Solute effects on the irreversible aggregation of serum albumin.

Thermal stress on bovine serum albumin (BSA) promotes protein aggregation through the formation of intermolecular beta-sheets. We have used light scattering and chromatography to study effects of (<1 M) Na(2)SO(4), NaSCN, sucrose, sorbitol and urea on the rate of the thermal aggregation. Both salts were strong inhibitors of BSA aggregation and they reduced both the size and number (concentration) of aggregate particles compared to non-ionic solutes (or pure buffer). Hence, the salts appear to suppress both nucleation- and growth rate. The non-electrolyte additives reduced the initial aggregation rate (compared to pure buffer), but did not significantly limit the extent of aggregation in samples quenched after 27 min. heat exposure (40-50% aggregation in all samples). The non-electrolytes did, however, modify the aggregation process as they consistently brought about smaller but more concentrated aggregates than pure buffer. The results are discussed along the lines of linkage- and transition state theories. In this framework, the rate of the aggregation process is governed by the equilibrium between a thermally denatured state (D) and the transition state D( not equal). Thus, the effect of a solute relies on its preferential interactions with respectively D and D( not equal). The current results do not show any correlation between the solutes' preferential interactions with native BSA and their effect on the rate of aggregation. This suggests that non-specific, "Hofmeister-type" interactions, which scale with the solvent accessible surface area, are of minor importance. Rather, salt induced suppression of aggregation is suggested to depend on the modulation of specific electrostatic forces in the D( not equal) state.

The effect of high pressure on the functional properties of pork myofibrillar proteins.

Complementary methodologies were used to analyse the pressure-induced modification and functionality of myofibrillar proteins from pork meat pressurised at 200, 400, 600, or 800 MPa (10 min, 5 or 20 °C). Pressure at 400 MPa was found to be the threshold for loss of solubility, and the structural proteins, myosin and actin, lost their native solubility due to aggregation. The results from the extraction of proteins with different reagents targeting the disruption of specific molecular interactions suggested that pressure-induced aggregation was caused mainly by hydrogen bonding during pressurisation and not hydrophobic interactions nor disulphide cross-links. Furthermore, the soluble proteins were exposed to remarkable structural changes already at 200 MPa and lost their native functionality. The modification of the proteins in pressurised meat affected the water binding sites of the myofibrillar proteins and, thereby, the interactions between proteins and water molecules, and distribution between myofibrillar and extra-myofibrillar compartments.

Light scattering coupled with reversed phase chromatography to study protein self-association under separating conditions.

An on-line method, coupling reversed phase chromatography with static light scattering, was developed to determine the association state of freshly eluted proteins. Under downstream process conditions, human insulin desB30 and human insulin AspB28 were tested at concentrations up to 8.5mg/mL. The refractive index increment (dn/dc) for insulin was found to depend strongly on the solvent used. A refractive index increment of 0.184±0.003mL/g was found in an aqueous buffer, pH 7.4, whereas the value was 0.155±0.003mL/g in 30%, w/w ethanol. The methodology combines on-line SLS and UV measurements with the pre-determined refractive index increment values. The developed on-line method was verified by standard off-line measurements establishing the association state at concentrations between 0.2 and 6.0mg/mL. The equipment was calibrated utilizing insulin under conditions reported to ensure either monomer or hexamer forms. The self-association of human insulin desB30 was found to be strongly suppressed in 30%, w/w ethanol at pH 7.4 in which the monomer predominates. When stabilized by zinc ions in 30%, w/w ethanol at pH 7.4, an average association number of 3.7 was found. These data demonstrate the effect of ethanol to lower strongly the energy advantage by protein self-association. Potassium chloride and/or calcium chloride in the eluents were found to be of no consequence to the association state.

Droplet surface properties and rheology of concentrated oil in water emulsions stabilized by heat-modified beta-lactoglobulin B.

Effects of substituting native beta-lactoglobulin B (beta-lactoglobulin) with heat-treated beta-lactoglobulin as emulsifier in oil in water emulsions were investigated. The emulsions were prepared with a dispersed phase volume fraction of Phi=0.6, and accordingly, oil droplets rather closely packed. Native beta-lactoglobulin and beta-lactoglobulin heated at 69 degrees C for 30 and 45 min, respectively, in aqueous solution at pH 7.0 were compared. Molar mass determination of the species formed upon heating as well as measurements of surface hydrophobicity and adsorption to a planar air/water interface were made. The microstructure of the emulsions was characterized using confocal laser scanning microscopy, light scattering measurements of oil droplet sizes, and assessment of the amount of protein adsorbed to surfaces of oil droplets. Furthermore, oil droplet interactions in the emulsions were quantified rheologically by steady shear and small and large amplitude oscillatory shear measurements. Adsorption of heated and native beta-lactoglobulin to oil droplet surfaces was found to be rather similar while the rheological properties of the emulsions stabilized by heated beta-lactoglobulin and the emulsions stabilized by native beta-lactoglobulin were remarkably different. A 200-fold increase in the zero-shear viscosity and elastic modulus and a 10-fold increase in yield stress were observed when emulsions were stabilized by heat-modified beta-lactoglobulin instead of native beta-lactoglobulin. Aggregates with a radius of gyration in the range from 25 to 40 nm, formed by heating of beta-lactoglobulin, seem to increase oil droplet interactions. Small quantities of emulsifier substituted with aggregates have a major impact on the rheology of oil in water emulsions that consist of rather closely packed oil droplets.