Environmental influences on bovine kappa-casein: reduction and conversion to fibrillar (amyloid) structures.

The caseins of milk form a unique calcium-phosphate transport complex that provides these necessary nutrients to the neonate. The colloidal stability of these particles is primarily the result of kappa-casein. As purified from milk, this protein occurs as spherical particles with a weight average molecular weight of 1.18 million. The protein exhibits a unique disulfide bonding pattern, which (in the absence of reducing agents) ranges from monomer to octamers and above on SDS-PAGE. Severe heat treatment of the kappa-casein (90 degrees C) in the absence of SDS, before electrophoresis, caused an increase in the polymeric distribution: up to 40% randomly aggregated high-molecular weight polymers, presumably promoted by free sulfhydryl groups (J. Protein Chem. 17: 73-84, 1998). To ascertain the role of the sulfhydryl groups, the protein was reduced and carboxymethylated (RCM-K). Surprisingly, at only 37 degrees C, the RCM-kappa-casein exhibited an increase in weight average molecular weight and tendency to self-association when studied at 3000 rpm by analytical ultracentrifugation. Electron microscopy (EM) of the 37 degrees C RCM sample showed that, in addition to the spherical particles found in the native protein, there was a high proportion of fibrillar structures. The fibrillar structures were up to 600 nm in length. Circular dichroism (CD) spectroscopy was used to investigate the temperature-induced changes in the secondary structure of the native and RCM-kappa-caseins. These studies indicate that there was little change in the distribution of secondary structural elements during this transition, with extended strand and beta turns predominating. On the basis of three-dimensional molecular modeling predictions, there may exist a tyrosine-rich repeated sheet-turn-sheet motif in kappa-casein (residues 15-65), which may allow for the stacking of the molecules into fibrillar structures. Previous studies on amyloid proteins have suggested that such motifs promote fibril formation, and near-ultraviolet CD and thioflavin-T binding studies on RCM-kappa-casein support this concept. The results are discussed with respect to the role that such fibrils may play in the synthesis and secretion of casein micelles in lactating mammary gland.