PRESSURE-INDUCED AGGREGATION OF BETA-LACTOGLOBULIN IN PH 7.0 BUFFERS

Solutions of a beta-lactoglobulin isolate in water, in potassium phosp hate buffer (20 or 50 mmoL/L), and in pressure-resistant buffers, at a protein concentration of 25 g/kg and at pH 7.0, were processed at 150 , 250, 350 or 450 MPa and 25 degrees C, for 15 min, then stored at 4 d egrees C, usually for 24 h, before analysis. Bis-Tris (20 or 50 mmol/L ) and bis-Tris-propane (10, 20 or 50 mmol/L) were selected as pressure -resistant buffers, while the pH of water or phosphate buffer is known to decrease reversibly by 0.2-0.3 pH unit per 100 MPa. After pressuri zation, nitrogen solubility at pH 4.7 and 7.0, and aggregation pattern s by polyacrylamide gel electrophoresis were determined. Both indicate d that aggregation of beta-lactoglobulin was more extensive in pressur e-resistant buffers than in phosphate buffer or in water. Electrophore tic patterns also revealed the progressive formation of dimers to hexa mers and of higher polymers of beta-lactoglobulin as a function of the type and molarity of buffer and of the pressure level. All high molec ular weight aggregates and most oligomers disappeared when pressurized solutions were treated with beta-mercaptoethanol (15 mL/L) prior to e lectrophoresis. Thus pressure induced the formation of-intermolecular S-S bonds, especially when the pH was maintained close to neutral. Pre vious studies with thermally-induced beta-lactoglobulin gels had shown that SH/S-S interchange reactions were enhanced at or above neutralit y (C) 1995 Academic Press Limited