Surface hydrophobicity and aggregation of .beta.-lactoglobulin heated near neutral pH

The surface hydrophobicity of beta-lactoglobulin (betaLG) was investigated by binding of 8-anilino-1-naphthalenesulfonate (ANS) or retinol (RET). Analysis of ANS-betaLG binding at pH 7.5 gave a low number of sites (n = 0.03-0.4) and a high dissociation constant (KD = 2.0-6.5 X 10(-5) M), suggesting a low affinity of betaLG for ANS. Analysis of RET-betaLG binding at pH 7.5 indicated one type of site. n tended to 0.9 when the RET/betaLG ratio increased (with a KD 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 betaLG. The affinity of native betaLG for retinol decreased when the pH was lowered from 7.5 to 6.5, probably as a result of the well-known betaLG conformational change and the higher proportion of dimers. Heating 1% betaLG 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 betaLG 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 betaLG 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