Comparison of the Chaperoning Action of Glycerol and β-Casein on Aggregation of Proteins in the Presence of Crowding Agent

beta-Casein is one of the major components of the milk micelles of most mammals and has been shown to exhibit in vitro chaperone-like activity. Glycerol is a chemical chaperone belonging to the polyol family, which increases protein stability and inhibits protein aggregation. These prompted us to compare the chaperone-like activity of beta-casein and glycerol. In this study, the effect of beta-casein and glycerol on folding of the target proteins (ovotransferrin, insulin and a-lactalbumin) in the presence of dextran, as a macromolecular crowding agent, is examined using visible absorption spectroscopy, intrinsic fluorescence spectroscopy, 1-anilino-8-naphthalene sulfonic acid fluorescence binding and near CD spectroscopy. In the presence of dextran, the rate and extent of aggregation of target proteins was enhanced and beta-casein was less effective in preventing the aggregation and precipitation of target proteins. These data support the hypothesis that beta-casein interacts more effectively with slowly aggregating rather than rapidly aggregating target proteins. It is proposed that dextran-induced changes to protein conformation and the rate of intermolecular association are in a kinetic competition with the chaperoning action of beta-casein; however our results demonstrated the higher activity of glycerol, as a chemical chaperone, than beta-casein on the folding of target proteins, especially in the presence of dextran. This is likely due to the stabilizing effect of glycerol on protein structure and environment. The implications for the in vivo functions of beta-casein and glycerol, based on their exhibiting such in vitro chaperone-like activities, are discussed.