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We have continued our work on understanding the role of cosolvents on enzyme precipitation and on the morphology of the obtained aggregates. This work has involved two graduate students and one undergraduate student. Their work has significantly contributed to the completion of two PhD dissertations. These two graduate students are currently employed in a chemical company (Alcon Research Ltd) and University (postdoctoral position at MIT). Moreover, PRF funds has allowed the PI’s research group to publish, so far, six papers in Journals with impact factor higher than 4.0. The PI reports below the specific results obtained for the September 2008-September 2009 period.

Role of hydration and preferential hydration of macromolecules. Hydration (the number of water molecules bound to a macromolecule) and preferential hydration (the excess of water molecules surrounding a macromolecule compared to bulk composition) of macromolecules are two distinct properties of their multicomponent aqueous solutions. They are involved in the thermodynamic stability of synthetic and biological macromolecules, and in the catalytic activity of enzymes. We have experimentally investigated ternary diffusion in a macromolecule-osmolyte-water system in order to characterize and compare these two independent properties of macromolecules. Specifically, we report the four diffusion coefficients for the poly(ethylene glycol)-di(ethylene glycol)-water system at 25ºC using Rayleigh interferometry. In this work, the molecular weight of poly(ethylene glycol) (PEG) is 200-fold higher than that of di(ethylene glycol) (DEG). This ratio is comparable to that between proteins and low molecular-weight osmolytes. This system has been selected because both solutes are neutral hydrated species with similar chemical properties and very different size. Hence, the observed ternary-diffusion behavior can be directly related to solute hydration and size ratio and is not complicated by other factors such as ionic interactions usually encountered in protein systems. Using our ternary diffusion coefficients, we have found that PEG hydration is slightly smaller than its preferential hydration. The observed difference can be attributed to PEG-DEG excluded-volume interactions. Our experimental results also enable us to reveal that Onsager cross-transport coefficients are large and negative. This implies that this transport coefficient should not be neglected in multicomponent-diffusion theoretical models even when ionic interactions or chemical association between the solute species are absent. This work provides the basis for understanding coupled diffusion in more complex aqueous systems such as those containing charged enzyme proteins in the presence of salts, osmolytes and organic solvents. This work has been published in Phys. Chem. Chem. Phys., 2009, DOI: 10.1039/b910152g.

Enzyme diffusion in multicomponent mixtures. Enzyme diffusion coefficients are important kinetic parameters for characterizing reaction kinetics in non-equilibrium systems and the kinetics of phase transitions.  Many enzyme solutions involve both the use of salts and polymer additives, especially for precipitation protocols. Dynamic light scattering is the main technique used to characterize diffusion coefficients of macromolecules. However it provides pseudo-binary diffusion coefficients in multicomponent mixtures.  For the first time, we have rigorously investigated quaternary diffusion in an enzyme-polymer-salt-water system. Specifically, we have measured the nine quaternary diffusion coefficients required for describing the lysozyme-poly(ethylene glycol)-NaCl-water system at pH 4.5 and 25 ºC using precision Rayleigh interferometry. Lysozyme is a model protein for protein-crystallization and enzymology studies. We find that the enzyme diffusion coefficient decreases as polymer concentration increases at a given salt concentration. This behavior can be quantitatively related to the corresponding increase in fluid viscosity only at low polymer concentration. However, at high polymer concentration (250 g/L), enzyme diffusion is enhanced compared to the corresponding viscosity prediction. Here the application of the Stokes-Einstein equation to extract the enzyme hydrodynamic radius may becom inaccurate.  We also find that an enzyme concentration gradient induces significant salt diffusion from high to low enzyme concentration. This effect increases in the presence of poly(ethylene glycol). Finally, we have evaluated systematic errors associated with measurements of enzyme diffusion coefficients by dynamic light scattering. This work describes enzyme diffusion in crowded environments and provide guidance for further theoretical developments in the field of enzymology. This work has been published in J. Phys. Chem. B, 2009, Articles ASAP.

 Enzyme aggregation at the liquid-liquid interface. For the enzyme laccase, we have experimentally shown that the liquid-liquid interface between two aqueous solutions can be used to induce enzyme aggregation. Specifically, we have prepared a biphasic system by mixing ammonium sulfate and poly(ethylene glycol) with water. Due to the net repulsive interactions between ammonium sulfate and poly(ethylene glycol), the first phase is rich in ammonium sulfate and the second phase is rich in poly(ethylene glycol). We have then added purified laccase at very low concentration to the biphasic system. Using an enzymatic assay, we have discovered that laccase concentration becomes enriched at the liquid-liquid interface. No laccase aggregation was observed in two coexisting aqueous phases taken separately. The formation of enzymatically-active laccase films at the interface becomes visible at relatively high laccase concentration. Interestingly, these films are birefringent under polarized light. We have hypothesized that 1) the liquid-liquid interface is depleted of both precipitants due to net repulsive interactions between ammonium sulfate and poly(ethylene glycol), 2) enzyme molecules diffuses towards high to low precipitant concentration due to protein-precipitant net repulsive interactions. These two hypotheses should explain protein adsorption at the water-rich liquid-liquid interface. This work is currently in progress.