UBC Theses and Dissertations
Separation of K- and a-caseins by gel filtration Nakahori, Chuichi
Studies were conducted to observe the changes in gel filtration elution profiles of casein fractions due to temperature, pH, sodium dodecyl sulfate (SDS) concentrations, and salts. Prom these results, the optimum conditions for separation were: I) elution with 5xI0ˉ³M borate buffer at pH 10 and room temperature to separate K-casein from acid casein; 2) elution with IxIO ˉ²M phosphate buffer at pH II and 4 C to separate K-casein from skimmilk; 3) elution with IxIOˉ³M SDS solution at room temperature to separate K- casein from acid casein and skimmilk; 4) elution with 5x10ˉ⁴M SDS solution at room temperature to separate α[subscript]si-casein from acid casein. Casein fractions were characterized for purity by acrylamide gel electrophoresis, sedimentation velocity centrifugation, stability of casein fractions in the presence of calcium ions, and sialic acid content. Compared to chemical preparations of casein fractions a reasonably pure K-casein fraction was obtained by gel filtration when eluted with buffers at pH 10 and pH II. Casein fractions obtained by SDS elution yielded α[subscript]si and K-caseins almost as pure as chemically prepared and purified caseins. However, considerable decrease in stabilizing ability was observed in K-casein eluted with SDS solution. It is possible that the decrease in stability is due to a binding of SDS with casein as the sulfur content of casein fractions prepared by the SDS methods was higher than that of chemically prepared casein fractions. There was slight variation in the sialic acid content of K-casein obtained by the different methods of preparations.
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