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Partial Purification of An Oxygen Scavenging Cell Membrane Fraction for use in Anaerobic Biochemical Reactions
Author(s) -
Jacobson KB,
Manos RE,
Wadzinski FA
Publication year - 1987
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1987.tb00485.x
Subject(s) - chemistry , autoxidation , chromatography , membrane , anaerobic exercise , enzyme , tetrahydrobiopterin , oxygen , biochemistry , size exclusion chromatography , cofactor , organic chemistry , biology , physiology
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.