Purification and characterization of an alkaline phosphatase from Halobacterium salinarium Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/7h149s90d

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  • The halophilic alkaline phosphatase of Halobacterium salinarium has been purified to a specific activity of 3,000-3,200 units per mg of protein. The purification scheme consisted of acetone and ammonium sulfate fractionations, and molecular sieving and adsorption chromatography on Sephadex G -50. The enzyme was stabilized during purification by the presence of 0.01 M manganous chloride. The purified preparation exhibited a constant specific activity across the final protein peak on Sephadex G-50 and appeared well over 95% pure upon disc-gel electrophoresis. Amino acid analysis of the purified enzyme revealed a composition very similar to other halophilic proteins which have been studied. The phosphatase was similarly high in the acidic amino acids, glutamic and aspartic acids. A detailed kinetic study revealed the enzyme's obligatory requirement of manganous ion for activity. Its affinity for this metal ion was found to be strongly pH and salt concentration dependent. The salt concentration dependency was interpreted as reflecting the charge shielding of electrostatic interactions on the enzyme surface which when unshielded distort the conformational features of the manganese binding site. When saturated with manganese, the enzyme exhibited an optimal activity at pH 8.5. Stability studies on the apoenzyme demonstrated that the stability of this species was strongly dependent on the nature and concentration of the stabilizing salt. The apoenzyme was stabile only in sodium and ammonium sulfates and in sodium and potassium chlorides. The salt concentrations required for apoenzyme stability were all well in excess of 3 N. The function of high salt concentration in the stabilization of the alkaline phosphatase was interpreted as being the result of salting-out effects of salt solutions. These effects were believed to diminish solvent-protein interactions thereby strengthening the intramolecular interactions required for the conformational stability of the enzyme.
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