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Alpha sl-casein-k-carrageenan interaction : a physicochemical study Skura, Brenton James


κ-Carrageenan, a sulfated polygalactan, stabilizes α[sub sl]-casein, the calcium sensitive protein of bovine milk, from precipitation by Ca⁺⁺. The α[sub sl]-casein-κ -carrageenan interaction, in calcium-free systems, was investigated employing sedimentation velocity, sedimentation equilibrium, viscosity, frontal chromatography and fluorescence polarization experiments. κ -Carrageenan stabilization of α[sub sl]-casein was also investigated in systems containing 0.01 M Ca⁺⁺. Schlieren patterns of α[sub sl]-casein- κ -carrageenan mixtures in calcium free systems during sedimentation velocity ultracentrifugation revealed a large α[sub sl]-casein containing interaction peak followed by a slower sedimenting peak thought to be residual κ -carrageenan. The s[sub 20,w] of the interaction peak was greater than the s[sub 20,w]of α[sub sl]-casein alone, under identical conditions. It was concluded, on the basis of the effects of ionic strength, temperature, pH, and 6.0 M urea on the s[sub 20,w] and reduced viscosities of α[sub sl]-casein- κ -carrageenan interaction mixtures, that hydrogen bonding was involved in the observed α[sub sl]-casein- κ -carrageenan interaction. However, fluorescence polarization, frontal gel chromatography and molecular weight distributions calculated from sedimentation equilibrium patterns by a multiple regression technique revealed that α[sub sl]-casein and κ -carrageenan did not interact in calcium-free systems. Fluorescencepolarization studies employed both DNS- α[sub sl]-casein and DNS- κ -carrageenan as the labelled component. α[sub sl]-casein - κ -carrageenan mixtures (pH 6.6, μ = 0.08) eluted from a controlled pore glass (170 Å pore diameter) column as the individual components with elution volumes similar to those obtained when α[sub sl]-casein and κ -carrageenan were chromatographed separately. The molecular weight distributions for α[sub sl]-casein- κ -carrageenan mixtures (pH 6.6, μ = 0.08) contained a major peak in the molecular weight range corresponding to unreacted α[sub sl]-casein. The "interaction" revealed by sedimentation velocity and viscosity data was not a chemical interaction, but, rather a physical entrapment of α[sub sl]-casein by κ -carrageenan. Fluid flow through the capillary during viscosity measurements and the intense gravitational fields generated during sedimentation velocity ultracentriguation induced physical entanglement of the κ -carrageenan. As a consequence, α[sub sl]-casein - κ -carrageenan mixtures flowed as a "porous-plug" where α[sub sl]-casein, larger than the pores of the κ -carrageenan network, was trapped giving rise to the observed "interaction". Urea (6.0 M) dissociated the α[sub sl]-casein aggregates to the monomer state, with a molecular size smaller than the pores in the entangled carrageenan network, rather than dissociating an " α[sub sl]-casein - κ -carrageenan complex". Physical entanglement of α[sub sl]-casein within the κ -carrageenan system was not induced and an "interaction" was not observed during frontal gel chromatography, sedimentation equilibrium and fluorescence polarization measurements since an intense force was not applied to the system during these analyses. κ -Carrageenan stabilization of α[sub sl]-casein in the presence of 0.01 M Ca⁺⁺ (pH 6.6) decreased with increasing gravitational fields during the centrifugation step of the stabilization test employed. Increasing ionic strength inhibited the stabilization of α[sub sl]-casein by κ -carrageenan as did increasing the α[sub sl]-casein concentration from 1.5 mg/ ml to 10 mg/ml (pH 6.6, μ = 0.08). Thus, α[sub sl]-casein and κ -carrageenan did not chemically interact in calcium-free systems. Stabilization of α[sub sl]-casein by κ -carrageenan involved a physical entrapment of calcium- α[sub sl]-caseinate particles by κ -carrageenan.

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