Evidence of molten globule like state(s) of interferon gamma in acidic and sodium perchlorate solutions

Abstract

Recombinant porcine interferon gamma (IFN γ) at neutral pH is characterized by a tryptophan (Trp) fluorescence maximum around 343 nm and a rigid conformation, evidenced from tryptophan polarization values. Guanidine HCl shifts the protein emission spectra further to the red and decreases the fluorescence polarization values, indicating denatured IFN γ in these solutions. In acidic solutions (3 <pH <4), the emission spectra show a blue shift and lower tryptophan polarization. The midpoint of transition of these fluorescence properties occurs around pH 3.5–3.6. The protein in NaClO 4 solution at neutral pH is similarly characterized by a blue shift in the tryptophan fluorescence maxima and low polarization values.The extent of quenching of tryptophan fluorescence by acrylamide is less in acid and in NaClO 4 solutions of IFN γ compared to its native form. This indicates a lower accessibility of the tryptophan in the altered conformation of the protein. The emission spectra of IFN γ in NaClO 4 solution shows a decrease in the tryptophan fluorescence intensity with simultaneous shift of the emission spectra over time. The presence of two conformational forms of IFN γ in perchlorate solution is evidenced from an isofluorescent point at 315 nm. The change in the conformational state in perchlorate solution is characterized by first order kinetics. The dye anilinonaphthalene sulfonic acid does not bind either to the native IFN γ or to its denatured form. However, the dye binds to the acid form of IFN γ , as well as when the protein is present in NaClO 4 solution at neutral pH. These observations, together with the results from literature that IFN γ retains its secondary structure in acid solution to a considerable degree, would suggest that the protein exists as a molten globule-like state in acidic solution. Similarities of the protein fluorescence and 1-anilino-8-naphthalene-sulfonic-acid (ANS) binding properties of the protein in NaClO 4 and acid solutions indicate that IFN γ also exists in a molten globule-like state in perchlorate solution at neutral pH.