Partial purification of an oxygen scavenging cell membrane fraction for use in anaerobic biochemical reactions
Jacobson, K.B.; Manos, R.E.; Wadzinski, F.A.
Biotechnology and Applied Biochemistry 9(5): 368-379
1987
ISSN/ISBN: 0885-4513 PMID: 3314908 Document Number: 288847
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.