Estimation of flavin-containing monooxygenase activities in crude tissue preparations by thiourea-dependent oxidation of thiocholine.
The activity of flavin-containing monooxygenases in microsomes and whole homogenates is readily estimated by following the thiourea-dependent oxidation of thiocholine. NADPH- and oxygen-dependent flavin-containing monooxygenases catalyze the oxidation of thiourea to formamidine sulfenic acid, which oxidizes thiocholine to thiocholine disulfide. The latter reaction is quite rapid and never rate limiting even at concentrations of thiocholine below 30 microM. The loss of thiocholine in deproteinized aliquots of the reaction medium is measured colorimetrically with the thiol reagent, DTNB [5,5'-dithiobis(2-nitrobenzoate)]. In the absence of thiourea, thiocholine is not oxidized and its disulfide is not reduced at a detectable rate even in reactions containing 4-5 mg of liver or kidney homogenate protein per milliliter. In all tissues where both can be measured, rates of thiocholine oxidation and N,N-dimethylaniline N-oxygenation were virtually identical, which suggests that both activities are catalyzed by the same monooxygenase.
Guo
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Clayton Foundation Biochemical Institute, University of Texas, Austin 78712.
Ziegler
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DM
D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D002645:Chickens; D004220:Disulfides / Q000378:metabolism; D005260:Female; D005420:Flavoproteins / Q000032:analysis*; D006168:Guinea Pigs; D007413:Intestinal Mucosa / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000032:analysis*; D011817:Rabbits; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine; D013860:Thiocholine / Q000378:metabolism*; D013890:Thiourea / Q000378:metabolism*
D000814:Aniline Compounds; D004220:Disulfides; D005420:Flavoproteins; D009249:NADP; D013860:Thiocholine; C015157:N,N-dimethylaniline; D010105:Oxygenases; D013890:Thiourea; D010100:Oxygen