ DPPC/
SP-B in terms of the time course and/or magnitude of
surface tension lowering. This order of dynamic surface
activity was found at both phospholipid concentrations
studied (1 and 2.5 mg/ml). PPL/SP-B mixtures containing 0.75% by weight of apoprotein to phospholipid
reached minimum surface tensions ⬍1 mN/m almost
as rapidly as CLSE at a low phospholipid concentration
of 1 mg/ml (Fig. 4A). Mixtures of SL or DPPC with
0.75% SP-B also reached very low minimum surface
tensions ⬍1 mN/m at 1 mg phospholipid/ml (Figs. 2A,
3A). Minimum surface tension values ⬍1 mN/m were
also attained by mixtures of PPL/SP-B (0.5%) and
SL/SP-B (0.5%) at a phospholipid concentration of 1
mg/ml, and by DPPC/SP-B (0.5%) at a higher phospholipid concentration of 2.5 mg/ml (Figs. 2–4).
In addition to investigating surface activity in dispersed phospholipid/SP-B mixtures, apoprotein effects
were also studied directly in spread interfacial films in
the Wilhelmy balance. In particular, dynamic respreading during cycling was examined in films of
DPPC/SP-B and PPL/SP-B spread to an initial surface
concentration of 15 Å2/molecule. This surface concentration substantially exceeds monolayer coverage of
the interface and emphasizes film properties associ-
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Wilhelmy balance methods. ␲-A isotherms were measured
for solvent-spread interfacial films in a modified Wilhelmy
surface balance incorporating a Teflon trough and ribbon
barrier (49). Surfactants dissolved in hexane-ethanol (9:1,
vol/vol) were spread dropwise from a syringe at the surface of
a buffered subphase (10 mM HEPES, 1.5 mM CaCl2, 150 mM
NaCl, pH 7.0) in the balance trough. Surfactant films were
spread to a uniform initial “surface excess” concentration of
15 Å2/molecule to examine film behavior during dynamic
cycling in the collapse regime. Typical volumes of spreading
solvent were 0.4 ml, with a 10-min pause allowed for solvent
evaporation before dynamic cycling. Surface pressure ␲ (the
amount by which surface tension was lowered below that of
the pure subphase) was measured during cycling from the
force on a sand-blasted platinum Wilhelmy slide dipped into
the ribbon-confined interface. A second slide outside the
ribbon barrier assessed film leakage, which was absent in all
isotherm data reported. Cycling was between maximum and
minimum areas of 448 and 103 cm2 (compression ratio
4.35:1) at 5 min per cycle at 23 ⫾ 1°C. Respreading of
molecules squeezed out of the interfacial film during compression was assessed from collapse plateaus on the ␲-A
compression curves for cycles 1, 2, and 7, as defined previously
by Notter and co-workers (34, 37, 57). A value of zero for the
ratio of compression isotherm collapse plateaus on cycle 2/
cycle 1 or cycle 7/cycle 1 indicated no respreading between the
designated compressions, whereas a collapse plateau ratio of
one indicated complete respreading of film material.
Excised lung pressure-volume methods. Physiological activity was determined by measuring quasistatic pressurevolume (P-V) deflation mechanics in an excised rat lung
model at 37°C (3). The thermodynamic consistency of P-V
measurements in this model has been documented in multiple studies (see Ref. 34 for review). Adult Sprague-Dawley
male rats (400–550 g; Charles River, Wilmington, MA) were
given a lethal intraperitoneal bolus of pentobarbital sodium,
and lungs were excised and degassed under vacuum. The
lungs were attached to an artificial trachea in an environmental chamber and rapidly inflated with air (Harvard small
animal respirator, 24.7 ml/min). Inflation was to 30 cmH2O,
followed by a 10–15 min period of stress relaxation to standardize total lung capacity (TLC) and rule out air leakage.
The lungs were then deflated from TLC at a slow rate of 2.47
ml/min to define normal surfactant-sufficient P-V mechanics.
