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Two peptides, [f135–158] and [f135–162]-SH, were used to study the binding of the peptides to native ß-lactolobulin, as well as the subsequent effects on aggregation and gelation of ß-lactoglobulin. The binding of the peptide [f135–158] to ß-lactoglobulin at room temperature was confirmed by SELDI-TOF-MS. It was further illustrated by increased turbidity of mixed solutions of peptide and protein (at pH 7), indicating association of proteins and peptides in larger complexes. At pH below the isoelectric point of the protein, the presence of peptides did not lead to an increased turbidity, showing the absence of complexation. The protein–peptide complexes formed at pH 7 were found to dissociate directly upon heating. After prolonged heating, extensive aggregation was observed, whereas no aggregation was seen for the pure protein or pure peptide solutions. The presence of the free sulfhydryl group in [f135–162]-SH resulted in a 10 times increase in the amount of aggregation of ß-lactoglobulin upon heating, illustrating the additional effect of the free sulfhydryl group. Subsequent studies on the gel strength of heat-induced gels also showed a clear difference between these two peptides. The replacement of additional ß-lactoglobulin by [f135–158] resulted in a decrease in gel strength, whereas replacement by peptide [f135–162]-SH increased gel strength.

Processing of ovalbumin may result in proteins that differ more than 23°C in denaturation temperature while the structural fold is not significantly affected. This is achieved by 1) conversion of positive residues into negative ones (succinylation); 2) elimination of negative charges (methylation); 3) reducing the proteins hydrophobic exposure (glycosylation); 4) increasing the hydrophobic exposure (lipophilization); or by 5) processing under alkaline conditions and elevated temperature (Sovalbumin). The effect on the structural fold was investigated using a variety of biochemical and spectroscopic tools. The consequences of the modification on the thermodynamics of the protein was studied using differential scanning calorimetry and by monitoring the tryptophan fluorescence or ellipticity at 222 nm of protein samples dissolved in different concentrations of guanidine-HCl. The impact of the modification on the denaturation temperature scales for all types of modifications with a free energy change of about 1 kJ per mol ovalbumin per Kelvin (or 0.0026 kJ per mol residue per K). The nature of the covalently coupled moiety determines the impact of the modification on the protein thermodynamics. It is suggested that especially for lipophilized protein the water-binding properties are substantially lowered. Processing of globular proteins in a controlled manner offers great opportunities to control a desired functionality, for example, as texturizer in food or medical applications. © 2003 Wiley Periodicals. Chemicals/CAS: guanidine, 113-00-8, 25215-10-5, 50-01-1; ovalbumin, 77466-29-6; tryptophan, 6912-86-3, 73-22-3; Ovalbumin, 9006-59-1

Divalent calcium ions have been suggested to be involved in intermolecular protein-Ca2+-protein cross-linking, intramolecular electrostatic shielding, or ion-induced protein conformational changes as a trigger for protein aggregation at elevated temperatures. To address the first two phenomena in the case of beta-lactoglobulin, a combination of chemical protein modification, calcium-binding, and aggregation studies was used, while the structural integrity of the modified proteins was maintained. Although increasing the number of carboxylates on the protein by succinylation results in improved calcium-binding, calcium appears to be less effective in inducing protein aggregation. In fact, the larger the number of carboxylates, the higher the concentration of calcium that is required to trigger the aggregation. Lowering the number of negative charges on the protein surface via methylation of carboxylates reduces calcium-binding properties, but calcium-induced aggregation at low concentration is improved. Monovalent sodium ions cannot take over the specific role of calcium. The relation between net surface charge and number of calcium ions bound required to trigger the aggregation suggests that calcium needs to bind site specific to carboxylates with a threshold affinity. Subsequent site-specific screening of surface charges results in protein aggregation, driven by the partial unfolding of the protein at elevated temperatures, which is then facilitated by the absence of electrostatic repulsion.

