Interactions of triply phosphorylated human beta-casein: monomer characterization and hydrodynamic studies of self-association.
The triply phosphorylated form of human beta-casein comprises about 15% of that fraction and is thus a significant component about midway between the two extremes of zero and five phosphoryls. Its partial specific volume, v, of 0.74 +/- 0.01 and absorbancy, E1% 1 cm, 280 nm, of 6.2 +/- 0.2 are almost identical to the other human beta-caseins. Equilibrium dialysis gave an average of 3.1 +/- 0.4 major Ca2+ binding sites at 37 degrees C with Kdiss = 8.6 x 10(-4) M. Sedimentation and viscosity at low temperatures or in 3.3 M urea suggested a prolate ellipsoidal monomer with 1.4 g H2O/g protein, 10 nm in length and 1.4 nm in width. The concentrated charge of the phosphoryls may be near one end of the ellipsoid, allowing the molecules to align with the flow in the viscometer at low concentration but, due to intermolecular electrostatic interactions, not when concentration is high. This would provide a reason for the heretofore unexplained curvature in the plots of reduced viscosity, eta red, vs beta-casein protein concentration. Self-association increased with temperature. At 37 degrees C in low salt buffer, s20,W was 16 S, which increased to about 33 S as ionic strength, I, was increased to 0.2 and above. At the same time, eta red in low salt buffer decreased from about 22 ml/g at 4 degrees C to a constant value of about 5 ml/g above 23 degrees C. A similar value for eta red at 37 degrees C, which was almost independent of protein concentration, was obtained at I greater than 0.25, giving an extrapolated intrinsic viscosity value of [eta] = 4.0 ml/g. Using this value and assuming a spherical aggregate, calculations suggest a radius of 9 nm with about 48 monomers and 0.86 g H2O/g protein.
Sood
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Department of Biochemistry, Loma Linda University School of Medicine, California 92350.
Chang
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Slattery
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D002118:Calcium / Q000378:metabolism; D002364:Caseins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009994:Osmolar Concentration; D010766:Phosphorylation; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology; D014783:Viscosity
D002364:Caseins; D046911:Macromolecular Substances; D014508:Urea; D002118:Calcium