Extraction of proteins from material rich in anionic mucilages: partition and fractionation of vanadate-dependent bromoperoxidases from the brown algae Laminaria digitata and L. saccharina in aqueous polymer two-phase systems.
For various reasons extraction of proteins from plant material is difficult. In particular phenolic compounds and polyanionic cell-wall mucilages render conventional procedures of extraction and purification much more difficult. In this respect, aqueous polymer two-phase systems are presented as a powerful technique in extraction of vanadate-dependent bromoperoxidases from the brown macroalga Laminaria digitata, a seaweed extremely rich in mucilages. Little bromoperoxidase activity was obtained when fresh thallus material was extracted in Tris buffer. Extraction from freeze-dried and powdered material was more efficient but only satisfactory when partitioning in an aqueous polymer two-phase system was employed. Among several two-phase systems tested, one composed of poly(ethylene glycol) (PEG 1550) and potassium carbonate proved most successful (phase system-1). A rapid and efficient extraction procedure was developed with special regard for suitability in large scale processes. Staining for catalytic activity after PAGE revealed a pattern of several bromoperoxidase isoforms. Bromoperoxidases extracted in phase system-1 were fractionated into two groups of isoforms by partitioning in a second system (phase system-2) indicating that isoforms from both groups differ significantly in surface properties. Subsequently, one purification step by hydrophobic interaction chromatography was sufficient to remove residual non-peroxidase proteins as well as remaining polysaccharides from bromoperoxidases of both groups. Thus, consideration of aqueous two-phase systems as a technique for extraction and purification of plant proteins can be recommended, whenever inconveniant amounts of phenolic compounds, mucilages or pigments are present.
Jordan
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Institute for Pharmaceutical Biology, University of Bonn, F.R.G.
Vilter
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D000269:Adhesives / Q000737:chemistry*; D002254:Carbonates; D007795:Laminaria / Q000201:enzymology*; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification*; D010940:Plant Proteins / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D011134:Polysaccharides / Q000737:chemistry; D011188:Potassium; D012995:Solubility; D014638:Vanadates
D000269:Adhesives; D002254:Carbonates; D010940:Plant Proteins; D011134:Polysaccharides; D014638:Vanadates; D011092:Polyethylene Glycols; C037593:potassium carbonate; D010544:Peroxidases; C026195:bromide peroxidase; D011188:Potassium