STRUCTURE RELAXATION OF A VITRIFIED HIGH-PROTEIN FOOD, BEEF, AND THE PHASE-TRANSFORMATIONS OF ITS WATER-CONTENT

To gain insight into the molecular relaxations in mixtures of structur ally complex proteins in which both the intramolecular and intermolecu lar interactions dominate, the nature of the glass transition of vitri fied beef, the crystallization of its water content, the melting of th e thus formed ice, and the ice <-> water phase equilibrium have been i nvestigated by differential scanning calorimetry (DSC) during both hea ting and cooling of the material at different rates from 298 to 103 K. The endothermic feature associated with the onset of molecular mobili ty appeared over a broad temperature range acid resembled that observe d 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 c ross-linked polymer (J. Phys. Chem. 1990, 94, 2689), and hydrated low molecular weight poly-homopeptides (J. Phys. Chem. 1994, 98, 13780). T hese broad features are attributed to the onset of the availability of different configurations when thermal activation causes the populatio ns in the configurational substates to change almost continuously with changing temperature. This is tantamount to a very broad distribution of relaxation rimes or a broad distribution of energy barriers betwee n the various substates, which also involve R-bonded water. The remark able resemblance between the calorimetric features of the chemically c omplex (and containing a mixture of proteins with other ionic and orga nic materials) state and that of the simpler state of pure polymers, w here 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 b y intermolecular barriers rather than intramolecular barriers. Water a nd ice coexist at a thermodynamic equilibrium at all temperatures belo w 273 K. Their respective amounts have been mesured down to 255 K, and a formalism based on equilibrium thermodynamics has been developed. B y using this formalism, the value of the rate constant for the freezin g equilibrium, and the difference between the C-p of ice and solution, the DSC scans for the crystallization on cooling have been simulated. This formalism agrees with the experimental data. The temperature var iation of the equlibrium constant for protein-water <-> protein-ice co existence does not agree with that given by the Gibbs-Helmholtz equati on, which is a reflection of strong interactions between the water mol ecules and H-bonding protein segments as well as of the freeze concent ration of the dissolved ionic and nonionic impurities on cooling. Expe riments 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 (mel 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 stil l short at those temperatures where the slowest moving segments of the protein molecules lose their mobility during cooling, and regain duri ng heating, it seems that the motion of the protein's segments control s the rate of the crystallization process, at least after a certain lo w fractional concentration of uncrystallized water has been reached. T he thermodynamic processes observed, their interpretation, the methods of data analysis, and the formalism developed in this paper are as ap plicable 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 multiba rrier diffusion for which the barrier hight itself is time-variant.