Endogenous surfactant was removed by 15 lavages with cool
0.15 M NaCl, followed by degassing, reinflation to 30 cmH2O,
stress relaxation, and a second deflation curve to define the
surfactant-deficient state. A surfactant mixture in 0.15 M
NaCl was then instilled via the trachea at a uniform total
dose of 100 mg phospholipid/kg rat body wt, and a third P-V
deflation curve was measured with identical methods to
determine the degree of restoration of mechanics toward
normal. The concentration of instilled surfactant mixtures
was constant at 25 mg/ml. Percent volume recovery at
selected transpulmonary pressures was calculated as
100(Vsurf ⫺ Vdef)/(Vnorm ⫺ Vdef), where Vnorm is the volume of
the freshly excised surfactant-sufficient lungs, Vdef is the
volume of the lavaged surfactant-deficient lungs, and Vsurf is
the volume after surfactant instillation at the pressure of
interest.
Statistical analyses. All results are expressed as means ⫾
SE. Statistical analyses used the Student’s t-test for comparisons of discrete data points, and functional data were analyzed by one-way analysis of variance with Scheffé’s procedure identifying points of significant difference. Differences
were considered statistically significant if the probability of
the null hypothesis was ⬍0.05.
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CONTENT-DEPENDENT SP-B ACTIVITY
Fig. 1. Effects of increasing contents of surfactant protein (SP)-B on
the adsorption of dipalmitoyl phosphatidylcholine (DPPC), synthetic
lipids (SL), and column-purified lung surfactant phospholipids
(PPL). A: DPPC/SP-B mixtures; B: SL/SP-B mixtures; C: PPL/SP-B
mixtures. SP-B had consistent content-dependent effects in enhancing adsorption in mixtures with DPPC, SL, and PPL. Also shown for
comparison in C is calf lung surfactant extract (CLSE), which contains all of the hydrophobic components of lavaged calf lung surfactant. Adsorption measurements were made in a Teflon dish with a
stirred subphase at 37°C (see MATERIALS AND METHODS). Final subphase surfactant concentration was uniform at 0.063 mg phospholipid/ml. Data are means ⫾ SE for n ⫽ 3–5.
AJP-Lung Cell Mol Physiol • VOL
ated with compression past collapse. Representative
surface excess ␲-A isotherms for DPPC/SP-B films at
apoprotein contents of 0, 0.1, 0.25, 0.5, and 0.75% by
weight relative to phospholipid are shown in Fig. 5. As
SP-B content is raised, there is a successively larger
shift to the right in the second and/or seventh compression curves on these representative isotherms, indicative of increased film respreading. Respreading calculations for DPPC/SP-B and PPL/SP-B films based on
collapse plateau ratios from multiple isotherms at different apoprotein contents are given in Table 1. PPL/
SP-B films had significantly better respreading than
corresponding DPPC/SP-B films at fixed apoprotein
content, reflecting the much better respreading of the
complete mix of lung surfactant phospholipids relative
to DPPC alone. Very low SP-B contents of ⱕ0.1% by
weight improved respreading in films with DPPC,
whereas effects on PPL film respreading were small.
However, all phospholipid/SP-B films exhibited a similar conceptual pattern of increased dynamic respreading as SP-B content was incrementally increased to
0.75% (Table 1).
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Fig. 2. Effects of SP-B on dynamic surface tension lowering in
mixtures with DPPC. A: 1 mg phospholipid/ml; B: 2.5 mg phospholipid/ml. Surface tension lowering in DPPC/SP-B mixtures on a
pulsating bubble surfactometer (20 cycles/min, 50% area compression, 37°C) was successively improved as apoprotein content increased from 0.05 to 0.75% by weight. Data are means ⫾ SE for n ⫽
3–5.
CONTENT-DEPENDENT SP-B ACTIVITY
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DISCUSSION
Fig. 3. Effects of SP-B on dynamic surface tension lowering in
mixtures with SL. A: 1 mg phospholipid/ml; B: 2.5 mg phospholipid/
ml. Surface tension lowering during dynamic cycling is shown for
mixtures of SL/SP-B from pulsating bubble studies. Experimental
details are the same as in the legend to Fig. 2. The composition of SL is
given in MATERIALS AND METHODS. Data are means ⫾ SE for n ⫽ 3–5.
