Dynamic interfacial properties of human apolipoproteins A-IV and B-17 at the air/water and oil/water interface
2000; Elsevier BV; Volume: 41; Issue: 9 Linguagem: Inglês
10.1016/s0022-2275(20)33454-4
ISSN1539-7262
AutoresRichard B. Weinberg, Victoria R. Cook, Jeanine A. DeLozier, Gregory S. Shelness,
Tópico(s)Proteins in Food Systems
ResumoViscoelastic behavior of proteins at interfaces is a critical determinant of their ability to stabilize emulsions. We therefore used air bubble surfactometry and drop volume tensiometry to examine the dynamic interfacial properties of two plasma apolipoproteins involved in chylomicron assembly: apolipoprotein A-IV and apolipoprotein B-17, a recombinant, truncated apolipoprotein B. At the air/water interface apolipoproteins A-IV and B-17 displayed wide area-tension loops with positive phase angles indicative of viscoelastic behavior, and suggesting that they undergo rate-dependent changes in surface conformation in response to changes in interfacial area. At the triolein/water interface apolipoprotein A-IV displayed maximal surface activity only at long interface ages, with an adsorption rate constant of 1.0 × 10−3 sec−1, whereas apolipoprotein B-17 lowered interfacial tension even at the shortest interface ages, with an adsorption rate constant of 9.3 × 10−3 sec−1. Apolipoprotein A-IV displayed an expanded conformation at the air/water interface and a biphasic compression isotherm, suggesting that its hydrophilic amphipathic helices move in and out of the interface in response to changes in surface pressure. We conclude that apolipoproteins A-IV and B-17 display a combination of interfacial activity and elasticity particularly suited to stabilizing the surface of expanding triglyceride-rich particles. —Weinberg, R. B., V. R. Cook, J. A. DeLozier, and G. S. Shelness. Dynamic interfacial properties of human apolipoproteins A-IV and B-17 at the air/water and oil/water interface. J. Lipid Res. 2000. 41: 1419–1427. Viscoelastic behavior of proteins at interfaces is a critical determinant of their ability to stabilize emulsions. We therefore used air bubble surfactometry and drop volume tensiometry to examine the dynamic interfacial properties of two plasma apolipoproteins involved in chylomicron assembly: apolipoprotein A-IV and apolipoprotein B-17, a recombinant, truncated apolipoprotein B. At the air/water interface apolipoproteins A-IV and B-17 displayed wide area-tension loops with positive phase angles indicative of viscoelastic behavior, and suggesting that they undergo rate-dependent changes in surface conformation in response to changes in interfacial area. At the triolein/water interface apolipoprotein A-IV displayed maximal surface activity only at long interface ages, with an adsorption rate constant of 1.0 × 10−3 sec−1, whereas apolipoprotein B-17 lowered interfacial tension even at the shortest interface ages, with an adsorption rate constant of 9.3 × 10−3 sec−1. Apolipoprotein A-IV displayed an expanded conformation at the air/water interface and a biphasic compression isotherm, suggesting that its hydrophilic amphipathic helices move in and out of the interface in response to changes in surface pressure. We conclude that apolipoproteins A-IV and B-17 display a combination of interfacial activity and elasticity particularly suited to stabilizing the surface of expanding triglyceride-rich particles. —Weinberg, R. B., V. R. Cook, J. A. DeLozier, and G. S. Shelness. Dynamic interfacial properties of human apolipoproteins A-IV and B-17 at the air/water and oil/water interface. J. Lipid Res. 2000. 