Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

Membrane surface-charge titration probed by gramicidin A channel conductance.

We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the channel conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also influenced by lipid-dependent structural factors.     

Depolarization of dehydroergosterol in phospholipid bilayers

The behavior in phospholipid bilayers of low concentrations of dehydroergosterol, a fluorescent cholesterol mimic, has been examined by fluorometry and calorimetry. In contrast to many fluorescent membrane probes, dehydroergosterol shows a decrease in fluorescence anisotropy when the matrix phospholipid goes from the liquid-crystalline to the gel state. This was observed in three systems in which the matrix lipid was either dipalmitoyl- or dimyristoylphosphatidylcholine or dilauroylphosphatidylethanolamine. The decrease in anisotropy is the result of a large increase in the fluorescence life time of dehydroergosterol in these bilayer systems which is probably the result of thermal quenching of dehydroergosterol by neighboring molecules. The rotation of dehydroergosterol in these bilayers can be described in terms of the thermal coefficient of frictional resistance offered by the environment (Weber et al. (1984) Biochemistry 23, 6785-6788). The thermal coefficients are observed to change abruptly at the onset and completion temperatures of the gel to liquid-crystalline phase transition temperatures of the three matrix phospholipids. These changes are, however, much smaller than are the corresponding changes in the thermal coefficient observed for the fluorescent probe diphenylhexatriene in dilauroylphosphatidylethanolamine bilayers. The difference in behavior of the two fluorescent probes may be the result of lateral phase separation of dehydroergosterol similar to that reported for cholesterol in similar systems.     

Surface potentials measure ion concentrations near lipid bilayers during rapid solution changes.

We describe a puffing method for changing solutions near one surface of lipid bilayers that allows simultaneous measurement of channel activity and extent of solution change at the bilayer surface. Ion adsorption to the lipid headgroups and screening of the bilayer surface charge by mobile ions provided a convenient probe for the ionic composition of the solution at the bilayer surface. Rapid ionic changes induced a shift in bilayer surface potential that generated a capacitive transient current under voltage-clamp conditions. This depended on the ion species and bilayer composition and was accurately described by the Stern-Gouy-Chapman theory. The time course of solute concentrations during solution changes could also be modeled by an exponential exchange of bath and puffing solutions with time constants ranging from 20 to 110 ms depending on the flow pressure. During changes in [Cs+] and [Ca2+] (applied separately or together) both the mixing model and capacitive currents predicted [Cs+] and [Ca2+] transients consistent with those determined experimentally from: 1) the known Cs(+)-dependent conductance of open ryanodine receptor channels and 2) the Ca(2+)-dependent gating of ryanodine receptor Ca2+ channels from cardiac and skeletal muscle.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

Two-dimensional microelectrophoresis in supported lipid bilayers

We report the application of supported bilayers for two-dimensional microelectrophoresis. This method allows the lateral separation and accumulation of charged amphiphilic molecular probes in bilayers by application of an electric field parallel to the bilayer surface. Diffusion coefficient and mobility of the fluorescent probes are determined by observation of the fluorescence recovery after photobleaching (pattern bleaching). The diffusion coefficients and the mobilities of oppositely charged fluorescent probes in one bilayer can be determined independently from a single measurement. By analysis of the motion of charged and uncharged probes in one membrane one can distinguish between the motion caused by the electric field acting on the charge of individual probes and that caused by frictional forces due to electroosmosis.     

Probing the micelle/water interface by rapid laser-induced proton pulse

The laser-induced pH jump (Gutman, M. and Huppert, D.J. (1979) Biochem. Biophys. Methods 1, 9–19) has a time resolution capable of measuring the diffusion-controlled rate constant of proton binding. In the present study we employed this technique for measuring the kinetics of protonation-deprotonation of surface groups of macromolecules.The heterogeneous surface of proteins excludes them from serving as a simple model, therefore we used micelles of a neutral detergent (Brij 58) as a high molecular weight structure. The charge was varied by the addition of a low concentration of sodium dodecyl sulfate and the surface group with which the protons react was an adsorbed pH indicator (bromocresol green or neutral red).The dissociation of a proton from adsorbed bromocresol green is slower than that from free indicator. This effect is attributed to the enhanced stabilization of the acid form of the indicator in the pallisade region of the micelle. The $\text{p}\text{K}$ shift of bromocresol green adsorbed on neutral micelles is thus quantitatively accounted for by the decreased rate of proton dissociation. Indicators such as neutral red, which are more lipid soluble in their alkaline form, do not exhibit such decelerated proton dissociation in their adsorbed state nor a $\text{p}\text{K}$ shift on adsorption to neutral micelles.The protonation of an indicator is a diffusion-controlled reaction, whether it is free in solution or adsorbed on micelles. By varying the electric charge of the micelle this rate can be accelerated or decelerated depending on the total charge of the micelle. The micellar charge calculated from this method was corroborated by other measurements which rely only on equilibrium parameters.The high time resulation of the pH jump is exemplified by the ability to estimate the diffusion coefficient of protons through the hydrated shell of the micelle.     

