Structural Relaxation of a Vitrified High-Protein Food, Beef, and the Phase Transformations of Its Water Content

Abstract

Listen

Translate article

To gain insight into the molecular relaxations in mixtures of structurally complex proteins in which both the intramolecular and intermolecular interactions dominate, the nature of the glass transition of vitrified beef, the crystallization of its water content, the melting of the thus formed ice, and the ice ↔ water phase equilibrium have been investigated by differential scanning calorimetry (DSC) during both heating and cooling of the material at different rates from 298 to 103 K. The endothermic feature associated with the onset of molecular mobility appeared over a broad temperature range and resembled that observed for less complex proteins, e.g., hemoglobin, myoglobin, and lysozyme (Biophys. J. 1994, 66, 249), an interpenetrating network polymer (J. Polym. Sci., Part B: Polym. Phys. 1994, 32, 683), a water-containing cross-linked polymer (J. Phys. Chem. 1990, 94, 2689), and hydrated low molecular weight poly-homopeptides (J. Phys. Chem. 1994, 98, 13780). These broad features are attributed to the onset of the availability of different configurations when thermal activation causes the populations in the configurational substates to change almost continuously with changing temperature. This is tantamount to a very broad distribution of relaxation times or a broad distribution of energy barriers between the various substates, which also involve H-bonded water. The remarkable resemblance between the calorimetric features of the chemically complex (and containing a mixture of proteins with other ionic and organic materials) state and that of the simpler state of pure polymers, where segments of the same molecules interact mutually with the water H-bonded to it, underscores the fact that molecular degrees of freedom involved in configurational relaxations are controlled predominantly by intermolecular barriers rather than intramolecular barriers. Water and ice coexist at a thermodynamic equilibrium at all temperatures below 273 K. Their respective amounts have been mesured down to 255 K, and a formalism based on equilibrium thermodynamics has been developed. By using this formalism, the value of the rate constant for the freezing equilibrium, and the difference between the Cp of ice and solution, the DSC scans for the crystallization on cooling have been simulated. This formalism agrees with the experimental data. The temperature variation of the equlibrium constant for 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 as well as of the freeze concentration of the dissolved ionic and nonionic impurities on cooling. Experiments with samples containing different amounts of water have shown that the enthalpy of melting remains at 5.48 ± 0.74 kJ/mol, and that 0.011 (mol of water)/(g of sample) do not freeze on cooling at rates as low as 30 K/min. For higher cooling rates, the amount that remains unfrozen is more than that. Since the relaxation time of water is still short at those temperatures where the slowest moving segments of the protein molecules lose their mobility during cooling, and regain during heating, it seems that the motion of the protein's segments controls the rate of the crystallization process, at least after a certain low fractional concentration of uncrystallized water has been reached. The thermodynamic processes observed, their interpretation, the methods of data analysis, and the formalism developed in this paper are as applicable to simpler proteins and synthetic polymers as to the complex mixtures in which proteins are found in nature. Those configurational relaxations have been considered and illustrated in terms of a multibarrier diffusion for which the barrier hight itself is time-variant.