41: 1419–1427. Apolipoprotein A-IV (apoA-IV) is a 46-kDa plasma glycoprotein (1Weinberg R.B. Scanu A.M. The isolation and characterization of human apolipoprotein A-IV from lipoprotein depleted serum.J. Lipid Res. 1983; 24: 52-59Google Scholar), which makes it the largest member of a family of lipid-binding proteins that regulate plasma lipoprotein metabolism (2Luo C.C. Li W.H. Moore M.N. Chan L. The isolation and characterization of human apolipoprotein A-IV from lipoprotein depleted serum.J. Mol. Biol. 1986; 187: 325-340Google Scholar). ApoA-IV is synthesized by the intestinal enterocytes of mammalian species (3Weisgraber K.H. Bersot T.P. Mahley R.W. Isolation and characterization of an apoprotein from the d < 1.006 lipoproteins of human and canine lymph homologous with the rat A-IV apoprotein.Biochem. Biophys. Res. Commun. 1978; 85: 287-292Google Scholar) during lipid absorption (4Hayashi H. Nutting D.F. Fujimoto K. Cardelli J.A. Black D. Tso P. Transport of lipid and apolipoproteins apo A-I and apo A-IV in intestinal lymph of the rat.J. Lipid Res. 1990; 31: 1613-1625Google Scholar, 5Kalogeris T.J. Fukagawa K. Tso P. Synthesis and lymphatic transport of intestinal apolipoprotein A-IV in response to graded doses of triglyceride.J. Lipid Res. 1994; 35: 1141-1151Google Scholar), and enters the circulation on the surface of lymph chylomicrons (6Green P.H. Glickman R.M. Saudek C.D. Blum C.B. Tall A.R. Human intestinal lipoproteins: studies in chyluric subjects.J. Clin. Invest. 1979; 64: 233-242Google Scholar, 7Green P.H. Glickman R.M. Riley J.W. Quinet E. Human apolipoprotein A-IV: intestinal origin and distribution in plasma.J. Clin. Invest. 1980; 65: 911-919Google Scholar). ApoA-IV is a hydrophilic protein, and has the weakest lipid affinity of the human apolipoproteins (8Weinberg R.B. Spector M.S. Structural properties and lipid binding of human apolipoprotein A-IV.J. Biol. Chem. 1985; 260: 4914-4921Google Scholar, 9Weinberg R.B. Jordan M. Effects of phospholipid on the structure of human apolipoprotein A-IV.J. Biol. Chem. 1990; 265: 8081-8086Google Scholar, 10Weinberg R.B. Jordan M. Steinmetz A. Distinctive structure and function of human apolipoprotein variant, Apo A-IV-2.J. Biol. Chem. 1990; 265: 18372-18378Google Scholar). Consequently, its binding to lipoproteins is labile and sensitive to processes that alter the physical state of the lipoprotein surface (1Weinberg R.B. Scanu A.M. The isolation and characterization of human apolipoprotein A-IV from lipoprotein depleted serum.J. Lipid Res. 1983; 24: 52-59Google Scholar, 11DeLamatre J.G. Hoffmeier C.A. Lacko A.G. Roheim P.S. Distribution of apolipoprotein A-IV between the lipoprotein and lipoprotein-free fractions of rat plasma: possible role of lecithin-cholesterol acyltransferase.J. Lipid Res. 1983; 24: 1578-1585Google Scholar, 12Weinberg R.B. Spector M.S. Human apolipoprotein A-IV: displacement from the surface of triglyceride-rich particles by HDL2-associated C-apoproteins.J. Lipid Res. 1985; 26: 26-37Google Scholar, 13Bisgaier C.L. Sachdev O.P. Lee E.S. Williams K.J. Blum C.B. Glickman R.M. Distribution of apolipoprotein A-IV in human plasma.J. Lipid Res. 1987; 28: 693-703Google Scholar, 14Bisgaier C.L. Siebenkas M.V. Hesler C.B. Swenson T.L. Blum C.B. Marcel Y.L. Milne R.W. Glickman R.M. Tall A.R. Effect of a neutralizing monoclonal antibody to cholesterol ester transfer protein on the redistribution of apolipoproteins A-IV and E among human lipoproteins.J. Lipid Res. 1989; 30: 1025-1031Google Scholar, 15Lagrost L. Gambert P. Dangremont V. Athias A. Lallemant C. Role of