Mild isolation procedure discloses new protein structural properties of beta-lactoglobulin

To explore the potentially available functional properties of beta -lactogl obulin in, for example, the processing of food products, it is important to isolate the protein by a procedure that avoids all possible denaturing con ditions, such as low pH, high ionic strength, or low or elevated temperatur es that could cause the protein to undergo irreversible conformational chan ges. In this work, a mild isolation protocol for beta -lactoglobulin from b ovine milk is presented, applicable to semi large-scale isolations (50 to 2 00 g). The protein could be isolated with a high efficiency (>80%) and a go od purity (>98%). Biochemical characterization of the material demonstrated no lactosylation of the protein, nor the formation of irreversibly associa ted dimers. Also, no proteose peptones could be detected. The ability of be ta -lactoglobulin to undergo conformational changes is studied by far and n ear-ultraviolet circular dichroism and differential scanning calorimetry. A "global" unfolding of the protein is detected around 72 (tertiary level) a nd 77 degreesC (secondary level). The dimer-monomer dissociation occurring around 52 degreesC could also be monitored at a secondary structural level. Remarkably, a low temperature transition around 30 degreesC was observed, where approximately 10 beta -stranded residues unfold cooperatively, not be en reported previously. This low temperature transition is irreversible at temperatures higher than 35 degreesC or upon freezing the material at -20 d egreesC. The addition of 20% glycerol could prevent this irreversible confo rmational change. The effect of the low temperature transition on the prote in's functionality remains to be investigated.