κ-Casein interactions in the suspension of the two major calcium-sensitive human β-caseins

The possible effects of both the β-casein (β-CN) phosphorylation level and the κ-CN glycosylation level on micelle formation were studied using the doubly-phosphorylated form (β-CN-2P) and the quadruply- phosphorylated form (β-CN-4P) of human β-CN, along with bovine κ-CN to compare with previous studies using the more highly glycosylated human κ-CN. Addition of bovine κ-CN to human β-CN-2P, β-CN-4P, or a 1/1 (wt/ wt) mixture of the two was at κ/β molar ratios from 0.0 to ∼0.6 and micelles were reconstituted by addition of Ca⁺² either directly at 37°C for determination of the fraction suspended or at an initial temperature of 4° that was gradually increased to 37°C with the change in particle size monitored by turbidity measurements. Analysis of the data indicates that the 4P form requires more κ-CN for stabilization than the 2P form but that the mixture of the two is more like the 4P form in that lateral κ-κ interactions may enhance β-κ interactions and micelle formation. Above a κ/β 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 κ-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 κ-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 κ-CN may interact with the negatively charged phosphate esters in the β-CN moieties in addition to κ-β hydrophobic interactions.