cholesterol ester transfer protein (CETP) in the HDL conversion process as evidenced by using anti-CETP monoclonal antibodies.J. Lipid Res. 1990; 31: 1569-1575Google Scholar). We have proposed that this behavior allows apoA-IV to function as a barostat that maintains lipoprotein surface pressure and lipid packing within the critical limits required for maximal activity of enzymes and transfer proteins (16Weinberg R.B. Ibdah J.A. Phillips M.C. Adsorption of apolipoprotein A-IV to phospholipid monolayers spread at the air/water interface.J. Biol. Chem. 1992; 267: 8977-8983Google Scholar, 17Weinberg R.B. Cook V.R. Kussie P. Tall A.R. Interfacial properties of recombinant human cholesterol ester transfer protein.J. Biol. Chem. 1994; 269: 29588-29591Google Scholar, 18Weinberg R.B. Jones J.B. Lacko A.G. Pritchard P.H. Effect of interfacial pressure on the binding and phospholipase A2 activity of recombinant human lecithin-cholesterol acyltransferase.Biochem. Biophys. Res. Commun. 1995; 211: 840-846Google Scholar). Although a broad spectrum of physiologic functions has been proposed for apoA-IV (19Lefevre M. Roheim P.S. Metabolism of apolipoprotein A-IV.J. Lipid Res. 1984; 25: 1603-1610Google Scholar, 20Kalogeris T.J. Rodriquez M.D. Tso P. Control of synthesis and secretion of intestinal apolipoprotein A-IV.J. Nutr. 1997; 127: 537S-543SGoogle Scholar), a preponderance of evidence suggests that its primary biological role is in intestinal lipid absorption (20Kalogeris T.J. Rodriquez M.D. Tso P. Control of synthesis and secretion of intestinal apolipoprotein A-IV.J. Nutr. 1997; 127: 537S-543SGoogle Scholar). Furthermore, the observations that 1) Pluronic L-81, a hydrophobic surfactant, selectively and simultaneously blocks intestinal synthesis and secretion of apoA-IV and chylomicrons (21Tso P. Balint J.A. Bishop M.B. Rodgers J.B. Acute inhibition of intestinal lipid transport by Pluronic L-81 in the rat.Am. J. Phys. 1981; 241: G487-G497Google Scholar, 22Tso P. Gollamundi S.R. Pluronic L-81: a potent inhibitor of the transport of intestinal chylomicrons.Am. J. Phys. 1984; 247: G32-G36Google Scholar, 23Tso P. Balint J.A. Formation and transport of chylomicrons by enterocytes to the lymphatics.Am. J. Phys. 1986; 250: G715-G726Google Scholar); 2) enterocyte apoA-IV synthesis does not increase during absorption of short-chain fatty acids (24Kalogeris T.J. Monroe F. Demichele S.J. Tso P. Intestinal synthesis and lymphatic secretion of apolipoprotein A-IV vary with chain length of intestinally infused fatty acids in rats.J. Nutr. 1996; 126: 2720-2729Google Scholar), which are transported directly into portal blood rather than packaged into chylomicrons; and 3) plasma apoA-IV levels are decreased in abetalipoproteinemia (6Green P.H. Glickman R.M. Saudek C.D. Blum C.B. Tall A.R. Human intestinal lipoproteins: studies in chyluric subjects.J. Clin. Invest. 1979; 64: 233-242Google Scholar, 25Bisgaier C.L. Sachdev O.P. Megna L. Glickman R.M. Distribution of apolipoprotein A-IV in human plasma.J. Lipid Res. 1985; 26: 11-25Google Scholar) and hypobetalipoproteinemia (1Weinberg R.B. Scanu A.M. The isolation and characterization of human apolipoprotein A-IV from lipoprotein depleted serum.J. Lipid Res. 1983; 24: 52-59Google Scholar), genetic disorders in which chylomicron synthesis is impaired, together suggest that apoA-IV plays a specific role in the process of chylomicron assembly. Chylomicron assembly is the final and essential step in the absorption of dietary lipids. In the first stage of chylomicron assembly (26Innerarity T.L. Boren J. Yamanaka S. Olofsson