Adsorption properties of proteins at and near the air/water interface fromIRRAS spectra of protein solutions

A detailed study is performed using infrared reflection absorption spectros copy (IRRAS) to characterize the molecular behaviour of proteins at and nea r the air/water interface of protein solutions. IRRAS spectra of beta -case in solutions in H2O and D2O show spectral shifts and derivative-like featur es not commonly observed in monomolecular layer systems. They can be fully understood using optical theory. Fair agreement between experimental and si mulated IRRAS spectra over a broad spectral range (4000-1000 cm(-1)) is obt ained using a stratified layer model. An attenuated total reflection and tr ansmission spectrum is used to represent the protein extinction coefficient in H2O and D2O, respectively. It is shown that the derivative-like feature s observed result from the reflective properties of the proteins themselves . Furthermore, both concentration and film thickness could be fitted. At hi gh protein concentrations (100 mg/mL) the spectrum is that of a single homo geneous protein solution. At 0.1 mg/mL, beta -casein is accumulated at the surface in a thin layer of approximately 10 nm thickness, with a concentrat ion about 2500 times higher than in the sub-phase. At an initial concentrat ion of 10 mg/mL, the concentration in the surface layer is about 15 times h igher than in the subphase, while the thickness is about 30 nm.