Article

Journal of Protein Chemistry

, Volume 22, Issue 3, pp 259-273

Environmental Influences on Bovine κ-Casein: Reduction and Conversion to Fibrillar (Amyloid) Structures

  • Harold M. FarrellJr.Affiliated withUnited States Department of Agriculture, ARS, Eastern Regional Research Center Email author 
  • , Peter H. CookeAffiliated withUnited States Department of Agriculture, ARS, Eastern Regional Research Center
  • , Edward D. WickhamAffiliated withUnited States Department of Agriculture, ARS, Eastern Regional Research Center
  • , Edwin G. PiotrowskiAffiliated withUnited States Department of Agriculture, ARS, Eastern Regional Research Center
  • , Peter D. HoaglandAffiliated withUnited States Department of Agriculture, ARS, Eastern Regional Research Center

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Abstract

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 κ-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 κ-casein (90°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-κ). Surprisingly, at only 37°C, the RCM-κ-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°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-κ-caseins. These studies indicate that there was little change in the distribution of secondary structural elements during this transition, with extended strand and κ turns predominating. On the basis of three-dimensional molecular modeling predictions, there may exist a tyrosine-rich repeated sheet-turnsheet motif in κ-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-κ-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.

Casein structure molecular weight disulfide interactions amyloid fibrils