Formation of stable covalent dimer explains the high solubility at pH 4.6 of lactose-β-lactoglobulin conjugates heated near neutral pH

The solubility of lactose-beta-lactoglobulin conjugates at pH 4.6, after heating near neutral pH in phosphate buffer/0.116 M NaCl, was investigated by size exclusion chromatography and compared with unmodified protein. Heated conjugates in the temperature range 65-90 degrees C showed greater solubility at pH 4.6. The proportion of soluble protein increased with the number of bound lactose molecules. Total solubility was obtained for conjugates with nine lactose residues attached per monomer of beta-lactoglobulin. The protective effect of bound sugar toward precipitation was associated with the formation of soluble disulfide cross-linked dimers, highly accessible to trypsin digestion. These results suggested that bound lactose, through steric hindrance and high surface hydrophilicity, prevents the thiol-disulfide exchange reactions of the polymerization-aggregation process of lactose-beta-lactoglobulin conjugates.