title: Application of merged spectroscopic data combined with chemometric analysis for resolution of hemoglobin intermediates during chemical unfolding.
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.
authors.affiliation: Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
authors.forename: L
authors.initials: L
authors.lastname: Fotouhi
authors.affiliation: Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
authors.forename: S
authors.initials: S
authors.lastname: Yousefinejad
authors.affiliation: Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
authors.forename: N
authors.initials: N
authors.lastname: Salehi
authors.affiliation: Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran.
authors.forename: A A
authors.initials: AA
authors.lastname: Saboury
authors.affiliation: Department of Ophthalmology and Visual Sciences and McPherson Eye Research Institute, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA.
authors.forename: N
authors.initials: N
authors.lastname: Sheibani
authors.affiliation: Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran. Electronic address: moosavi@ut.ac.ir.
authors.forename: A A
authors.initials: AA
authors.lastname: Moosavi-Movahedi
mesh_terms: D002942:Circular Dichroism; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
chemical_list: D006454:Hemoglobins; C517773:ferrylhemoglobin; C100003:deoxyhemoglobin
keywords: Chemical unfolding; Chemometrics; Curve resolution; Folding intermediate; Hemoglobin; Spectroscopy