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 aggregation properties of zinc-free insulin have been studied using static and dynamic light scattering. The aggregation has been investigated as a function of three parameters, the concentration of sodium chloride (in the range 10-100 mM), the pH value (in the range pH 7.5-10.5), and the insulin concentration (1.8-13.4 mg/mL). The measured homodyne autocorrelation function was used to determine the apparent mean hydrodynamic diameter as well as the apparent weight-averaged molar mass of the insulin species in solution. A method of data analysis was employed, which allows the separation of light scattering contributions from the insulin oligomers and from irrelevant macromolecules and possible impurities present in the sample solutions. Also, a simple phenomenological equilibrium model describing the association of oligomers of insulin is presented. One aspect of this model is that it makes it possible to determine weight average molar masses corrected for virial effects on the Rayleigh ratio. This was necessary because virial effects cannot be isolated and corrected for by dilution since this would change the equilibrium distribution of oligomers. The basis of the model is a positive contribution to Gibbs free energy from charge repulsion depending on the protein charge and the number of monomers in the oligomers, and an assumed constant negative contribution to Gibbs free energy arising from either an entropic gain or hydrogen bonding upon association. The equilibrium model gives a good description of both the apparent weight average molar masses and the apparent hydrodynamic diameters, when the effect of the insulin concentration is taken into account by including virial effects arising from charge-charge repulsion (Donnan effect). The result shows that the association of insulin as a function of pH and ionic strength can be described by an effective charge equal to the charge derived from proton titration reduced by the number of sodium ions binding to insulin. At the lowest pH and highest salt concentration (pH 7.5, 100 mM NaCl, 12 mg/mL insulin), the weight average molar mass is close to that of the hexamer, and at the highest pH and lowest salt concentration (pH 10.5, 10 mM NaCl, 1.9 mg/mL), the weight average molar mass is close to that of the monomer. In all cases, however, a distribution of oligomers is present with a relative Gaussian width of about 30%.(ABSTRACT TRUNCATED AT 400 WORDS)

Aqueous mixtures of egg-yolk lecithin and the bile salt glycochenodeoxycholic acid sodium salt are studied using small-angle neutron scattering. Upon dilution, the shape and size of the aggregates change dramatically. This is due to very different critical micellar concentrations and spontaneous curvatures of lecithin and bile salt. At high concentrations, cylindrical micelles with a length of a few hundred angstroms are formed. As the samples are diluted, the length of the micelles first decreases and then increases by a factor of 3, their flexibility becomes noticeable, and the micelles can be described as semiflexible cylindrical micelles, also known as wormlike micelles. We have developed a mathematical model for the scattering of the wormlike micelles, which takes into account the intermicellar interaction effects. By the simultaneous fitting of the scattering data from a range of concentrations, the concentration-dependent growth law of the micelles can be parametrized. The obtained growth law of the mixed micelles is compared to the growth laws observed in simple micellar systems.

Analytical results are given for whey powders prepared on a commercial or semi-commercial scale by three companies. Altogether, five preparations enriched in ␤-lactoglobulin, four whey protein isolates and a fraction enriched in ␣-lactalbumin were analyzed for protein composition, including % ␤-lactoglobulin, ␣-lactalbumin, bovine serum albumin, casein (glyco) macropeptide and the main triglycerides. Protein composition was determined by high pressure gel permeation and reversed phase liquid chromatography and by capillary zone electrophoresis. The extent of modification of the native ␤-lactoglobulin structure was also measured through the degree of lactosylation and the fraction of accessible free sulphydryl groups. One significant finding was that the calculated recovery of protein following quantitation of the chromatogram or electropherogram was seldom above 90% and occasionally below 60% of that loaded onto the column or capillary, raising doubts as to the reliability of the analytical results. Extrapolation by linear regression to 100% recovery allowed estimates to be made of the true ␤-lactoglobulin composition of the samples. The nine samples could be placed into three distinct groups with estimat-ed true ␤-lactoglobulin weight % of 70.9 Ϯ 1.1, 62.0 Ϯ 3.4 and 39.5 Ϯ 4.9. Physico-chemical properties of the group of samples are reported elsewhere (Holt et al., 1999).The inter-laboratory comparisons involved the Royal Veterinary and Agricultural University (KVL), two laboratories of BDI (Lab1 and Lab2), NIZO food research (NIZO) and the Composition of whey protein isolates and fractions C. Holt et al. Figure 2 RP-HPLC analysis of commercial whey protein samples using the LRTL method. A. MDFwpi-1, B. MDFwpi-1a, C. MDFwpi-1b.Figure 3 Capillary zone electropherograms of selected samples as obtained by the KVL method. Within the b-Lg region, lactosylated forms have longer migration times than the native forms. on the Molecular Basis of the Aggregation, Denaturation, Gelation and Surface Activity of Whey Proteins (MADGELAS), CT96-1202. All other members of the MADGELAS group are thanked for their co-operation in the sample survey, of which this paper forms a part. The HRI work was supported by the Scottish Office Composition of whey protein isolates and fractions C. Holt et al.

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