Tyrosine/tyrosinate fluorescence at 700 MPa: A pressure unfolding study of chicken ovomucoid at pH 12 Original Research Article

The utility of tyrosine/tyrosinate fluorescence for pressure-unfolding studies of Trp-lacking proteins has been explored for the first time, with chicken ovomucoid (OVM) as target. A newly developed fluorescence spectrometer working in the range 0.1–700 MPa is employed for this purpose. At 25 °C at pH 12, all six Tyr residues give tyrosine emission at 306 nm, implying that all five Tyr residues are well buried at pH 12 in the folded OVM, except one giving “half-tyrosinate” emission at 325 nm. Upon increasing pressure, however, a distinct intermediate state, in which domains 1 and 2 are selectively unfolded, appears and increases up to 700 MPa. Extrapolated to 0.1 MPa, this intermediate lies 8.8 ± 2.6 kJ mol− 1 above the native state, characterized with a partial molar volume smaller by − 28.9 ± 7.4 ml mol− 1. At 5 °C at 700 MPa, even domain 3 gives a sign of cold denaturation.