2-DIMENSIONAL FOURIER-TRANSFORM NEAR-INFRARED SPECTROSCOPY STUDY OF HEAT DENATURATION OF OVALBUMIN IN AQUEOUS-SOLUTIONS

Heat-induced denaturation of ovalbumin in aqueous solutions has been i nvestigated by generalized two-dimensional (2D) Fourier transform near -infrared (FT-NIR) correlation spectroscopy. New insight has been gain ed into hydration and the unfolding process of secondary structures of ovalbumin by studying temperature-dependent correlation patterns in 2 D synchronous and asynchronous spectra, which are constructed from con centration-perturbed Nm spectra at different temperatures. The correla tion patterns have provided information about the correlation and phas e relationships between different absorption bands of ovalbumin and wa ter. The hydration of ovalbumin is almost unchanged from 45 to 67 degr ees C, where ovalbumin molecules are in a natively folded state. A sud den change in the hydration is detected in a narrow temperature range of 67-69 degrees C, where the unfolding of the ordered secondary struc tures starts. The hydration, again, remains nearly unchanged upon furt her heating to 80 degrees C, even though the unfolding process develop s progressively until the denatured state. The sudden change in the hy dration around 68 degrees C seems to be caused by the stabilization of slightly unstable hydrogen bonds in the folded state. The change may make the intramolecular hydrophobic cores of ovalbumin less condensed and more accessible to the solvent molecules. On the other hand, the d evelopment of unfolding from 69 to 80 degrees C results in band shifts for combination bands involving free NH stretching-amide IT (amide Am ), intramolecular hydrogen-bonded NH stretching-amide II (amide Bm) of ovalbumin. The present experiment demonstrates that the generalized 2 D NIR correlation spectroscopy is powerful in detecting subtle but val uable structural information about the protein denaturation in the aqu eous solution.