Casein Micelles. Formation and Structure.II

Studies of the formation and structure of casein micelles have revealed experimental facts which any model must accommodate. These are: (A) Prior to micelle formation at 37° there are present free calcium α^s-caseinate and a low weight ratio calcium α^s-k-caseinate interaction product. (B) For stability, micelles require a minimum level of calcium which is greater than that required to precipitate the free α^s-casein present. (C) The more centrifugable (larger) the micelle the lower its weight fractional content of k-casein. (D) Precipitates formed in the presence of k-casein and at calcium concentrations sufficient for micelle stability are different from calcium α^s-caseinate precipitates in being nonadherent and compacting to give white opaque pellets. These precipitates contain small amounts of k-casein. Calcium as-caseinate precipitates treated with K-casein become nonadherent. (E) Micelles are stable with respect to the close approach induced by sedimentation into a pellet. (F) In all cases micelles have a size distribution which depends strongly on the initial ratio, the calcium concentration on single aliquot addition, and the initial protein concentration. The apparent final states of the systems are dependent on the path. Path dependencies were found for assay supernatant protein, supernatant protein after ultracentrifugation, and a wide variety of micelle-precipitate combinations obtained either by single aliquot or incremental addition of calcium. (G) Complete stabilization in the region of the peak can be achieved at all α^s/K initial weight ratios up to 10 and micelle populations stable with respect to assay conditions may have weight ratios in excess of 10. (H) The size distribution of micelles can be altered by the addition of k-casein and probably by the addition of calcium. Size changes induced by k-casein addition occur rapidly (minutes) while those attending calcium addition take hours. k-Casein has some capacity to solubilize calcium α^s-caseinate precipitate but the resulting supernatant α^sK weight stabilization ratios are small (0.5-2). (I) Stabilizing capacity is available in some micelle systems after the systems have come to apparent final states. (J) A micelle distribution, once formed, is relatively stable to dilution with a buffer containing an appropriate calcium concentration. (K) Micelles are highly solvated and the solvation decreases as the initial ratio increases. (L) Although micelles require the presence of α^s-and k-caseins, they can incorporate variable amounts of β-casein. With these facts in mind, models of the micelles as single phase particles or large chemical compounds are unattractive. The following model is proposed. Micelles consist, in simplest form, of cores of calcium α^s-caseinate covered by a uniform coat of low weight ratio calcium α^s-k-caseinate. Calcium α^s-@r@ncaseinate in the core need not be in contact, therefore in exchange, with the environment. The carbohydrate moiety of k-casein is placed to the outside where little tendency to interaction is required. Internally the coat subunits have a strong interaction with core calcium α^s-caseinate and probably a lateral preference for each other. Surface components are in exchange with similar components in solution. Kinetically, random processes lead to the production of calcium as-caseinate particles and their acquisition of a coat. The calcium dependency of coat formation is greater than that of calcium α^s-caseinate precipitation. There is little free calcium k-caseinate or calcium α^s-caseinate in solution in contact with micelles. There may be variable amounts of excess coat material in the form of coreless micelles or other interaction products. The coat itself has a calcium instability and therefore a calcium dependency less than that of products having a lower weight ratio than the coat submit. The final member of the latter series could be a form of pure calcium k-caseinate. The solvation of micelles is reasonable from the solvation characteristics of components. The model accepts (β-casein to an extent. It places the carbohydrate moiety of k-casein to the outside where it is accessible, even in extraordinarily stable micelles, to the action of rennin.