Fluorescence Studies on Denaturation and Stability of Recombinant Human Interferon-Gamma

Petya Christova 1 , Kristina Todorova 2 , Ilijana Timtcheva 1 , Genoveva Nacheva 2 , Andrey Karshikoff 3  and Peter Nikolova 1
  • 1 Institute of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences, “Acad. G. Bonchev”, Str., bl. 9, 1113 Sofia, Bulgaria
  • 2 Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
  • 3 Department of Biosciences at Novum, Karolinska Institutet, Huddinge, Sweden

Unfolding/folding transitions of recombinant human interferon-gamma (hIFNγ) 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 ΔG0 = 13.4 kJ/mol) than urea (C* = 2.8 m and ΔG0 = 11.7 kJ/mol). The close ΔG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNγ is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNγ 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 hIFNγ, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNγ.

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A Journal of Biosciences: Zeitschrift für Naturforschung C (ZNC) is an international scientific journal for the emerging field of natural and natural-like products. ZNC publishes original research on the isolation, bio-chemical synthesis and bioactivities of natural products, their biochemistry, pharmacology, biotechnology, and biological activity and innovative developed computational methods for predicting their structure and/or function.

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