A bovine β-lactoglobulin hydrolysate, obtained by the hydrolysis by the Glu specific enzyme Bacillus licheniformis protease (BLP), was fractionated at pH 7.0 into a soluble and an insoluble fraction and characterized by LC-MS. From the 26 peptides identified in the soluble fraction, five peptides (A[f97-112] = [f115-128], AB[f1-45], AB[f135-157], AB[f135-158], and AB[f138-162]) bound to β-lactoglobulin at room temperature. After heating of β-lactoglobulin in the presence of peptides, eight peptides were identified in the pellet formed, three of them belonging to the previously mentioned peptides. Principle component analysis revealed that the binding at room temperature (to β-lactoglobulin) was related to the total hydrophobicity and the total charge of the peptides. The binding to the unfolded protein could not be attributed to distinct properties of the peptides. The presence of the peptides caused a 50% decrease in denaturation enthalpy (from 148 ± 3 kJ/mol for the protein alone to 74 ± 2 kJ/mol in the presence of peptides), while no change in secondary structure or denaturation temperature was observed. At temperatures85 °C, the addition of peptides resulted in a 30-40% increase of precipitated β-lactoglobulin. At pH6, no differences in the amount of aggregated β-lactoglobulin were observed, which indicates the lack of binding of peptides to β-lactoglobulin at those pH values as was also observed by SELDI-TOF-MS. Although only a few peptides were found to participate in aggregation, suggesting specificity, principal component analysis was unable to identify specific properties responsible for this.

Because the formation of protein/polysaccharide complexes is dominated by electrostatic interaction, polysaccharide charge density is expected to play a major role in the adsorption behavior of the complexes. In this study, pullulan (a non-charged polysaccharide) carboxylated to four different charge densities (fraction of carboxylated subunits: 0.1, 0.26, 0.51, and 0.56) was used to investigate the effect of charge density on the properties of mixed protein/polysaccharide adsorbed layers at air/water interfaces. With all pullulan samples, soluble complexes with β-lactoglobulin could be formed at low ionic strength, pH 4.5. It was shown that the higher was the pullulan charge density, the more the increase of surface pressure in time was retarded as compared to that for pure β-lactoglobulin. The retardation was even more pronounced for the development of the dilatational modulus. The lower dilatational modulus can be explained by the ability of the polysaccharides to prevent the formation of a compact protein layer at the air/water interface due to electrostatic repulsion. This ability of the polysaccharides to prevent "layer compactness" increases with the net negative charge of the complexes. If charge density is sufficient (≥0.26), polysaccharides may enhance the cohesion between complexes within the adsorbed layer. The charge density of polysaccharides is shown to be a dominant regulator of both the adsorption kinetics as well as the resulting surface rheological behavior of the mixed layers formed. These findings have significant value for the application of complex protein-polysaccharide systems. © 2007 American Chemical Society.

A number of relevant properties of Woodward's reagent K have been determined, such as the stability of the reactant and the optimal reaction conditions of the reactant with protein carboxylates. A Woodward's reagent K stock solution was stable at 4°C for prolonged time, whereas upon storage at 22°C, almost 20% of the reactive compound was lost within 1 week. The pH-dependency of the spontaneous degradation reaction of Woodward's reagent K was studied and was shown to be basemediated. A molar extinction coefficient of 3150 M-1 cm-1 at 269 nm for the enol ester resulting from the reaction between Woodward's reagent K and the protein carboxylates was established using the conditions laid out in this work. This value was validated using a variety of proteins that were modified by Woodward's reagent K. In addition, upon methylation of the carboxylates of a single protein, ovalbumin in this case, the degree of modification could be determined accurately and was confirmed by cation exchange chromatography elution profiles. Chemicals/CAS: ovalbumin, 77466-29-6; Carboxylic Acids; Esters; Isoxazoles; N-ethyl-5-phenylisoxazolium-3'-sulfonate, 4156-16-5; Ovalbumin, 9006-59-1; Proteins