The effect of surface charge density on valinomycin-K+ complex formation in model membranes

The model membrane approach was used to investigate the surface charge effect on the ion-antibiotic complexation process. Mixed monolayers of valinomycin and lipids were spread on subphases containing K+ or Na+. The surface charge density was modified by spreading ionizable valinomycin analogs on aqueous subphases of different pH or by changing the nature of the lipid (neutral, negatively charged) in the mixed film. Surface pressure and surface potential measurements demonstrated that a neutral lipid (phosphatidylcholine) or positively charged valinomycin analogs didn't enhance the anti-biotic complexing capacity. However, a maximal complexation is reached for a critical lipid concentration in the valinomycin-phosphatidylserine mixed film. The role of the surface charge on the valinomycin complexing properties was examined in terms of the Gouy-Chapman theory. As a consequence of the negative charge of the lipid monolayer, the K+ concentration near the surface is larger than the bulk concentration, by a Boltzmann factor. A good agreement was observed between the experimental results and the theoretical predictions. Conductance measurements of asymmetric bilayers containing a neutral lipid (egg lecithin) on one side and a negatively charged lipid (phosphatidyl-serine) on the other, confirm the role of the surface charge. Indeed, addition of K+ to the neutral side of the bilayer containing valinomycin had no effect on the conductance whereas addition of K+ to the charged side of the bilayer caused a 80-fold conductance increase.     

Characterization of reconstituted ATPase complex proteoliposomes prepared from the thermophilic cyanobacterium Synechococcus 6716

The preparation and some properties are described of proteoliposomes consisting of the ATPase complex and lipids from the thermophilic cyanobacterium Synechococcus 6716. In the proteoliposomes (about 200 nm in diameter) only a low amount of protein can be incorporated (protein/lipid ratio of 0.01 w/w) and they show very few protein particles on freeze-fracture replicas. The octyl glucoside and cholate dialysis method of reconstitution yielded stable proteoliposomes with a relatively low proton permeability. ATP hydrolysis and 32Pi/ATP exchange activities were about 400 and 120 nmol X min-1 X mg protein-1, respectively; the former was strongly stimulated by an uncoupler. ATP hydrolysis induces membrane energization as monitored by membrane-potential- and surface-potential-indicating probes and by different pH indicators trapped inside the vesicles. The probes used were a membrane-bound fluorescent aminoacridine, which monitors surface charge-density changes, the native carotenoids and added oxonol VI for monitoring electrical potential in the membrane and the pH indicators neutral red and cresol red. The different rise kinetics of these probes indicate that proton accumulation upon ATP hydrolysis involves at least two steps: a membrane-localized potential charge and proton transfer followed by a much slower acidification of the bulk intravesicular space. Internal neutral red and cresol red seem to discriminate between proton translocation to the internal interface and bulk space, respectively.     

Voltage-dependent translocation of R18 and DiI across lipid bilayers leads to fluorescence changes.

