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Chemical, rheological and ultrastructural properties of a major alkali-soluble protein of rapeseed Gill, Thomas Allan

Abstract

A 12S glycoprotein was isolated from commercial rapeseed meal (Brassica campestris) and examined by chemical, microscopical, and rheological methods. The molecular weight of the protein was estimated to be 129,200 daltons by conventional sedimentation equilibrium ultracentrifugation although the presence of higher molecular weight material was detected in the preparation. The 12S protein was found to be oligomeric, dissociating, into low molecular weight fragments in the presence of urea or sodium dodecyl sulfate. The protein aggregate was separated into subunits with apparent molecular weights of approximately 42,000, 37,600, 30,100, 17,400 and 12,200 by SDS gel electrophoresis. Electrophoretic patterns of non-reduced and reduced samples indicated the presence of intermolecular disulfide bonds although the cystine content was low. The 12S protein contained 12.9% (w/w) carbohydrate and reacted strongly when oxidized and treated with Schiff reagent. PAS-treated SDS gels indicated that most of the carbohydrate was present in one low molecular weight fragment. SDS immunoelectrophoretic analysis suggested that the glyco-peptide portion is located on the surface of the complex. Although the isolate contained a high molecular weight contaminant (17S), immunoelectrophoretic analysis resulted in the formation of one homogeneous pair of precipitin arcs. This would suggest that the 12S protein self-associates to form aggregates of higher molecular weight. In an attempt to separate the 17S and 12S fractions by gel filtration, a 33.9S protein was isolated, presumably the product of a self-associating system. SDS electrophoretic patterns of the 33.9S and 12S proteins were similar. Histochemical studies revealed that the 12S glycoprotein was present in some but not all of the cells of the intact rapeseed kernel. Schiff-positive aleurones were distributed randomly throughout the kernels. Transmission electron microscopy of negatively-stained specimens revealed that the protein was morula-shaped with a maximum particle diameter of 120Å The 12S rapeseed protein formed gels when dispersions of this material were heated. The rheological and ultra-structural examination of this phenomenon revealed that gel structure depended upon pH and ionic strength but to a lesser degree on low levels of urea or dithiothreitol.

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