In Situ FTIR ATR Spectroscopic Study of the Interaction of Immobilized Human Serum Albumin with Cholate in Aqueous Environment

Fourier transform infrared (FTIR) attenuated total reflection (ATR) spectroscopy is increasingly used for the investigation of processes at or near interfaces. Exposing HSA dissolved in phosphate buffered saline (PBS) to a clean germanium (Ge) multiple internal reflection element (MIRE) led to the formation of a stable compact monolayer with a surface concentration of (5.31 ± 0.49) × 10 −12 mol cm −2 . Using a flow-through cuvette enabled in situ the monitoring of the HSA layer preparing and the interaction of this layer with a 5 mM solution of cholate in PBS. In order to achieve optimum sensitivity, two enhanced spectroscopic techniques have been applied: single-beam-sample-reference (SBSR) technique and concentration-modulated excitation (c-ME). Solutions of cholate and pure PBS were pumped alternatively with frequency ω through the sample compartment containing the immobilized HSA monolayer. Spectral changes due to interaction between cholate and HSA were now marked by the same frequency ω and could therefore be separated from the dominating unaffected part of the spectrum by phase-sensitive detection (PSD). It was found that at 5.0 mM bulk concentration of cholate 6.1 ± 1.0 cholate molecules were bonded by one immobilized HSA molecule. Furthermore, an increase of α-helical structures in HSA was initiated by cholate bonding.