The effects of microviscosity, bound water and protein mobility on the radiolysis and sonolysis of hen egg white

There is a great deal of work on proteins in aqueous solutions aimed at understanding their radiological and ultrasonic damage and the associated biochemical impacts. However, how these data are used and interpreted when proteins (and other biomacromolecules) are organized in three-dimensional structures (as in real biological systems) has been explored to a much lesser extent even though in such structural arrangements important and unique physicochemical effects exist. In this Dissertation, the effects of such physicochemical properties, including microviscosity, bound water, and protein mobility, on the radiolytic and sonolytic denaturation of hen egg white were studied. To improve our understanding of the molecular level effects of radiation and ultrasound on protein biomacromolecular gels (as a step towards complex biological systems), the thick fraction of egg white is used as a convenient and economical model system for the luminal mammalian mucous membrane as both are hydrogels (∼90% water) that contain highly glycosylated, cysteine-rich and hydrated proteins.;In this Dissertation, measurement of rate constants for diffusion-controlled reactions were used to investigate the microlevel rheological properties of macromolecular aqueous solutions and protein gels; however, the kinetics of reactions with highly charged reactants were found to be complicated by ionic effects. We developed a method to give a second-order rate constant where these ionic effects were not operative (Krise et al. 2010. Phys. Chem. Chem. Phys. 12: 7695). As a precursor to studies in biological macromolecular aqueous gels (hen egg white thick fraction), we assessed the macro- and microscale rheological properties of a simple polymeric model system, poly(vinyl alcohol) (PVA) solutions [up to 10% (w/w)], using diffusion-controlled reaction rate constants and electric conductivity measurements. From these measurements, PVA aqueous solutions can be envisioned as dynamic systems comprising hydrated PVA molecules (two or less water molecules per OH group) and "interconnected water pools" (located between macromolecules), the rheological properties of which are very similar to that of bulk water (Krise et al. 2011. J. Phys. Chem. B. 115: 2759). These studies were extended to assess the physicochemical properties of the thick fraction of egg white. Measurements of the rate constants of diffusion-controlled reactions occurring within the gel (and corresponding activation energies) and electric conductivity revealed that the thick fraction of egg white can be envisioned as a three-dimensional network comprising hydrated protein molecules (held by intermolecular S-S bridges) surrounded by water pools and channels (of non-uniform diameters) that have a microviscosity very similar to that of bulk water (Krise and Milosavljevic. 2011. Biomacromolecules. 12: 2351).;With the physicochemical properties of the thick and thin fractions of hen egg white now more thoroughly characterized, a deeper understanding of early events in radiation chemistry observed in these proteinaceous matrixes was obtained. From pulse radiolysis experiments, the hydrated electron absorption spectra (measured at the end of the pulse) and radiolytic yield indicate that bound water does not affect the hydrated electron yield. Due to the very low microviscosity, hydrated electrons are as reactive and mobile in thick fraction hydrogel as in bulk water. Steady-state gamma radiolysis studies on thick and thin fraction (a true protein solution) revealed that immobility of egg white proteins disfavors reactions that result in molecular mass change.;To understand the effects of ultrasound on protein gels at the molecular level, the thick fraction of egg white was sonicated and its immeasurably high viscosity decreased significantly (to 2.5 - 4.0 mPa·s) with a 12 minute sonication, indicating that the three-dimensional protein network was degraded by ultrasound. Differential scanning calorimetry, sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and turbidity measurements confirm that inter-protein S-S bridges rather than the primary structure of constituent proteins are disrupted by ultrasound. Despite the relatively large concentration of hydroxyl radical produced in the sonication time range applied, no protein agglomeration or scission was observed which can be attributed to the antioxidant properties of glucose in the thick fraction; therefore, hydrodynamic sheer (from the collapse of bubbles produced by cavitation) was the operative mechanism of ultrasonic degradation of the thick fraction of hen egg white in the 55 kHz ultrasonic bath used.