Casein Micelles. Formation and Structure. I

Casein interactions which take place prior to and during micelle formation have been studied using either a variety of mixtures of α^s,-and K-caseins or First Cycle casein or Solubilized Skim Milk. Techniques have involved analytical and preparative ultracentrifugation and, for systems to which calcium was added, measurements of supernatant protein at 37° after assay centrif-ugation for 1 min. at 400g. In the absence of calcium at pH 7 and °, α^s-and k-caseins show little or no tendency to interact and interaction products will not develop after urea or alkaline treatment. At 20°, while interaction products are normally absent, they appear after urea or alkaline treatment. At 37° a variety of condition-sensitive interaction products normally exist. The addition of calcium to give low concentrations produces no visible effect on solutions at pH 7 α^s-casein or mixtures of α^s-and k-caseins. Depending on protein concentration, precipitation of calcium α^s-caseinate is initiated from both at the same calcium concentration: at 0.004 M calcium for 5 mg.jml. of α^s-casein and at 0.005 M calcium for 10 mg.jml. of α^s-casein. Precipitation from mixtures is thereafter progressively retarded by the presence of k-casein and to an extent corresponding to a weight ratio near unity just before micelle formation is initiated. Thus, the only interaction products that can survive the addition of small amounts of calcium, or which are formed just prior to micelle formation, must have weight ratios near unity. That the behavior of α^s-K-casein mixtures is not an artifact of preparative procedures is shown by a similar behavior on the part of either First Cycle casein or of Solubilized Skim Milk. The addition of calcium, in single aliquots to calcium-free mixtures of α^s-and k-caseins, to give concentrations between 0.007 (depending on protein concentration) and 0.02 M leads to increasing micelle formation and complete stabilization if the initial α^s/k weight ratio, Ri, is 10 or less. Thus, a pronounced dip, having descending and ascending limbs, appears in the plot of supernatant protein vs. calcium concentration. Progressively above Ri = 10 assay centrifugate increases, but the amount of protein remaining α^ssupernatant micelles greatly exceeds the sum of initial k-casein plus calcium as-caseinate solubility. It is apparent from the occurrence of the dip that micelle-forming reactions have a higher calcium dependency than calcium α^s-caseinate precipitation. The dip also presents evidence consistent with low weight ratio interaction products, since the dip decrements as Ri decreases but is small and still apparent at Ri = 2.5. If calcium is added incrementally to solutions containing α^s-casein or α^s-k-casein mixtures at Ri ˂ 10 the de.