Pressure-Temperature Stability, Ca2+ Binding, and Pressure-Temperature Phase Diagram of Cod Parvalbumin: Gad m 1

Fish allergy is associated with IgE-mediated hypersensitivity reactions to parvalbumins, which are small calcium-binding muscle proteins and represent the major and sole allergens for 95% of fish-allergic patients. We performed Fourier transform infrared and tryptophan fluorescence spectroscopy to explore the pressure-temperature (p-T) phase diagram of cod parvalbumin (Gad m 1) and to elucidate possible new ways of pressure-temperature inactivation of this food allergen. Besides the secondary structure of the protein, the Ca2+ binding to aspartic and glutamic acid residues was detected. The phase diagram was found to be quite complex, containing partially unfolded and molten globule states. The Ca2+ ions were essential for the formation of the native structure. A molten globule conformation appears at 50 degrees C and atmospheric pressure, which converts into an unordered aggregated state at 75 degrees C. At >200 MPa, only heat unfolding, but no aggregation, was observed. A pressure of 500 MPa leads to a partially unfolded state at 27 degrees C. The complete pressure unfolding could only be reached at an elevated temperature (40 degrees C) and pressure (1.14 GPa). A strong correlation was found between Ca2+ binding and the protein conformation. The partially unfolded state was reversibly refolded. The completely unfolded molecule, however, from which Ca2+ was released, could not refold. The heat-unfolded protein was trapped either in the aggregated state or in the molten globule state without aggregation at elevated pressures. The heat-treated and the combined heat- and pressure-treated protein samples were tested with sera of allergic patients, but no change in allergenicity was found.