S.O. Biosynthesis of apolipoprotein B-48-containing lipoproteins.J. Biol. Chem. 1996; 271: 2353-2356Google Scholar) apoB-48 is transcribed in the rough endoplasmic reticulum of intestinal enterocytes and is cotranslationally lipidated with a small amount of triglyceride by microsomal triglyceride transfer protein to form 100-Å-diameter nascent particles. Absence or truncation of apoB-48 (27Young S.G. Cham C.M. Pitas R.E. Burri B.J. Connoly A. Flynn L. Pappu A.S. Wong J.S. Hamilton R.L. Farese R.V. A genetic model for absent chylomicron formation: mice producing apo B in the liver, but not in the intestine.J. Clin. Invest. 1995; 96: 2932-2946Google Scholar), or absence or inhibition of microsomal triglyceride transfer protein (28Gordon D.A. Recent advances in elucidating the role of microsomal triglyceride transfer protein in apolipoprotein B lipoprotein assembly.Curr. Opin. Lipid. 1997; 8: 131-137Google Scholar), arrests this process. In the second stage, the nascent chylomicron particles, which already have apoA-IV on their surface (29Kumar N.S. Mansbach C.M. Prechylomicron transport vesicle: isolation and partial characterization.Am. J. Phys. 1999; 276: G378-G386Google Scholar), are transported to the Golgi apparatus, where they acquire additional triglyceride molecules and expand to diameters of 5,000–10,000 Å. Pluronic ethylene-propylene copolymer surfactants specifically block this stage (21Tso P. Balint J.A. Bishop M.B. Rodgers J.B. Acute inhibition of intestinal lipid transport by Pluronic L-81 in the rat.Am. J. Phys. 1981; 241: G487-G497Google Scholar, 22Tso P. Gollamundi S.R. Pluronic L-81: a potent inhibitor of the transport of intestinal chylomicrons.Am. J. Phys. 1984; 247: G32-G36Google Scholar, 23Tso P. Balint J.A. Formation and transport of chylomicrons by enterocytes to the lymphatics.Am. J. Phys. 1986; 250: G715-G726Google Scholar). Finally, mature chylomicrons are exocytosed from the basolateral cell membrane into the mesenteric lymphatics, from where they ultimately reach the circulation. The thermodynamic requisites for the dispersion of hydrophobic dietary lipids within the intracellular aqueous milieu as large, stable particles are much the same as for the formation of an oil-in-water macroemulsion (30Rosen M.J. Emulsification by surfactants.in: Surfactants and Interfacial Phenomena. 2nd edition. John Wiley & Sons, New York1989: 304-322Google Scholar). In this regard, the elastic behavior of proteins at interfaces is a critical determinant of their ability to stabilize foams and emulsions (30Rosen M.J. Emulsification by surfactants.in: Surfactants and Interfacial Phenomena. 2nd edition. John Wiley & Sons, New York1989: 304-322Google Scholar). Although the interaction of plasma apolipoproteins with lipid monolayers has been extensively studied by surface balance techniques (16Weinberg R.B. Ibdah J.A. Phillips M.C. Adsorption of apolipoprotein A-IV to phospholipid monolayers spread at the air/water interface.J. Biol. Chem. 1992; 267: 8977-8983Google Scholar, 31Krebs K.E. Phillips M.C. Sparks C.E. A comparison of the surface activities of rat plasma apolipoproteins C-II, C-III-0, and C-III-3.Biochim. Biophys. Acta. 1983; 751: 470-473Google Scholar, 32Krebs K.E. Ibdah J.A. Phillips M.C. A comparison of the surface activities of human apolipoprotein A-I and A-II at the air/water interface.Biochim. Biophys. Acta. 1988; 959: 229-237Google Scholar, 33Ibdah J.A. Phillips M.C. Effects of lipid composition and packing on the adsorption of apolipoprotein A-I to lipid monolayers.Biochemistry. 