We show that the lipophilic, cationic fluorescent dyes R18 and Dil translocate from one monolayer of a phospholipid bilayer membrane to the other in a concentration and voltage-dependent manner. When the probes were incorporated into voltage-clamped planar membranes and potentials were applied, displacement currents resulted. The charged probes sensed a large fraction of the applied field. When these probes were added to only one monolayer, displacement currents were symmetrical around 0 mV, indicating that the probes distributed equally between the two monolayers. Charge translocation required that the bilayer be fluid. When membranes were in a condensed gel phase, displacement currents were not observed; raising the temperature to above the gel-liquid crystalline transition restored the currents. Translocation of R18 was also shown by fluorescence measurements. When R18 was in the bilayer at high, self-quenching concentrations, voltage pulses led to voltage-dependent fluorescence changes. The kinetics of the fluorescence changes and charge translocations correlated. Adding the quencher I- to one aqueous phase caused fluorescence to decrease or increase when voltage moved R18 toward or away from the quencher at low, nonquenching concentrations of R18. In contrast to R18, Dil incorporated into bilayers was a carrier fo I-, and hence I- altered Dil currents. Voltage-driven translocations allow R18 and Dil to be used to probe membrane potential changes.     

Correlated fluorescence-atomic force microscopy of membrane domains: structure of fluorescence probes determines lipid localization

Coupling atomic force microscopy (AFM) with high-resolution fluorescence microscopy is an attractive means of identifying membrane domains by both physical topography and fluorescence. We have used this approach to study the ability of a suite of fluorescent molecules to probe domain structures in supported planar bilayers. These included BODIPY-labeled ganglioside, sphingomyelin, and three new cholesterol derivatives, as well as NBD-labeled phosphatidylcholine, sphingomyelin, and cholesterol. Interestingly, many fluorescent lipid probes, including derivatives of known raft-associated lipids, preferentially partitioned into topographical features consistent with nonraft domains. This suggests that the covalent attachment of a small fluorophore to a lipid molecule can abolish its ability to associate with rafts. In addition, the localization of one of the BODIPY-cholesterol derivatives was dependent on the lipid composition of the bilayer. These data suggest that conclusions about the identification of membrane domains in supported planar bilayers on the basis of fluorescent lipid probes alone must be interpreted with caution. The combination of AFM with fluorescence microscopy represents a more rigorous means of identifying lipid domains in supported bilayers.     

Fluorescent probes for asymmetric lipid bilayers: Synthesis and properties in phosphatidyl choline liposomes and erythrocyte membranes

Summary We have synthesized three sets of fluorescent probes which we believe will be useful in studies of asymmetric membranes and have studied their interactions with model lipid bilayers and erythrocyte membranes. The probes were designed to partition preferentially into one face of a lipid bilayer with asymmetrically disposed phospholipids and to report lipid transitions in that monolayer.We synthesized more than twenty probes containing anthroyl-, dansyl-, or pyrene rings with acidic, basic, and neutral functional groups and alkyl spacers of various lengths. The interactions of these probes with liposomes of phosphatidyl choline and with erythrocyte membranes were characterized to determine whether probe insertion was asymmetric, how deeply the probe penetrated the bilayer, and whether the probe reflected thermotropic phase transitions in model membranes. The set of variously charged anthroyl esters, analogs of local anaesthetics, appears to be promising for studies of asymmetric membranes.Fluorescent probes have been used extensively to provide information on the lipid regions of biological membranes. Membrane fluidity, a composite of molecular packing and motion of acyl chains in lipid bilayers, has been assessed with a variety of fluorescent probes, the fluorescence of which undergoes some measurable change at the temperature of the membrane's thermotropic phase transition. A large number of fluorescent probes have been used for this purpose. Bashford, Morgan and Radda (Bashford, C.L., Morgan, C.G., Radda, G.K. 1976;Biochim. Biophys. Acta 426: 157) and Thulborn and Sawyer (Thulborn, K.R., Sawyer, W.H. 1978;Biochim. Biophys. Acta 511: 125) synthesized several fatty acid derivatives in which an anthracene group is attached (in ester linkage) along the acyl chain at various positions, and have shown that this set of probes may be useful in probing membrane fluidity at differentdepths within the bilayer.This report describes the synthesis and properties of several sets of amphipathic fluorescent probes, which may partition unequally into the two faces of an asymmetric lipid bilayer, and may therefore provide information about membranes complementary to that obtainable with existing probes.     

