title: Characterization and analysis of binding of Thioflavin T with partially folded and native states of α-lactalbumin protein by calorimetric and spectroscopic techniques.
abstract: We have analysed binding of Thioflavin T (ThT) with molten globule (MG) and native (N) states of α-lactalbumin (α-LA) by using calorimetry and spectroscopy. ThT has been widely used for detection of amyloid fibrils from enhancement of its fluorescence emission intensity. Instead of the spectral changes of ThT, we, rather, monitored the changes occurring in the spectral properties of the MG and N states upon interaction with ThT, from fluorescence, absorbance and circular dichroism (CD) spectroscopy. Our novel and most important observation is non-fluorescent mode of binding of ThT to the MG state of α-LA suggesting that the mechanism of binding is distinctly different from that of association with the protein fibrils. CD and DLS (Dynamic Light Scattering) results show the absence of any major structural and size changes in the protein upon ThT binding. The thermodynamic parameters of binding of ThT with the MG and N states of α-LA obtained from Isothermal Titration Calorimetry (ITC) experiments show the formation of stable complex between ThT and α-LA (both MG and N states) and also provide insights on the probable mode of interaction of ThT with the MG and N states of α-LA.
authors.affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India.
authors.forename: Moumita
authors.initials: M
authors.lastname: Dasgupta
authors.affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India. Electronic address: nandk@chem.iitb.ac.in.
authors.forename: Nand
authors.initials: N
authors.lastname: Kishore
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002151:Calorimetry; D002417:Cattle; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013057:Spectrum Analysis; D013816:Thermodynamics; D013844:Thiazoles / Q000378:metabolism*
chemical_list: D052160:Benzothiazoles; D013844:Thiazoles; C009462:thioflavin T; D007768:Lactalbumin
keywords: Binding thermodynamics; Isothermal titration calorimetry; Molten globule state; Thioflavin T; α–Lactalbumin