Lars Øgendal

Characterization and Isolation of Intermediates in β-Lactoglobulin Heat Aggregation at High pH

The early stages of heat induced aggregation at 67.5 degrees C of beta-lactoglobulin were studied by combined static light scattering and size exclusion chromatography. At all conditions studied (pH 8.7 without salt and pH 6.7 with or without 60 mM NaCl) we observe metastable heat-modified dimers, trimers, and tetramers. These oligomers reach a maximum in concentration at about the time when large aggregates (1000-4000 kg/mol) appear, after which they decline in concentration. By isolating the oligomers it was demonstrated that they rapidly form aggregates upon heating in the absence of monomeric protein, showing that these species are central to the aggregation process. To our knowledge this is the first time that intermediates in protein aggregation have been isolated. At all stages of aggregation the dominant oligomer was the heat-modified dimer. Whereas the heat-modified oligomers are formed at a higher rate at pH 8.7 than at pH 6.7, the opposite is the case for the formation of aggregates from the metastable oligomers indicating cross-linking via disulfide bridges for the oligomers and noncovalent interaction in the formation of the aggregates. The data suggest that an aggregate nucleus is formed from four oligomers. For protein concentrations of 10 or 20 g/l a heat-modified monomer can be observed until about the time when the maximum in concentration appears of the heat-modified dimer. The disappearance of this heat-modified monomer correlates to the formation of dimers (trimers and tetramers).

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.

Kinetics of the renneting reaction followed by measurement of turbidity as a function of wavelength

In order to describe the kinetics of rennet coagulation, measurements of turbidity as a function of wavelength were used to determine the weight-average degree of polymerization, x¯ w , during renneting of milk at three different concentrations of enzyme and three concentrations of casein, including the normal casein concentration of milk. The change of x¯ w as a function of time was described using Von Smoluchowskis equation, testing a number of expressions for the aggregation rate constant, k ij . The best description was achieved when k ij was taken as a function of an energy barrier against aggregation that was diminished by the proteolysis of κ-casein. The initial value of the energy barrier partly depended on the casein concentration, and had a value >25 k B T at normal casein concentration, where k B is Boltzmanns constant and T the absolute temperature. When the proteolysis of κ-casein was complete, the energy barrier was reduced to 11 k B T and was independent of casein concentration.

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.