1988; 27: 7155-7162Google Scholar, 34Ibdah J.A. Krebs K.E. Phillips M.C. The surface properties of apolipoproteins A-I and A-II at the air/water interface.Biochim. Biophys. Acta. 1989; 1004: 300-308Google Scholar, 35Ibdah J.A. Lund-Katz S. Phillips M.C. Molecular packing of high density and low density lipoprotein surface lipids and apolipoprotein A-I binding.Biochemistry. 1989; 28: 1126-1133Google Scholar), these approaches afford little insight into their dynamic behavior in the setting of rapidly changing interfacial geometry. Therefore, we used air bubble surfactometry and drop volume tensiometry to examine the dynamic interfacial properties of apoA-IV at the air/water and oil/water interface. As an important point of comparison we also studied the dynamic interfacial properties of apoB-17, a truncated form of human apolipoprotein B that includes the aminoterminal α1 helical domain that is essential for the initiation of triglyceride-rich particle assembly (36Ingram M.F. Shelness G.S. Folding of the aminoterminal domain of apolipoprotein B initiates microsomal triglyceride transfer protein-dependent lipid transfer to nascent very low density lipoprotein.J. Biol. Chem. 1997; 272: 10279-10286Google Scholar) and is soluble in aqueous buffer. l-α-Dimyristoyl phosphatidylcholine (DMPC) and triolein (Sigma, St. Louis, MO) were >99% pure by thin-layer chromatography on silica gel. DMPC was diluted to 0.1 mg/ml in high performance liquid chromatography-grade chloroform (Aldrich, Milwaukee, WI) and stored under nitrogen at −20°C. Phospholipid concentration was confirmed by phosphorus assay (37Bartlett G.R. Phosphorus assay in column chromatography.J. Biol. Chem. 1959; 234: 466-468Google Scholar). Pluronic surfactants L-81, L-84, and 25R4 were obtained from BASF (Mount Olive, NJ). ApoA-IV was isolated from lipoprotein-depleted plasma obtained from donors with the A-IV-1/1 genotype by adsorption to a phospholipid-triglyceride emulsion (38Weinberg R.B. Hopkins R.A. Jones J.B. Purification, isoform characterization, and quantitation of human apolipoprotein A-IV.Methods Enzymol. 1996; 263: 282-296Google Scholar). ApoA-I was isolated from high density lipoproteins (39Shore B. Shore V. Isolation and characterization of polypeptides of human serum lipoproteins.Biochemistry. 1969; 8: 4510-4516Google Scholar). Both apolipoproteins were purified by anion-exchange high pressure liquid chromatography, and were homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Apolipoprotein concentration was determined by the bicinchoninic acid protein assay (40Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Protein assay using bicinchoninic acid.Anal. Biochem. 1985; 150: 76-85Google Scholar). The FLAG epitope (DYKDDDDK) was appended to the amino-terminal 17% of human apoB (amino acids 1–772 of apoB-100) by polymerase chain reaction. The modified apoB construct was subcloned into the vector pCMV5 and sequenced to confirm its identify. ApoB-17F was excised from the vector, using the flanking EcoRI (5′) and KpnI (3′) restriction enzyme sites, and ligated to EcoRI- and KpnI-digested pFastBac1 (Life Technologies, Rockville, MD). This plasmid was transposed into DH10Bac competent cells; recombinant baculovirus DNA was isolated and transfected into Sf9 cells at a density of 9 × 105 cells/35-mm dish. Virus was harvested after 72 h and titered by plaque assay. Viral stock was amplified by infecting a 1-liter culture of 3.2 × 106 Sf9 cells/ml in suspension in Sf00-II medium (Life Technologies) at a multiplicity of infection of 0.5. After 48 h the cells were pelleted, and the supernatant (250 