Physiological activity experiments addressed the effects of tracheally instilled SL/SP-B mixtures on P-V
deflation mechanics in excised, lavaged rat lungs. The
lipid mixture in these excised lung studies had a broad
distribution of lipid components superficially related to
those in lavaged lung surfactant (SL ⫽ 40:40:10:5:2.5:
2.5 DPPC/egg PC/egg PG/egg PI/egg PE/Sph ⫹ 5%
cholesterol). As found in biophysical studies, the activity of SL/SP-B mixtures in improving surfactant-deficient P-V deflation mechanics increased as apoprotein
content was raised from 0.1 to 0.75% (Fig. 6, Table 2).
Small but measurable improvements in surfactantdeficient mechanics were present for SL/SP-B mixtures
with low apoprotein contents of 0.1–0.2% by weight
relative to phospholipid. SL/SP-B (0.3% by weight) had
a significantly increased activity in improving surfactant-deficient mechanics, and activity at low transpulmonary pressures was increased further in SL/SP-B
(0.5%) and SL/SP-B (0.75%) mixtures. The activity of
SL/SP-B (0.75% by weight) in lavaged excised rat lungs
approached but did not fully equal that of CLSE (Fig. 6,
Table 2).
AJP-Lung Cell Mol Physiol • VOL
Fig. 4. Effects of SP-B on dynamic surface tension lowering in
mixtures with PPL. A: 1 mg phospholipid/ml; B: 2.5 mg phospholipid/
ml. Surface tension lowering during dynamic cycling on the pulsating bubble apparatus is shown for PPL/SP-B mixtures. Experimental
details are the same as in Figs. 2 and 3. Data are means ⫾ SE for n ⫽
3–5.
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This study has examined the content-dependent effects of surfactant protein SP-B on the adsorption,
dynamic surface behavior, and pulmonary mechanical
activity of lipids. Biophysical and physiological studies
on three different lipid/SP-B mixtures were consistent
in demonstrating substantial activity differences as a
function of apoprotein content. At fixed SP-B content,
adsorption and dynamic surface tension lowering for
the mixtures studied were generally ordered as PPL/
SP-B ⬇ SL/SP-B ⬎ DPPC/SP-B. However, all mixtures
gave similar patterns of surface activity changes with
SP-B content. Very low SP-B contents ⱕ0.1% by weight
had a measurable effect in increasing the adsorption,
dynamic surface tension lowering, and film respreading of DPPC, SL, and/or column-purified lung surfactant phospholipids (Figs. 1–4, Table 1). However, surface activity successively increased as SP-B content
was incrementally raised to 0.75% by weight relative to
phospholipid. The adsorption and dynamic surface tension lowering of PPL/SP-B (0.75%) were only slightly
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CONTENT-DEPENDENT SP-B ACTIVITY
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Fig. 5. Surface pressure-area (␲-A)
isotherms for mixtures of DPPC with
bovine SP-B. A–E: ␲-A isotherms for
solvent-spread films containing SP-B
contents of 0.1–0.75% by weight relative to DPPC. Graphs show the compression curves for cycles 1, 2, and 7,
and the expansion curve for cycle 1.
Respreading ratios calculated from
multiple isotherms at fixed SP-B content are given in Table 1. All films were
spread to an initial concentration of 15
Å2/molecule at the air-water interface
in a Wilhelmy balance at 23°C (see
MATERIALS AND METHODS).
less than CLSE, a highly active surfactant extract
containing the complete mix of lipids and hydrophobic
proteins from lavaged calf lung surfactant (18, 21, 33,
34, 38) (Figs. 1C and 4). Mixtures of SL/SP-B (0.75%)
and DPPC/SP-B (0.75%) also adsorbed rapidly and
reached low minimum surface tensions ⬍1 mN/m during dynamic cycling at a low phospholipid concentration of 1 mg/ml in pulsating bubble studies (Figs. 1–3).
Mixtures of PPL, SL, or DPPC with 0.5% SP-B had
reduced rates of adsorption and dynamic surface tension lowering, but did reach minimum surface tensions
⬍1 mN/m during prolonged cycling (Figs. 1–4).
Wilhelmy balance experiments on dynamic respreading in cycled interfacial films also displayed the
AJP-Lung Cell Mol Physiol • VOL
content-dependent effects of SP-B in mixtures with
phospholipids (Fig. 5, Table 1). Variations in respreading as a function of SP-B content in interfacial films at
room temperature in the Wilhelmy balance were conceptually similar to those in adsorption and pulsating
bubble studies on surfactant dispersions at 37°C. Even
very low SP-B contents ⱕ0.1% by weight impacted the
respreading of DPPC, and this film property then improved further as levels of SP-B were increased from
0.1 to 0.75% (Table 1). Previous studies have also
documented the very poor respreading of pure DPPC in
cycled surface films (e.g., Refs. 37, 52), which may
make the effects of low levels of SP-B more apparent.
