title: Fluorescence studies on denaturation and stability of recombinant human interferon-gamma.
abstract: Unfolding/folding transitions of recombinant human interferon-gamma (hIFNgamma) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy. At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 M and deltaG0 = 13.4 kJ/mol) than urea (C* = 2.8 M and deltaG0 = 11.7 kJ/mol). The close deltaG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNgamma is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNgamma remains native in the pH range of 4.8-9.5. Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified. Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNgamma, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNgamma.
authors.affiliation: Institute of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev, Str., bl. 9, 1113 Sofia, Bulgaria.
authors.forename: Petya
authors.initials: P
authors.lastname: Christova
authors.forename: Kristina
authors.initials: K
authors.lastname: Todorova
authors.forename: Ilijana
authors.initials: I
authors.lastname: Timtcheva
authors.forename: Genoveva
authors.initials: G
authors.lastname: Nacheva
authors.forename: Andrey
authors.initials: A
authors.lastname: Karshikoff
authors.forename: Peter
authors.initials: P
authors.lastname: Nikolov
mesh_terms: D002021:Buffers; D003001:Cloning, Molecular; D004355:Drug Stability; D004926:Escherichia coli; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry*; D011489:Protein Denaturation; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence
chemical_list: D002021:Buffers; D011994:Recombinant Proteins; D007371:Interferon-gamma