Attenuated total reflection (ATR)/FT-IR spectra were measured for human serum albumin (HSA) in aqueous solutions (pH 6.6) with concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 wt % over a temperature range of 45–80 °C. Generalized two-dimensional (2D) correlation spectroscopy was employed to explore concentration and heat-induced structural variations of HSA in aqueous solutions. To generate 2D correlation spectra, the raw spectra were subjected to the appropriate pretreatment procedure involving ATR correction, subtraction of the spectrum of an aqueous solution, and smoothing. The synchronous and asynchronous correlation spectra were calculated for the concentration-dependent IR spectral variations in the amide I region at various temperatures. The two-dimensional ATR/IR correlation spectra greatly enhance band separation in the region and provide information about the correlation between the amide bands of HSA arising from the same and different secondary structure components. Based on the correlation investigated and previously proposed relationship between the secondary structure elements and the amide band frequencies, we have proposed the detailed assignments in the amide I region at 45 and 80 °C. The proposed assignments are compared with those based on the results of second derivative and Fourier self-deconvolution (FSD) of the ATR/IR spectra. The asynchronous spectrum generated from the concentration-dependent spectral variations at 45 °C show that side chains, the random coil, and extended chains are more sensitive than the α-helices and β-turns to the concentration change. On the other hand, the corresponding spectrum at 80 °C reveals that the conformation changes in side chains and β-turns (or β-strands) of HSA start before those in extended chain, random coil structures, and α-helices.
Two-Dimensional Infrared Spectroscopy of Antiparallel β-Sheet Secondary Structure
