Monolayer and Brewster angle microscopy study of human serum albumin—Dipalmitoyl phosphatidyl choline mixtures at the air–water interface

The aim of this study is to deepen the understanding of the behavior of human serum albumin (HSA) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixed monolayers. For this purpose, different amounts of DPPC were spread at 25 °C on the water surface containing a monolayer of HSA. Surface film balance and Brewster angle microscopy techniques have been used to analyze the structural and energetic characteristics (structure, topography, thickness, miscibility and interactions) of these mixtures. HSA/DPPC mixed monolayers exhibit two phase transitions evidenced by two discontinuities in the corresponding π–A isotherms and by two minimum values in the compressional modulus (Cs−1)–surface pressure (π) curves. The plot of the molecular areas occupied by the mixed monolayers as function of the mass fraction of DPPC shows the absence of deviations from linearity, a typical behavior for ideal or inmiscible system. This result was confirmed from the values calculated for the free energy of excess (ΔGexc), which are practically zero whatever the composition of the mixtures and the surface pressures at which ΔGexc values were calculated. In addition, relative thickness values of HSA/DPPC mixed monolayers showed the existence of an exclusion surface pressure (πexc), below which the monolayer is composed of a mixture of both components, while above πexc the HSA molecules are squeezed out the interface, but not totally. In fact, although in this region DPPC domains predominate at the interface, the existence of protein molecules in a packing “loops” configuration can be observed in BAM images. Moreover, relative thickness measurements confirm this hypothesis.

Surface pressure (π)–area (A) isotherms for HSA, DPPC and their mixtures spread at the air/water interface at 25 °C and BAM images of HSA/DPPC mixture monolayers for 0.5 mass fractions of DPPC in different zones of the isotherm.Figure optionsDownload as PowerPoint slideHighlights
► Procedure for obtaining mixed films to quantify the possible HSA/DPPC interactions.
► Spreading different amounts of phospholipid molecules on a monolayer of HSA.
► Relative thickness of mixed monolayers showed the existence of a sharp decrease.
► BAM images showed the existence of protein packed “loops”.
► BAM images confirm the immiscibility of the film components.