Bimodal distribution and fluorescence response of environment-sensitive probes in lipid bilayers

A remarkable heterogeneity is often observed in the spectroscopic properties of environment-sensitive fluorescence probes in phospholipid bilayers. To explain its origin, we provided a detailed investigation of the fluorescence excitation and emission spectra of 4'-dimethylamino-3-hydroxyflavone (probe F) in bilayer vesicles with the variations of fatty acid composition, polar heads, temperature, and cholesterol content. Probe F, due to excited-state intramolecular proton transfer, exhibits two bands in emission that are differently sensitive to intermolecular interactions-thereby allowing us to distinguish universal (dipole-dipole) and specific (H-bonding) interactions within the bilayer. Spectroscopic, quenching, and anisotropy data suggest the presence of two forms of probe F at different locations in the bilayer: an H-bond free form located below sn(1)-carbonyls and an H-bonded form located at the polar membrane interface. We provide a quantitative analysis of the distribution of the probe between these two locations as well as the polarity of these locations, and show that both the distribution and the polarity contribute to the probe response. Moreover, analysis of literature data on other environment-sensitive probes (Prodan, Laurdan, Nile Red, NBD lipids, etc.) in lipid bilayers allows us to suggest that the bimodal distribution in the lipid bilayer is probably a general feature of low-polar molecules with polar groups capable of H-bonding interactions.     

The effect of the lipid bilayer state on fluorescence intensity of fluorescein-PE in a saturated lipid bilayer

Fluorescein-PE is a fluorescence probe that is used as a membrane label or a sensor of surface associated processes. Fluorescein-PE fluorescence intensity depends not only on bulk pH, but also on the local electrostatic potential, which affects the local membrane interface proton concentration. The pH sensitivity and hydrophilic character of the fluorescein moiety was used to detect conformational changes at the lipid bilayer surface. When located in the dipalmitoylphosphatidylcholine (DPPC) bilayer, probe fluorescence depends on conformational changes that occur during phase transitions. Relative fluorescence intensity changes more at pretransition than at the main phase transition temperature, indicating that interface conformation affects the condition in the vicinity of the membrane. Local electrostatic potential depends on surface charge density, the local dielectric constant, salt concentration and water organisation. Initial increase in fluorescence intensity at temperatures preceding that of pretransition can be explained by the decreased value of the dielectric constant in the lipid polar headgroups region related in turn to decreased water organisation within the membrane interface. The abrupt decrease in fluorescence intensity at temperatures between 25 degrees C and 35 degrees C (DPPC pretransition) is likely to be caused by an increased value of the electrostatic potential, induced by an elevated value of the dielectric constant within the phosphate group region. Further increase in the fluorescence intensity at temperatures above that of the gel-liquid phase transition correlates with the calculated decreased surface electrostatic potential. Above the main phase transition temperature, fluorescence intensity increase at a salt concentration of 140 mM is larger than with 14 mM. This results from a sharp decline of the electrostatic potential induced by the phosphocholine dipole as a function of distance from the membrane surface.     

Effect of phosphorylation of phosphatidylinositol on myelin basic protein-mediated binding of actin filaments to lipid bilayers in vitro

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also assemble actin filaments and tether them to lipid bilayers through electrostatic interactions. Here we investigate the effect of increased negative charge of the lipid bilayer due to phosphorylation of phosphatidylinositol (PI) on MBP-mediated binding of actin to the lipid bilayer, by substituting phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate for PI in phosphatidylcholine/phosphatidylglycerol lipid vesicles. Phosphorylation of PI caused dissociation of the MBP/actin complex from the lipid vesicles due to repulsion of the negatively charged complex from the negatively charged membrane surface. An effect of phosphorylation could be detected even if the inositol lipid was only 2mol% of the total lipid. Calcium-calmodulin dissociated actin from the MBP-lipid vesicles and phosphorylation of PI increased the amount dissociated. These results show that changes to the lipid composition of myelin, which could occur during signaling or other physiological events, could regulate the ability of MBP to act as a scaffolding protein and bind actin filaments to the lipid bilayer.     

Electrostatic interactions at charged lipid membranes. Measurement of surface pH with fluorescent lipoid pH indicators.

The 5-dimethylaminonapthalene-1-sulfonyl (dansyl) chromophore attached to the polar head groups of lipids has been used as a fluorescent lipoid pH indicator to evaluate the interfacial pH in lipid-water lamellar systems prepared from negatively charged lipids. The pH in the vicinity of the charged lipid bilayers is different from the pH of the bulk aqueous phase and the difference is a function of the electrolyte concentration in the aqueous phase and of the lipid packing in the bilayer. At a fixed electrolyte concentration in the aqueous phase, the observed interfacial pH is 0.6 to 0.7 pH units lower above the thermal phase transition of the lipid than it is below this temperature. A quantitative interpretation of the results is given on the basis of the Gouy-Chapman theory. The results indicate that the dansyl chromophore is located in front of the charged surface and its distance from this surface increases with a decrease in lipid packing.     

