Two-dimensional near-infrared correlation spectroscopy studies on protein denaturation

We have studied thermal and acid denaturation of ovalbumin by use of genera lized two-dimensional(2D) near-infrared(NIR) correlation spectroscopy. NIR spectroscopy provides unique information about the hydration and structure of proteins which infrared and Raman spectroscopy do not offer. However, th ere observed a number of bands due to overtones and combination modes in th e NIR region, so that spectral analysis in the NIR region is not always str aightforward. Thus, we have employed 2D correlation spectroscopy to analyze the NIR spectra. By using 2D correlation analysis we have been able to sep arate bands due to water and protein amide groups. We have calculated synch ronous and asynchronous correlation spectra of ovalbumin aqueous solutions by use of its concentration as the perturbation from 45 degrees C to 80 deg rees C at an interval of 2 degrees C and from pH 5.8 to pH 2.4 at an interv al of pH 0.2. The 2D correlation analysis have suggested that the sudden ch ange in the hydration around 68 degrees C causes the unfolding process of t he secondary structure and that in the acid denaturation the hydration chan ges markedly around pH 3.0, leading to the gelation. Our studies have demon strated that 2D correlation NIR spectroscopy holds considerable promise in the studies of hydration and secondary structure change of proteins.