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

Abstract

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 Ca(2+) 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 Ca(2+) ions were essential for the formation of the native structure. A molten globule conformation appears at 50 °C and atmospheric pressure, which converts into an unordered aggregated state at 75 °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 °C. The complete pressure unfolding could only be reached at an elevated temperature (40 °C) and pressure (1.14 GPa). A strong correlation was found between Ca(2+) binding and the protein conformation. The partially unfolded state was reversibly refolded. The completely unfolded molecule, however, from which Ca(2+) 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.

Solution and high-pressure NMR studies of the structure, dynamics, and stability of the cross-reactive allergenic cod parvalbumin Gad m 1

“…Our data showed that the structural features of parvalbumins can also be correlated to IgE cross‐reactivity. As documented in the literature, cod parvalbumin is more closely related to the parvalbumins of carp than of mackerel . Van do et al .…”

“…The reduction of the IgE‐binding ability is protein‐specific. Cyp c 1 from carp and Sco j 1 from Pacific mackerel were observed to be very sensitive to Ca 2+ ‐depletion, while only small changes in allergenicity were observed for Gad c 1 . Therefore, regions of the protein beyond the ion‐binding sites may contribute to IgE‐epitopes.…”

Protein Denaturation on p-T Axes – Thermodynamics and Analysis

“…The phase diagram of cod parvalbumin is also quite exotic (Fig. 2.8) (Somkuti et al 2012). Although the overall elliptic shape can be recognized on the diagram, a number of states appear besides the usual folded and unfolded states.…”

“…In the meantime several practical applications appeared: pressure treatment of biological systems, like microorganisms and complex food systems were studied and some of these studies led to the development of products which were successfully commercialized Yamakura et al 2005;Rastogi et al 2007;Yaldagard et al 2008;Knorr et al 2011). Simultaneously more and more proteins were experimentally investigated and characterized under pressure as part of basic research projects (Brandts et al 1970;Hawley 1971;Panick et al 1999;Smeller et al 1999;Lassalle et al 2000;Meersman et al 2002Meersman et al , 2005Smeller 2002Smeller , 2009Maeno et al 2009;Somkuti et al 2012Somkuti et al , 2013a.…”

“…He also analyzed the data obtained on ribonuclease-A by Brandts (Brandts et al 1970). Since then a number of proteins were studied under pressure, and the complete p-T phase diagrams have been determined for several of them (Zipp and Walter 1973;Panick et al 1999;Lassalle et al 2000;Meersman et al 2005;Smeller 2002;Maeno et al 2009;Somkuti et al 2011Somkuti et al , 2012Somkuti et al , 2013a. This review summarizes the present knowledge about the p-T phase diagram of protein unfolding.…”