Kappa-casein interactions in the suspension of the two major calcium-sensitive human beta-caseins

The possible effects of both the beta-casein (beta-CN) phosphorylation level and the kappa-CN glycosylation level on micelle formation were studied using the doubly-phosphorylated form (beta-CN-2P) and the quadruply-phosphorylated form (beta-CN-4P) of human beta-CN, along with bovine kappa-CN to compare with previous studies using the more highly glycosylated human kappa-CN. Addition of bovine kappa-CN to human beta-CN-2P, beta-CN-4P, or a 1/1 (wt/wt) mixture of the two was at kappa/beta molar ratios from 0.0 to approximately 0.6 and micelles were reconstituted by addition of Ca+2 either directly at 37 degrees C for determination of the fraction suspended or at an initial temperature of 4 degrees that was gradually increased to 37 degrees C with the change in particle size monitored by turbidity measurements. Analysis of the data indicates that the 4P form requires more kappa-CN for stabilization than the 2P form but that the mixture of the two is more like the 4P form in that lateral kappa-kappa interactions may enhance beta-kappa interactions and micelle formation. Above a kappa/beta molar ratio of about 0.2, the caseins were fully suspended into reconstituted micelles. However, micelle size decreased at a higher ratio, indicating that the kappa-CN probably occupies a surface position and may regulate micelle size by its relative abundance. A comparison with published results suggests that the higher glycosylation level of human kappa-CN may protect a larger surface area and result in smaller micelles. Changes in reconstituted micelle size with pH indicate that positively charged groups in the kappa-CN may interact with the negatively charged phosphate esters in the beta-CN moieties in addition to kappa-beta hydrophobic interactions.