ml) was made 1 mm phenylmethylsulfonyl fluoride, 0.05% sodium azide, 0.5% Triton X-100, and applied at 3 ml/min to a 2-ml bed volume of anti-FLAG monoclonal antibody M2-conjugated agarose beads (Sigma). Most apoB-17 appeared in the effluent (Fig. 1A, lanes 1 and 2), presumably because the amount of protein present exceeded the column binding capacity; the flowthrough fraction was reapplied under the same conditions. The column was washed with phosphate-buffered saline (PBS), and apoB-17 was eluted with PBS containing FLAG peptide at 150 μg/ml (Fig. 1A, lanes 4–6). Eluate was concentrated with Centricon-10 centrifugal concentrators (Amicon-Millipore, Danvers, MA). ApoB-17 concentration was determined by densitometry of 8% SDS-polyacrylamide gels stained with Coomassie blue, using bovine serum albumin as a mass standard. Multilamellar vesicles were made by adding DMPC in CHCl3 to a glass flask, removing the solvent, suspending the lipid in PBS with 0.05% azide, and vortexing. ApoB-17 (200 μg/ml) was incubated in the absence or presence of vesicles (lipid:protein weight ratio, 20:1) at 24°C with gentle inversion, and the optical density at 325 nm versus buffer was measured at 0, 0.25, and 20 h. Aliquots were adjusted to 1.25 g/ml with solid KBr, and subjected to density gradient centrifugation in a Beckman (Palo Alto, CA) TL-100 rotor (36Ingram M.F. Shelness G.S. Folding of the aminoterminal domain of apolipoprotein B initiates microsomal triglyceride transfer protein-dependent lipid transfer to nascent very low density lipoprotein.J. Biol. Chem. 1997; 272: 10279-10286Google Scholar). Protein was precipitated from the top d < 1.25 g/ml and bottom d > 1.25 g/ml fractions with 10% trichloroacetic acid and quantitated by 8% SDS-PAGE with Coomassie blue staining. Dynamic interfacial behavior at the air/water interface was examined with a pulsating bubble surfactometer (Electronetics, Amherst, NY). This instrument sinusoidally oscillates a tiny air bubble in a plastic chamber filled with aqueous sample, records bubble pressure (P) as a function of bubble radius (r), and calculates interfacial tension (γ) from the Young-Laplace equation, ΔP = 2γ/r (41Enhorning G. Pulsating bubble technique for evaluating pulmonary surfactant.Appl. Physiol. 1977; 43: 198-203Google Scholar). Studies were conducted at 25°C with apolipoproteins at 0.4 mg/ml and Pluronic detergents at 0.02% (v/v) in 50 mm Tris, 100 mm NaCl, pH 7.5. Interfacial tension at minimum bubble radius reached stable values within 15 min of the start of bubble cycling. Thereafter, surface area (A)-tension (γ) loops were recorded at 20 cycles/min. Sur face pressure at the air/water interface was taken as γbuffer − γsample. Absolute elasticity (ϵ) was calculated as Δγ/(ΔA/A), and the viscoelastic component (ϵv) was calculated as ϵ sin(ϕ), where ϕ is the phase angle of the γ-A loops (42Lucassen J. Van Den Tempel M. Dynamic measurements of dilational properties of a liquid interface.Chem. Eng. Sci. 1972; 27: 283-1291Google Scholar, 43Benjamins J. Cagna A. Lucassen-Reynders E.H. Viscoelastic properties of triacylglycerol/water interfaces covered with proteins.Colloids Surfaces. 