SURFACE HYDROPHOBICITY AND AGGREGATION OF BETA-LACTOGLOBULIN HEATED NEAR NEUTRAL PH

The surface hydrophobicity of beta-lactoglobulin (beta-LG) was investigated by binding of 8-anilino-1-naphthalenesulfonate (ANS) or retinol (RET). Analysis of ANS-beta-LG binding at pH 7.5 gave a low number of sites (n = 0.03-0.4) and a high dissociation constant (K(D) = 2.0-6.5 X 10(-5) M), suggesting a low affinity of beta-LG for ANS. Analysis of RET-beta-LG binding at pH 7.5 indicated one type of site. n tended to 0.9 when the RET/beta-LG ratio increased (with a K(D) between 0.12 X 10-7 and 1.7 X 10(-7) M). This result is consistent with the existence of one strong hydrophobic binding site for retinol in beta-LG. The affinity of native-beta-LG for retinol decreased when the pH was lowered from 7.5 to 6.5, probably as a result of the well-known beta-LG conformational change and the higher proportion of dimers. Heating 1%-beta-LG solutions also decreased affinity for retinol. At pH 6.5, this decrease took place after 3 or 6 min at 75 or 90-degrees-C but required at least 6 min at 90-degrees-C at pH 7.5. However, hydrophobic interaction chromatography indicated a smaller loss of native-beta-LG after heating at pH 6.5 than at pH 7.5. Gel permeation chromatography (at pH 6.0) revealed that progressive heating at pH 7.5 caused the dissociation of beta-LG dimers into monomers and a subsequent aggregation into oligomers. Heating at pH 6.5 caused the formation of high molecular weight soluble aggregates (labile to SDS). The lesser affinity for retinol probably resulted from these aggregation phenomena.