Partial purification of an oxygen scavenging cell membrane fraction for use in anaerobic biochemical reactions.
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.
Jacobson
K B
KB
Biology Division, Oak Ridge National Laboratory, Tennessee 37831.
Manos
R E
RE
Wadzinski
F A
FA
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
D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; D001708:Biopterins; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase; C003402:sapropterin; D010100:Oxygen