Real time evaluation of composition and structure of concanavalin A adsorbed on a polystyrene surface

In situ qualitative and quantitative evaluations of adsorbed submonolayers and multilayers of the protein Concanavalin A (ConA) on a polystyrene surface were carried out by attenuated total reflection infrared spectroscopy. The influence of pH and adsorption time on the composition and structure of the adsorbed protein layers was investigated by comparison of the experimental spectra with simulated spectra of hypothetical multilayer systems with the assumed composition, thickness, and structure. This methodology allows the differentiation of observed spectral changes that result from pure optical effects, associated with the passing of an incident beam through the multilayer system, from the chemical and structural changes caused by physicochemical interactions of proteins with polymer surfaces. This represents significant progress since small variations in the band positions or intensities of amide I and amide II infrared absorbance bands have an important interpretation consequence. The applied methodology significantly reduces the misinterpretation of recorded spectra of protein layers and is rewarded by a deep insight of the structure and composition of the samples. The composition, structure, and kinetics of the adsorption of ConA and hydration level of the adsorbed layers were evaluated in detail. Competitive adsorption of bovine serum albumin on pre-adsorbed ConA layers also was investigated to characterize the ConA surface distribution. Parallel studies using X-ray photoelectron spectroscopy support the conclusions drawn from infrared spectroscopic investigations on ConA molecular distributions at the polymer surface. Two-step models that describe ConA submonolayer formation at pH 4.8 and multilayer formation at pH 7.8 are proposed.