Interactions Leading to Formation of Casein Submicelles

Gel chromatography was employed in studies of interactions of soluble whole casein that was prepared by dissociation of casein micelles with ethylenediaminetetraacetate. With increasing protein concentration at pH 6.6 and 37~C, components of whole casein associate to polymers that approach molecular radii with apparent upper limit of 9.4 -+ .4 nm. With decreasing protein concentration, K-casein dissociates from the other casein components. This was shown by analysis of the eluted protein boundary by get electrophoresis and radial immunodiffusion. The peak maximum elution volume and the broad, skewed character of the separated K-casein peak indicate that in whole casein K-casein exists in a size distribution of disulfide bonded polymers. This apparently suggests that SH-K-casein monomers aggregate independently of the other casein components in the growth of casein submicelles, and additional studies with the purified casein components support this concept. However, after disulfide bond reduction with dithiothreitol, chromatography of whole casein over the same concentration range did not result in separation of SH-K-casein polymers, because all of the casein was eluted under one peak. These findings show that, in vivo, casein submicelles could be formed by interaction of SH-K-casein monomers with those of a s-and /3-casein, followed by S-S-K-casein polymer formation through oxidation after milking.