1996; 114: 245-254Google Scholar). Dynamic interfacial behavior at the oil/water interface was studied with a Kruss USA (Charlotte, NC) DVT-10 drop volume tensiometer. This instrument measures the interfacial tension between two immiscible liquids by pumping the lighter phase through a tungsten carbide capillary into a cuvette filled with the heavy phase and timing the drop interval with a photocell. At the instant of drop detachment the separation force equals the attachment force: Vdrop(σH − σL)g = γπd, where σH is the density of the heavy phase, σL is the density of the light phase, g is the gravitational constant, and d is the orifice diameter. Because the infusion rate is set, the drop interval gives Vdrop and yields γ (44Mollet C. Touhami Y. Hornof V. A comparitive study of the effects of ready-made and in-situ formed surfatants on interfacial tension measured by drop volume tensiometry.J. Colloid Interface Sci. 1996; 178: 523-530Google Scholar). Triolein (σ = 0.91 g/ml) served as the light phase and 41.3 mm Tris, pH 7.5 (σ = 0.9994 g/ml), served as the heavy phase. Apolipoproteins were studied at 10 μg/ml and Pluronic detergents at 0.02% (v/v) at light-phase flow rates of 5.0–0.02 ml/h. Rate constants were calculated by fitting the data to a Langmuir derived adsorption equation γ(t) = γEexp[A exp(–kt)] (45Xu Y. Dynamic interfacial tension between bitumen and aqueous sodium hydroxide solutions.Energ. Fuel. 1995; 9: 148-154Google Scholar). The effect of apoA-IV on the adsorption of cholesterol to the oil/water interface was studied by measuring the inter facial tension of increasing concentrations of free cholesterol ([CH]) dissolved in triolein, in the absence and presence of apoA-IV in the aqueous phase, at a light-phase flow rate of 0.2 ml/h. Maximal interfacial cholesterol concentration (Γ) was calculated from the Gibbs adsorption equation, Γ = −(dγ/dln[CH])/RT (46Handa T. Saito H. Tanaka I. Kakee A. Tanaka K. Miyajima K. Lateral interactions of pig apolipoprotein A-I with egg yolk phosphatidylcholine and with cholesterol in mixed monolayers at the triolein-saline interface.Biochemistry. 1992; 31: 1415-1420Google Scholar). Mean molecular area (A)-surface pressure (Π) isotherms of apoA-IV and apoA-I at the air/water inter face were determined with a KSV 5000 Langmuir film balance (KSV Instruments, Helsinki, Finland), equipped with a computer-controlled compression barrier and a Wilhelmy plate electrobalance, and enclosed in a cabinet maintained at 25°C and 70–75% relative humidity. A 150 × 250 mm Teflon trough was filled with degassed 50 mm Tris, 100 mm NaCl, pH 7.5, or 2 m KCl, 5 mm Tris, pH 7.5, and the buffer surface was cleaned by vacuum aspiration. Apolipoprotein solutions diluted to 0.10 mg/ml in buffer with 30% (v/v) ethanol were layered on the surface with a glass rod to initial pressures of 1.25 g/ml (Fig. 1B, lanes 1 and 2); however, when apoB-17 was incubated with DMPC vesicles, most of the apoB-17 floated the d < 1.25 g/ml top fraction (Fig. 1B, lanes 3 and 4). These data establish that recombinant apoB-17 can bind to large multilamellar DMPC vesicles to form smaller vesicles and discoidal recombinant lipoproteins, as previously described (49Herscovitz H. Hadzopoulou-Cladaras M. Walsh M.T. Cladaras C. Zannis V.I. Small D.M. Expression, secretion, and lipid-binding characterization of the N-terminal 17% of apolipoprotein B.Proc. Natl. Acad. Sci. USA. 1991; 88: 7313-7317Google Scholar). Apolipoproteins and Pluronic detergents bound to the air/water interface as evidenced by a decrease in bubble surface tension. Of the apolipoproteins, apoB-17 displayed the lowest interfacial tension and highest surface pressure at minimum (resting) bubble radius (Table 1). The FLAG peptide at equivalent concentrations demonstrated no surface activity; this suggests that FLAG epitope did not contribute to the interfacial behavior of recombinantapoB-17, although we cannot rule out the possibility that it might alter the three-dimensional structure of apoB-17 