Vitrification and structural relaxation of a water-swollen protein, wheat gluten and the thermodynamics of its water-protein<->ice equilibrium

To understand the chemical physics of molecular motions in a structurally simple, vegetable native protein containing absorbed water, in which both intermolecular and intramolecular interactions occur, the nature of the glass-transition and structural relaxation of vitrified 52.8 wt.% water swollen wheat gluten have been studied by differential scanning calorimetry (DSC), as has the water <-> ice phase transformation. Experiments performed during both cooling and heating and with samples of different thermal histories show a broad endothermic feature beginning at ca. 160 K which is barely interrupted by a partial-crystallization exotherm at ca. 250 K. The endothermic features resembled those observed for several simpler hydrated proteins (Biophys. J., 1994, 66, 249; J. Phys. Chem., 1994, 98, 13780), a hydrated crosslinked polymer (J. Phys. Chem., 1990, 94 2689) and a dry interpenetrating network polymer blend (J. Polym. Sci. B, 1992, 32, 683). Their broadness is a result of the closely spaced multiplicity of small but sharp mini-endotherms and has its origin in the onset of different configurational substrates that become available to the protein's structure as the temperature is increased. The remarkable similarity of these features amongst a broad class of materials is a reflection of the predominant role of the intermolecular energy barriers in determining the structural relaxation kinetics. This has been described in terms of a time-dependent potential-energy surface, which represent a hierarchy of molecular and segmental motions. Ice and freeze-concentrated solution coexist at a thermodynamic equilibrium at T < 273 K. Their respective amounts have been measured between 260 and 273 K and formalism based on equilibrium thermodynamics has been developed, and the DSC scans for cooling simulated. This formalism agrees with the data. The temperature variation of the equilibrium constant for the protein-water <-> protein-ice coexistence does not agree with that given by the Gibbs-Helmholtz equation, which is a reflection of strong interactions between the water molecules and H-bonding protein segments. The interpretation in terms of the role of protein dynamics in the crystallization of water, and the formalism developed, are general and useful for studies on other complex biomaterials.