The complete mix of rigid and fluid surfactant phos-
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CONTENT-DEPENDENT SP-B ACTIVITY
Table 1. Effects of SP-B on the respreading of DPPC
and PPL in dynamically cycled surface films
Respreading (Collapse Plateau) Ratios
Cycle 2/Cycle 1
Cycle 7/Cycle 1
DPPC
⫹ 0.05% SP-B
⫹ 0.1% SP-B
⫹ 0.25% SP-B
⫹ 0.5% SP-B
⫹ 0.75% SP-B
0.35 ⫾ 0.02
0.44 ⫾ 0.02
0.50 ⫾ 0.02
0.52 ⫾ 0.01
0.51 ⫾ 0.02
0.52 ⫾ 0.02
0.10 ⫾ 0.0
0.10 ⫾ 0.01
0.15 ⫾ 0.01
0.25 ⫾ 0.01
0.32 ⫾ 0.03
0.37 ⫾ 0.03
PPL
⫹ 0.05% SP-B
⫹ 0.1% SP-B
⫹ 0.25% SP-B
⫹ 0.5% SP-B
⫹ 0.75% SP-B
0.54 ⫾ 0.01
0.56 ⫾ 0.01
0.61 ⫾ 0.01
0.64 ⫾ 0.02
0.72 ⫾ 0.01
0.73 ⫾ 0.05
0.19 ⫾ 0.01
0.20 ⫾ 0.01
0.23 ⫾ 0.01
0.26 ⫾ 0.03
0.30 ⫾ 0.02
0.34 ⫾ 0.01
Data are means ⫾ SE for n ⫽ 4–5. Respreading calculations are
based on collapse plateau ratios (34, 36, 52) calculated from dynamic
surface pressure-area isotherms on the Wilhelmy balance at 23°C
(MATERIALS AND METHODS). A collapse plateau ratio of zero indicates
no respreading between the two designated cycles, whereas a ratio of
1 indicates perfect respreading.
pholipids in PPL has much higher film respreading
than DPPC (57). Despite this, low SP-B contents of
0.1–0.25% by weight also generated measurable respreading improvements in films with PPL, with larger
increases as apoprotein content was raised further
(Table 1).
The content-dependent activity of SP-B was also
apparent in physiological measurements on the effects
of instilled SL/SP-B mixtures in excised, lavaged rat
lungs. The ability of SL/SP-B mixtures to improve
surfactant-deficient P-V deflation mechanics increased
as SP-B content increased from 0.1 to 0.75% by weight
(Fig. 6, Table 2). SL/SP-B mixtures with apoprotein
contents of 0.75% had very substantial mechanical
activity, although the degree of P-V restoration did not
fully equal that of CLSE. Mixtures of SL/SP-B (0.5 and
0.3%) also had significant activity in the excised lungs.
The mechanical activity of SL/SP-B (0.3%) appeared to
be greater relative to SL/SP-B (0.2%) than found in
adsorption and dynamic surface activity studies on
corresponding SL/SP-B mixtures (Fig. 6 vs. Figs. 1B
and 3). However, physiological data were consistent
with biophysical findings in showing an overall pattern
of increasing activity for SL/SP-B mixtures as apoprotein content increased. The excised rat lung model in
our experiments has previously been used with success
to correlate the pulmonary activity of lung surfactants
with their surface activity and inhibition (e.g., 17, 21,
22, 38). The rat lung model has also been shown in
multiple studies to respond to the instillation of whole
and extracted lung surfactant materials that improve
in vivo pulmonary function in other animal models of
surfactant deficiency and dysfunction (see Ref. 34 for
review).
The physiological range of SP-B contents in endogenous lung surfactant from animals of different species
and age is not known precisely. CLSE used in comparison studies here has been reported to have a content of
AJP-Lung Cell Mol Physiol • VOL
SP-B of ⬃0.9% by weight relative to phospholipid (39).
