CHARACTERIZING THE SECONDARY HYDRATION SHELL ON HYDRATED MYOGLOBIN, HEMOGLOBIN, AND LYSOZYME POWDERS BY ITS VITRIFICATION BEHAVIOR ON COOLING AND ITS CALORIMETRIC GLASS-]-LIQUID TRANSITION AND CRYSTALLIZATION BEHAVIOR ON REHEATING

For hydrated metmyoglobin, methemoglobin, and lysozyme powders, the fr eezable water fraction of between similar to 0.3-0.4 g water/g protein up to similar to 0.7-0.8 g water/g protein has been fully vitrified b y cooling at rates up to similar to 1500 K min(-1) and the influence o f cooling rate characterized by x-ray diffractograms. This vitreous bu t freezable water fraction started to crystallize at similar to 210 K to cubic ice and at similar to 240 K to hexagonal ice. Measurements by differential scanning calorimetry have shown that this vitreous but f reezable water fraction undergoes, on reheating at a rate of 30 K min( -1), a glass-->liquid transition with an onset temperature of between similar to 164 and similar to 174 K, with a width of between similar t o 9 and similar to 16 degrees and an increase in heat capacity of betw een similar to 20 and similar to 40 J K-1 (mol of freezable water)(-1) but that the glass transition disappears upon crystallization of the freezable water. These calorimetric features are similar to those of w ater imbibed in the pores of a synthetic hydrogel but very different f rom those of glassy bulk water. The difference to glassy bulk water's properties is attributed to hydrophilic interaction and H-bonding of t he macromolecules' segments with the freezable water fraction, which t hereby becomes dynamically modified. Abrupt increase in minimal or cri tical cooling rate necessary for complete vitrification is observed at similar to 0.7-0.8 g water/g protein, which is attributed to an abrup t increase of water's mobility, and it is remarkably close to the thre shold value of water's mobility on a hydrated protein reported by Kimm ich et al. (1990, Biophys. J. 58:1183). The hydration level of similar to 0.7-0.8 g water/g protein is approximately that necessary for comp leting the secondary hydration shell.