Attenuated total reflection (ATR)/infrared (IR) spectra were measured for human serum albumin (HSA) in aqueous solutions over a pH range of 5.0-3.2. Generalized two-dimensional (2D) correlation analysis was applied to the amide I region of the spectra to investigate pH-dependent changes in secondary structures and in hydrogen bondings of side chains of HSA. The synchronous and asynchronous 2D spectra were generated from the pi-I-dependent spectral variations for the three states of HSA, the N isomeric form (pH 5.0-4.4), the N-F transition (pH 4.6-3.8), and the F isomeric form (pH 3.8-3.0). The most interesting finding in the 2D spectra is identification of four bands at 1740, 1715, 1705, and 1696 cm(-1) due to a C=O stretching mode of free and hydrogen bonded (weak, medium, and strong) COOK groups of HSA. The 2D correlation analysis provides unambiguous evidence for the existence at least the four C=O bands, demonstrating its powerful deconvolution ability. The asynchronous spectrum of the N form is characterized by a rather broad cross-peak centered at (1715, 1654) cm(-1). The sign of the cross-peak indicates that protonation of COO- groups of glutamic (Glu) and aspartic (Asp) acid residues occurs at: pH's higher than those of structural changes in the a-helices. In the N-F transition, it seems that the formation of free COOK groups and those with the hydrogen bonds of the medium strength occurs is linked with changes in secondary structures of HSA. The asynchronous spectrum indicates that the formation of the strongly hydrogen-bonded COOK groups upon the protonation of COO- groups plays a key role in the initiation of the N-F transition, where mainly alpha -helices ndergo the conformational changes.-The synchronous and asynchronous spectra of the F form show that beta -strands and beta -turns of HSA change significantly in this pH region.