Our data indicate that lower SP-B contents of 0.75%
(and to a lesser extent 0.5%) also generate significant
improvements in the adsorption, dynamic surface activity, and physiological activity of lipids. If molecular
weights of 750 and 7,500 are used for phospholipids
and monomeric SP-B, respectively, a weight content of
0.5% SP-B relative to phospholipid is equivalent to a
molar ratio of 5 ⫻ 10⫺4. This indicates that SP-B
significantly affects phospholipid activity when one
molecule of peptide monomer is present among 2,000
phospholipid molecules. If dimer SP-B rather than
monomer SP-B is assumed to be the active functional
form, the number of phospholipid molecules per molecule of active protein is twice as large. The ability of
SP-B to enhance the surface activity of phospholipids
at such molecular ratios is striking and is consistent
with prior research indicating the crucial functional
relevance of this surfactant apoprotein in endogenous
and exogenous lung surfactants.
Although both of the hydrophobic lung surfactant
proteins are known to enhance phospholipid activity,
multiple studies indicate that SP-B is more effective
than SP-C on a weight and molar basis (13, 25, 34, 40,
41, 44, 46, 48, 56, 58). SP-B has been shown to be more
active than SP-C in increasing both the adsorption and
dynamic surface tension lowering of phospholipids (13,
44, 46, 48, 56, 58). The improved adsorption of mixtures of phospholipids with SP-B is consistent with the
finding that this apoprotein has a fourfold higher capacity for binding lipid vesicles to interfacial films on a
weight basis compared with SP-C (40, 41). Mixtures of
phospholipids with SP-B vs. SP-C also have an improved ability to resist inhibition by serum albumin
Fig. 6. Effects of SL/SP-B mixtures on pressure-volume deflation
mechanics in surfactant-deficient rat lungs. Volume fraction is relative to total lung capacity. Lungs were excised from adult rats, and
mechanics were measured during quasistatic deflation in the normal
(surfactant-sufficient) state, after depletion of endogenous surfactant
by lavage (surfactant-deficient), and after tracheal instillation of
SL/SP-B mixtures or CLSE (100 mg/kg rat body wt) (see MATERIALS
AND METHODS). SP-B contents are weight % relative to phospholipid.
Calculated % volume recoveries at selected transpulmonary pressures are given in Table 2. Data for each mixture are means ⫾ SE for
n ⫽ 3.
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Surfactant
Mixture
L903
L904
CONTENT-DEPENDENT SP-B ACTIVITY
Table 2. Percent volume recoveries at selected transpulmonary deflation pressures for SL/SP-B
mixtures in surfactant-deficient rat lungs
Calculated percent volume recovery at pressure, cmH2O
Surfactant mixture
4
6
8
10
Synthetic lipids
⫹ 0.1% SP-B
⫹ 0.2% SP-B
⫹ 0.3% SP-B
⫹ 0.5% SP-B
⫹ 0.75% SP-B
CLSE
18.0 ⫾ 1.5
31.2 ⫾ 2.4
29.0 ⫾ 2.6
66.2 ⫾ 1.5*
79.2 ⫾ 0.5†
83.6 ⫾ 1.5†
92.6 ⫾ 1.4‡
14.8 ⫾ 1.1
28.2 ⫾ 2.7
31.2 ⫾ 4.9
71.8 ⫾ 1.7*
79.3 ⫾ 0.6†
81.3 ⫾ 1.3†
92.3 ⫾ 1.5‡
14.5 ⫾ 3.6
30.9 ⫾ 3.1
36.9 ⫾ 3.7
78.1 ⫾ 1.4*
82.1 ⫾ 1.3
83.3 ⫾ 1.2†
93.6 ⫾ 1.8‡
11.7 ⫾ 1.5
37.4 ⫾ 3.5
50.6 ⫾ 3.6
78.6 ⫾ 1.7*
81.7 ⫾ 0.3
80.6 ⫾ 0.8
94.3 ⫾ 1.2‡
Data are means ⫾ SE for n ⫽ 3–5. SL ⫽ 40:40:10:5:2.5:2.5 DPPC/egg PC/egg PG/egg PI/egg PE/Sph ⫹ 5% cholesterol. Percent volume
recovery is calculated from P-V deflation data as given in MATERIALS AND METHODS. Instilled surfactant dose was 100 mg phospholipid/kg rat
body wt. * Significantly different (P ⬍ 0.05) relative to mixtures containing 0.1–0.2% SP-B by weight; † Significantly different (P ⬍ 0.05) from
mixtures containing 0.1–0.3% SP-B by weight. ‡ Significantly different (P ⬍ 0.05) from all SL/SP-B mixtures.
