Adsorption of Egg Phosphatidylcholine to an Air/Water and Triolein/Water Bubble Interface: Use of the 2-Dimensional Phase Rule To Estimate the Surface Composition of a Phospholipid/Triolein/Water Surface as a Function of Surface Pressure

Abstract

Phospholipid monolayers play a critical role in the structure and stabilization of biological interfaces including all membranes, the alveoli of the lung, fat droplets in adipose tissue, and lipoproteins. The behavior of phospholipids in bilayers and at an air-water interface is well understood. However, the study of phospholipids at oil-water interfaces is limited due to technical challenges. In this study, egg-phosphatidylcholine (EPC) was deposited from small unilamellar vesicles onto a bubble of either air or triolein (TO) formed in a low salt buffer. The surface tension (γ) was measured using a drop tensiometer. We observed that EPC binds irreversibly to both interfaces and at equilibrium exerts 12 and 15 mN/m of pressure (Π) at an air and TO interface, respectively. After EPC was bound to the interface, the unbound EPC was washed out of the cuvette and the surface was compressed to study the Π/area relationship. To determine the surface concentration (Γ), which cannot be measured directly, compression isotherms from a Langmuir trough and drop tensiometer were compared. The air-water interfaces had identical characteristics using both techniques, thus Γ on the bubble can be determined by overlaying the two isotherms. TO and EPC are both surface active so in a mixed TO/ EPC monolayer both molecules will be exposed to water. Since TO is less surface active than EPC, as Π increases the TO is progressively ejected. To understand the Π/area isotherm of EPC on a TO bubble, a variety of TO-EPC mixtures were spread at the air-water interface. The isotherms show an abrupt break in the curve caused by the ejection of TO from the monolayer into a new bulk phase. By overlaying the compression isotherm above the ejection point with a TO bubble compression isotherm, Γ can be estimated. This allows determination of Γ of EPC on a TO bubble as a function of Π.

Surface behavior of apolipoprotein A-I and its deletion mutants at model lipoprotein interfaces

“…Approximately 250 ml or 150 ml of fresh buffer was exchanged for removing POPC or proteins, respectively. These exchange volumes removed more than 99.9% of the POPC or the proteins in the aqueous phase ( 65 ). POPC once adsorbed on the TO/W interface does not desorb, so ␥ does not change while removing POPC SUVs.…”

“…When ␥ reached ‫ف‬ 25 mN/m, the excess POPC SUVs in the aqueous phase were removed by the buffer exchange procedure (see following method) and ␥ stayed constant. When ␥ was about 25 mN/m, the coverage of POPC on the interface was about 37% ( 65 ). Then different amounts of protein stocks were added to the aqueous phase to obtain different protein concentrations.…”

Surface Tensiometry of Apolipoprotein B Domains at Lipid Interfaces Suggests a New Model for the Initial Steps in Triglyceride-rich Lipoprotein Assembly

“…Thus, the conformationally flexible interfacial structure of the ␣HD allows the ␤␣1 superdomain to remodel as the composition and surface pressure of bound protein changes during initial lipoprotein assembly. (20). Protein was then added to the bulk solution (black arrow), but no change in pressure (black line) was observed as the high surface concentration of POPC (⌫ POPC , where ⌫ is surface concentration) prevented ␣HD from binding.…”

“…After reaching the equilibrium surface pressure (⌸ EQ ), the excess unbound protein was removed from the bulk solution by flowing 220 ml of peptidefree bulk buffer through the cuvette. This "washout" effectively depleted the bulk peptide by Ͼ99.9% (16,20). After the washout, the drop was slowly and linearly expanded at a rate of ϳ2.7 mm 2 /min, held at a large area for ϳ1000 s, then linearly compressed at a rate of ϳ2.7 mm 2 /min.…”

“…The triolein/POPC/water interface was made by adsorbing POPC SUVs to a triolein/ water interface followed by a washout (20). After the washout of POPC, the surface was compressed to increase ⌸ I .…”