Human beta-casein was separated according to the extent of phosphorylation and the fully phosphorylated moiety was characterized. Fully phosphorylated human beta-casein makes up to 13-15% of the beta-casein fraction. It has a partial specific volume, v, of 0.754 +/- 0.008 and an absorbancy, E1(1%)cm,280 nm of 6.4 +/- 0.2. Sedimentation and viscosity data yield a solvation of 2.9 g H2O/g protein and an axial ratio of about 5 for the monomer. This would be consistent with a prolate ellipsoid of 10 nm length and 2 nm width. There is one strong binding site for Ca2+ for each organic phosphate ester in the molecule. The protein will precipitate at room temperature upon the addition of either 10 mM Ca2+ or greater than 1 M NaCl. Increasing the temperature from 4 to 37 degrees C causes an apparent conformational change and an increase in protein aggregation which is further increased by the addition of NaCl at this temperature until a limiting size is reached at about 0.25 M NaCl. This limiting size polymer contains 95-105 monomers and is nearly spherical with a radius of about 15 nm and a solvation of 3 g H2O/g protein. If this polymer were the submicelle of human casein, it could account for the abnormally high solvation of human casein micelles but their small average size would be more difficult to reconcile without additional information concerning K-casein association. The addition of Ca2+ to the system introduces association patterns which are more complex and not easily assessed.
Information on the structure of human casein micelles has been obtained from dissociation of beta-casein (CN). Two approaches were used: cooling at 4 degrees C and addition of EDTA. An initial loss of about 80% of the protein optical density occurred upon cooling to 4 degrees C. Dissociation was time dependent, and at > or = 24 h about 10% remained. However, mean size and voluminosity of micelles increased, as indicated by laser light scattering and viscosity measurements. This process was reversible, and 95% of the protein reentered the micelles upon incubation for 3 h at 37 degrees C. Upon cooling, amounts of nonphosphorylated beta-CN increased, and singly phosphorylated beta-CN levels were almost constant relative to the total beta-CN in micelles. Upon addition of EDTA (0 to 5 mM), the forms with three to five phosphates were the major dissociating constituents; EDTA that was added by dialysis produced similar results but at lower concentrations. These data suggest that, in the absence of significant amounts of alpha s1-CN, nonphosphorylated and singly phosphorylated human beta-CN may form a framework, as proposed for alpha s1-CN for bovine milk, along with the colloidal calcium phosphate for the development of the final micelle structure by addition of the more highly phosphorylated forms. The results also indicate that human casein micelles have a less rigid structure than those of other species.
Because previous purification procedures for human kappa-casein may have caused the loss of some carbohydrate, relatively gentle methods were used. The protein was isolated by a four-step procedure which included isoelectric precipitation of whole casein, gel chromatography on Sephadex G-200 in the presence of SDS, removal of the SDS with Extracti-Gel D, and FPLC chromatography on Mono Q with buffers containing 6 M urea. The purified protein was nearly identical in amino acid composition to that found earlier by amino acid analysis and peptide sequencing and a molar extinction coefficient of 11.2 +/- 0.1 was determined on the basis of amino acid analysis with a norleucine internal standard. Hydrolysis, acylation, and methylsilylation of the carbohydrate, followed by gas chromatographic analysis on a fused silica column, yielded approximately 5% fucose, 17% galactose, 18% N-acetylglucosamine, 8% N-acetylgalactosamine and 7% sialic acid, totaling almost 55% by weight. The percentages from two different donors were almost the same. About 1 mole phosphorus per mole of kappa-casein was also detected. Using low-speed sedimentation equilibrium methods, a molecular weight of only 33,400 was obtained for human kappa-casein, suggesting carbohydrate lability. Human beta-casein with four phosphoryls was stabilized against precipitation by 10 mM Ca+2 ions at a level greater than 95% when the molar ratio of kappa/beta exceeded 0.15.
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