AJP-Lung Cell Mol Physiol • VOL
face films (6, 12, 40–42). Human SP-B monomer contains not only hydrophobic amino acids but also polar
residues, including 10 basic Arg/Lys residues that are
positively charged at neutral pH. This may facilitate
specific interactions of SP-B with anionic phospholipids as well as zwitterionic phosphatidylcholines in lung
surfactants. Due to its overall amphipathic structure,
SP-B likely occupies a relatively peripheral position in
phospholipid bilayers where it can interact with both
head groups and hydrophobic chains (1, 10, 40, 42, 43,
53). SP-B can order the head-group region of lipid
bilayers and increase the gel-to-liquid crystal transition temperatures of fluid phospholipids (1, 2, 30, 53,
54). SP-B also broadens phase transition width, indicating an ability to disrupt or reduce order in at least a
portion of the bilayer (1, 2, 23, 24, 30, 43). The extensive molecular biophysical interactions between SP-B
and phospholipids support a crucial role for this apoprotein in surfactant biophysical function consistent
with the activity findings here.
In summary, this study shows the striking contentdependent impact of SP-B on the surface and physiological activity of lipids. Even low SP-B contents of
ⱕ0.1% by weight had measurable effects in increasing the adsorption, dynamic surface tension lowering,
and/or film respreading of DPPC, mixed synthetic lipids, and column-purified lung surfactant phospholipids. The magnitude of surface activity improvements
associated with SP-B increased significantly as apoprotein content was incrementally raised to 0.75% by
weight in phospholipid mixtures. Physiological studies
in lavaged excised rat lungs also showed that SP-B had
content-dependent activity in improving the ability of
instilled lipid-SP-B mixtures (0.1–0.75%) to reverse
surfactant-deficient P-V deflation mechanics. Activity
results were consistent with the ability of SP-B to
influence the biophysical function of phospholipids in a
molar ratio of 1:2,000 in endogenous and exogenous
lung surfactants.
This work was supported in part by National Heart, Lung, and
Blood Institute Grant HL-56176.
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(48, 56). These biophysical findings correlate directly
with physiological activity studies. Exogenous surfactants containing lipids combined with SP-B have been
found to give greater improvements in pulmonary mechanics and function in animals than corresponding
mixtures of lipids with SP-C (45). Supplementation
with SP-B or related synthetic peptides has also been
shown to improve the surface and physiological activity
of the clinical exogenous surfactant Survanta, which
contains SP-C but has only very low levels of SP-B (28,
39, 55). The high surface and physiological activity of
SP-B vs. SP-C is consistent with the lethal nature of
congenital SP-B deficiency in humans (14, 19, 31, 32).
Lethal respiratory failure also occurs in “knockout”
mice lacking the SP-B gene (9), and compliance abnormalities exist even in mice that are SP-B (⫹/⫺) heterozygotes (8, 50).
Although not a primary focus of study, our experiments did address the interactions of SP-B with different phospholipids. The finding that mixtures of DPPC/
SP-B had substantial surface and physiological
activity, which increased with SP-B content, indicates
the ability of this amphipathic apoprotein to interact
strongly with disaturated phosphatidylcholine molecules. However, the fact that PPL/SP-B mixtures consistently had better surface activity suggests that the
complete mix of phospholipids in lung surfactant is
important in interacting with SP-B. This mix of lung
surfactant phospholipids includes unsaturated as well
as saturated phosphatidylcholines plus multiple anionic phospholipids (34). The detailed roles and importance of specific phospholipid molecules in interacting
with SP-B require more comprehensive investigation
in future research.
The current study focused on defining quantitative
improvements in activity associated with different contents of SP-B in dispersions and films and did not
address functional molecular biophysical mechanisms.
Substantial prior research has investigated the molecular biophysical interactions of SP-B with phospholipids. SP-B is known to increase phospholipid aggregation, disrupt and fuse phospholipid bilayers and
vesicles, and promote phospholipid insertion into sur-
CONTENT-DEPENDENT SP-B ACTIVITY
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