title: Partial purification of an oxygen scavenging cell membrane fraction for use in anaerobic biochemical reactions.
abstract: Anaerobic conditions are necessary to prevent the autoxidation of tetrahydrobiopterin. An enzymatic method for achieving such an anaerobic condition is shown to be obtained by a lactate oxidase activity in the membrane of Escherichia coli when the enzyme concentration exceeds 0.05 unit/ml. A procedure is described for partial purification of the membrane fragment that involves salt precipitation and gel sieve chromatography on Bio-Gel A-50m. The rate of removal of oxygen is used to define the enzyme activity, to determine stability during storage, and to define conditions for stabilization of tetrahydrobiopterin. A second assay procedure that measures the rate of reduction of resazurin is described and is useful when the enzyme concentration is low.
authors.affiliation: Biology Division, Oak Ridge National Laboratory, Tennessee 37831.
authors.forename: K B
authors.initials: KB
authors.lastname: Jacobson
authors.forename: R E
authors.initials: RE
authors.lastname: Manos
authors.forename: F A
authors.initials: FA
authors.lastname: Wadzinski
mesh_terms: D000693:Anaerobiosis; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D004566:Electrodes; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000648:ultrastructure*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism; D010100:Oxygen / Q000032:analysis / Q000378:metabolism*; D014966:Xanthenes
chemical_list: D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; D001708:Biopterins; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase; C003402:sapropterin; D010100:Oxygen