Available online 29 July 2013
Author(s): Akihiro Maeno , Hiroshi Matsuo , Kazuyuki Akasaka
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 0.1-700MPa is employed for this purpose. At 25°C at pH12, all six Tyr residues give tyrosine emission at 306nm, implying that all five Tyr residues are well buried at pH12 in the folded OVM, except one giving “half-tyrosinate” emission at 325nm. Upon increasing pressure, however, a distinct intermediate state, in which domains 1 and 2 are selectively unfolded, appears and increases up to 700MPa. Extrapolated to 0.1MPa, this intermediate lies 8.8±2.6kJmol-1
above the native state, characterized with a partial molar volume smaller by -28.9±7.4mlmol-1
. At 5°C at 700MPa, even domain 3 gives a sign of cold denaturation.