Fluorescence Monitoring of Flavins During Preparation of Respiratory Membranes

ABSTRACT

Azotobacter vinelandii large and small membrane particles were examined by fluorescence spectroscopy through purification to qualitatively monitor contamination by non-respiratory flavin. Flavin was analyzed by observing the effects of reduction by dithionite or NAD(P)H and subsequent oxidation. Flavin of the large particles did not change significantly with purification on a sucrose gradient. The small particle or R3 fraction contained relatively large amounts of non-respiratory flavin. Small particles eluted from a Sepharose CL-6B column with a fluorescence peak but still contained contaminating flavin. After centrifugation on a sucrose gradient, the flavin of these particles was essentially the same as the large particles. This method is an improvement over just observation of fluorescence intensity for monitoring flavoprotein purity of membrane particle preparations.