Abstract
Using tetradecyltrimethylammonium bromide (TTAB) as a surfactant denaturant, and augmentation of different spectroscopic data, helped to detect the intermediates of hemoglobin (Hb) during unfolding process. UV-vis, fluorescence, and circular dichroism spectroscopy were used simultaneously to monitor different aspects of hemoglobin species from the tertiary or secondary structure points of view. Application of the multivariate curve resolution-alternating least square (MCR-ALS), using the initial estimates of spectral profiles and appropriate constraints on different parts of augmented spectroscopic data, showed good efficiency for characterization of intermediates during Hb unfolding. These results indicated the existence of five protein species, including three intermediate-like compounds in this process. The unfolding pathway in the presence of TTAB included conversion of oxyhemoglobin into deoxyhemoglobin, and then ferrylhemoglobin, ferrihemoglobin or aquamethemoglobin, which finally transformed into hemichrome. This is the first application of chemometric analysis on the merged spectroscopic data related to chemical denaturation of a protein. These types of analysis in multisubunit proteins not only increase the domain of information, but also can reduce the ambiguities of the obtained results.