in a manner that affects its surface activity. Surface pressures at minimum (resting) bubble radius for apoA-IV and apoA-I were lower, and were similar to values determined by surface balance techniques (16Weinberg R.B. Ibdah J.A. Phillips M.C. Adsorption of apolipoprotein A-IV to phospholipid monolayers spread at the air/water interface.J. Biol. Chem. 1992; 267: 8977-8983Google Scholar, 32Krebs K.E. Ibdah J.A. Phillips M.C. A comparison of the surface activities of human apolipoprotein A-I and A-II at the air/water interface.Biochim. Biophys. Acta. 1988; 959: 229-237Google Scholar, 33Ibdah J.A. Phillips M.C. Effects of lipid composition and packing on the adsorption of apolipoprotein A-I to lipid monolayers.Biochemistry. 1988; 27: 7155-7162Google Scholar, 34Ibdah J.A. Krebs K.E. Phillips M.C. The surface properties of apolipoproteins A-I and A-II at the air/water interface.Biochim. Biophys. Acta. 1989; 1004: 300-308Google Scholar, 35Ibdah J.A. Lund-Katz S. Phillips M.C. Molecular packing of high density and low density lipoprotein surface lipids and apolipoprotein A-I binding.Biochemistry. 1989; 28: 1126-1133Google Scholar). Of the Pluronic detergents, L-81 displayed the highest surface activity, and reduced interfacial tension more effectively than any of the apolipoproteins; L-84 and 25R4, which are more hydrophilic, were less surface active. With bubble pulsation, the ΔA-γ loops for apoA-IV and apoB-17 displayed large changes in surface tension with changes in surface area, indicative of high elasticity at the air/water interface, and positive phase angles, indicative of viscous behavior (Fig. 2). These data suggest that apoA-IV and apoB-17 undergo rate-dependent changes in surface conformation in response to changes in interfacial area. These properties are characteristic of foaming and emulsifying agents that stabilize expanding interfaces (30Rosen M.J. Emulsification by surfactants.in: Surfactants and Interfacial Phenomena. 2nd edition. John Wiley & Sons, New York1989: 304-322Google Scholar). Conversely, the ΔA-γ loops for apoA-I and the Pluronic detergents were flat, consistent with little viscoelastic behavior. This property, particularly when combined with high surface activity, as with L-81, is characteristic of antifoaming agents that destroy interfaces and induce phase separation (30Rosen M.J. Emulsification by surfactants.in: Surfactants and Interfacial Phenomena. 2nd edition. John Wiley & Sons, New York1989: 304-322Google Scholar).TABLE 1.Dynamic interfacial properties at the air/water interfaceγΠεϕεvmN/mmN/mmN/mdegmN/mApoB-1734.037.819.076.318.4ApoA-I39.832.07.612.41.6ApoA-IV42.529.321.728.010.2Pluronic L-8133.338.52.8NDNDPluronic L-8441.330.52.8NDNDPluronic 25R444.027.82.7NDNDγ, Interfacial tension at minimum (resting) bubble surface area; Π, surface pressure at minimum (resting) bubble surface area; Π, elasticity; ϕ, γ-area loop phase angle; εv, viscous elastic component; ND, not determined. Open table in a new tab γ, Interfacial tension at minimum (resting) bubble surface area; Π, surface pressure at minimum (resting) bubble surface area; Π, elasticity; ϕ, γ-area loop phase angle; εv, viscous elastic component; ND, not determined. Apolipoproteins and Pluronic detergents bound to the oil/water interface as evidenced by a decrease in oil drop interfacial tension below 30 mN/m, the interfacial tension between pure triolein and buffer (Fig. 3). ApoB-17 lowered interfacial tension at the shortest interface ages, with maximal reduction at times >300 sec. Pure FLAG p
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