Abstract
The membrane-bound NADPH:O2 oxidoreductase of human neutrophils has been solubilized in approximately 70% yield and purified on concanavalin A-Sepharose and gel sieving columns of varying bed volumes and sieving ranges. The half-life of the solubilized oxidoreductase stored at 2-4 degrees C in the presence of 25% glycerol at pH 8.6 is approximately 30 h. The oxidoreductase contains a flavoprotein identifiable by its fluorescence spectrum for FAD which binds weakly to concanavalin A-Sepharose and elutes from gel sieving columns at a molecular weight range of approximately 51,000. This flavoprotein accounts for approximately 70% of the total FAD content found in granular membrane fractions recovered from activated neutrophils. Recovery of oxidoreductase activity from both concanavalin A-Sepharose affinity and gel sieving columns is affected by the resolution of the flavoprotein free of the cytochrome b component of the oxidoreductase. The resolved flavoprotein and cytochrome b appear unable to catalyze either NADH nor NADPH oxidase activities with O2, ferricyanide, or nitroblue tetrazolium salt serving as electron acceptors.
Highlights
From the Clinical Pathology Service,Veterans Administration Medical Center, and Department of Biochemistu, Oregon Health Sciences University, Portland, Oregon 97201
The membrane-bound NADPH:O2 oxidoreductase of Attempts at purifying and characterizing the oxidoreduchuman neutrophils has been solubilized in approxitase free of the neutrophil membrane with respect to recovermately 70% yieldandpurified on concanavalin A- able enzyme activity have not been successful.The enzyme is Sepharose andgel sieving columns ofvarying bed vol- well-known to be quite unstable once freed of the membrane umes and sieving ranges
The lability of the oxidoreductase freed of the concanavalin A-Sepharosaeffinity and gel sieving col- membrane might be accounted for in terms of dissolution and umns is affected by the resolution of the flavoprotein uncoupling of its individual protomeric redox components free of the cytochrombe component of the oxidoreduc- from one another
Summary
Vol 263, No 12, Issue of April 25, pp. 5617-5623, 1988 Printed in U.S.A. ISOLATION OF ITS CATALYTICALLY INACTIVE CYTOCHROME b AND FLAVOPROTEIN REDOX CENTERS*. The membrane-bound NADPH:O2 oxidoreductase of Attempts at purifying and characterizing the oxidoreduchuman neutrophils has been solubilized in approxitase free of the neutrophil membrane with respect to recovermately 70% yieldandpurified on concanavalin A- able enzyme activity have not been successful.The enzyme is Sepharose andgel sieving columns ofvarying bed vol- well-known to be quite unstable once freed of the membrane umes and sieving ranges. The lability of the oxidoreductase freed of the concanavalin A-Sepharosaeffinity and gel sieving col- membrane might be accounted for in terms of dissolution and umns is affected by the resolution of the flavoprotein uncoupling of its individual protomeric redox components free of the cytochrombe component of the oxidoreduc- from one another. Principal dioxygen products of the oxidoreductase appear to be superoxide and, to a lesser extent, hydrogen peroxide [1]
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