Fluorescence lifetimes of carbocyanine lipid analogues in phospholipid bilayers

The fluorescence lifetimes for the 1,1'-dialkyl-3,3,3',3'-tetramethylindocarbocyanine (CNdiI) dyes (N = 12, 18, and 22) in a variety of lipid bilayer membranes were measured. Effects of bilayer physical state, probe chain length, probe concentration, charge, lipid head group, and cholesterol concentration were examined. Even in single-phase membranes these probes did not exhibit single-exponential decays. Rather, the data were fit by biexponential decays with lifetimes of approximately 0.3-0.4 and approximately 0.9-1.3 ns with no significant improvement in chi 2 convergence with the addition of a third component. Average lifetimes were dependent upon lipid phase and to a lesser degree surface charge and the phospholipid head group. In dipalmitoyl-phosphatidylcholine (DPPC)-cholesterol membranes, the C18diI lifetime was sensitive to membrane reorganizations at both 20 and approximately 33 mol % cholesterol. In egg phosphatidylcholine (EPC) bilayers, the C18diI lifetime was essentially independent of its concentration below 1:10(3).     

Effect of head group and curvature on binding of the antimicrobial peptide tritrpticin to lipid membranes

In this work we examine the interaction between the 13-residue cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) and model membranes of variable lipid composition. The effect on peptide conformational properties was investigated by means of CD (circular dichroism) and fluorescence spectroscopies. Based on the hypothesis that the antibiotic acts through a mechanism involving toroidal pore formation, and taking into account that models of toroidal pores imply the formation of positive curvature, we used large unilamellar vesicles (LUV) to mimic the initial step of peptide-lipid interaction, when the peptide binds to the bilayer membrane, and micelles to mimic the topology of the pore itself, since these aggregates display positive curvature. In order to more faithfully assess the role of curvature, micelles were prepared with lysophospholipids containing (qualitatively and quantitatively) head groups identical to those of bilayer phospholipids. CD and fluorescence spectra showed that, while TRP3 binds to bilayers only when they carry negatively charged phospholipids, binding to micelles occurs irrespective of surface charge, indicating that electrostatic interactions play a less predominant role in the latter case. Moreover, the conformations acquired by the peptide were independent of lipid composition in both bilayers and micelles. However, the conformations were different in bilayers and in micelles, suggesting that curvature has an influence on the secondary structure acquired by the peptide. Fluorescence data pointed to an interfacial location of TRP3 in both types of aggregates. Nevertheless, experiments with a water soluble fluorescence quencher suggested that the tryptophan residues are more accessible to the quencher in micelles than in bilayers. Thus, we propose that bilayers and micelles can be used as models for the two steps of toroidal pore formation.     

Recombination of human erythrocyte apoprotein and lipid. I. Interaction of apoprotein and lipid at the air-water interface

The formation and stabilization of a complex between total erythrocyte apoprotein and monolayers of total erythrocyte lipid as measured by changes of surface pressure (Δπ) and rate of change of surface pressure (dπ/dt) was studied as a function of pH, ionic strength, and lipid surface pressure. Penetration of apoprotein into lipid monolayers was favored by conditions in which lipid and apoprotein were oppositely charged. Once the interaction was completed, the resultant surface complex was resistant to large changes in subphase pH and ionic strength as shown by the insensitivity of Δπ to these parameters. The dπ/dt, however, showed strong dependence on pH and ionic strength, but not on lipid surface pressure. A sharp decrease in dπ/dt around pH 3.5–4.5 is associated with the change in apoprotein charge from (+) to (?). Comparison of complex formation between apoprotein and bovine serum albumin, cytochrome c, and human hemoglobin suggests that erythrocyte apoprotein was specialized in its interaction with erythrocyte lipids. The data show that formation of an apoprotein-lipid complex at the air-water interface has both electrostatic and hydrophobic components. This contradicts results from other laboratories studying erythrocyte membrane recombination by bulk methods.