Hydrocarbon-coated Sepharoses. Use in the purification of glycogen phosphorylase

Hydrophobic and biospecific chromatography in the purification of maltodextrin phosphorylase from E. Coli

“…This was achieved by reaction of the saccharide with sulfanilamide to yield its N-glycosylamine derivative which was then coupled through its amide nitrogen to CNBr-activated agarose [18]. It should be noted that saccharides bind to CNBr-activated agarose also through their hydroxyl groups [8], therefore there is some heterogeneity in the mode of binding of the saccharides to the agarose (especially in the case of polysaccharides which contain many hydroxyl groups that compete with the amide nitrogen). Fig.3 illustrates the biospecific purification of maltodextrin phosphorylase on Seph-SA-dextrin which results in an essentially pure enzyme with a specific activity of 11.7 units/mg (about 2.5-fold higher than the one reported previously [2].…”

“…This paper describes a new method for the purification of the enzyme which makes use of hydrophobic [8,9] and biospecific affinity chromatography [10,11]. The procedure results in an overall 78-fold purification and yields an enzyme with a specific activity about 2.5-fold higher than the one reported previously.…”

Omega-aminoalkyl agaroses in the resolution of enzymes involved in regulation of glutamine metabolism.

“…Cell-free extracts of the frozen cell paste (300 g wet weight) were prepared by homogenization in 600 ml of a buffer composed of 10 mM imidazole-HCI, 10 mM 2-mercaptoethanol, and 0.1 mM K2Mg EDTA, pH 7.0. This homogenization (30 sec in a Waring blender) was followed by passage (twice) through a French pressure cell at 12000 lbs./inch2 (83000 kPa) and centrifugation for 1 (18,19). One unit of adenylyltransferase activity is arbitrarily defined as the amount of enzyme required to decrease the glutaminesynthetase-catalyzed formation of y-glutamylhydroxamate by 1 ,mol (AA540nm = 0.36)/min at 370.…”

“…The term "hydrophobic chromatography" was introduced (1,2) to describe a new approach to the resolution, purification, and probing of biomolecules using homologous series of hydrocarbon-coated agaroses. The individual columns in each series differ in the length of their hydrocarbon side chains and in their ability to retard or retain different molecules in a mixture.…”

“…The individual columns in each series differ in the length of their hydrocarbon side chains and in their ability to retard or retain different molecules in a mixture. Such columns were shown to be very effective in the purification of several proteins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Their discrimination power may involve several types of interactions (hydrophobic, electrostatic, hydrogen bonding, dipoledipole, etc).…”