Exogenous Flavin Research Articles

Mediating the performance of anaerobic granular sludge by exogenous flavin supplementation: Flavin binding capacity and behavior, interspecies electron transfer, and methane production

Although flavins are known as effective electron mediators, the binding capacity of exogenous flavins by anaerobic granular sludge (AGS) and their role in interspecies electron transfer (IET) remains unknown. In this study, AGS was mediated by using three exogenous flavins of riboflavin (RF), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Results showed that the total amounts of flavins associated with extracellular polymeric substance (EPS) of AGS increased by 2.03–2.42 and 3.83–4.94 folds, after exposure to 50 and 200 μM of exogenous flavins, respectively. A large portion of FMN and FAD was transformed into RF by AGS. Exogenous flavin mediation also stimulated the production of EPS and cytochrome c (c-Cyts) as well as cytochrome-bound flavins. The increased abundance of these electron mediators led to a reduced electrochemical impedance of EPS and improved extracellular electron transfer capacity. The methane production of AGS after mediation with exogenous RF, FMN, and FAD increased by 19.03–31.71%, 22.86–26.04%, and 28.51–33.44%, respectively. This study sheds new light on the role of exogenous flavins in promoting the IET process of a complex microbial aggregate of AGS.

Enhanced small green fluorescent proteins as a multisensing platform for biosensor development

Engineered light, oxygen, and voltage (LOV)-based proteins are able to fluoresce without oxygen requirement due to the autocatalytic incorporation of exogenous flavin as a chromophore thus allowing for live cell imaging under hypoxic and anaerobic conditions. They were also discovered to have high sensitivity to transition metal ions and physiological flavin derivatives. These properties make flavin-binding fluorescent proteins (FPs) a perspective platform for biosensor development. However, brightness of currently available flavin-binding FPs is limited compared to GFP-like FPs creating a need for their further enhancement and optimization. In this study, we applied a directed molecular evolution approach to develop a pair of flavin-binding FPs, named miniGFP1 and miniGFP2. The miniGFP proteins are characterized by cyan-green fluorescence with excitation/emission maxima at 450/499nm and a molecular size of ∼13kDa. We carried out systematic benchmarking of miniGFPs in Escherichia coli and cultured mammalian cells against spectrally similar FPs including GFP-like FP, bilirubin-binding FP, and bright flavin-binding FPs. The miniGFPs proteins exhibited improved photochemical properties compared to other flavin-binding FPs enabling long-term live cell imaging. We demonstrated the utility of miniGFPs for live cell imaging in bacterial culture under anaerobic conditions and in CHO cells under hypoxia. The miniGFPs' fluorescence was highly sensitive to Cu(II) ions in solution with Kd values of 67 and 68nM for miniGFP1 and miniGFP2, respectively. We also observed fluorescence quenching of miniGFPs by the reduced form of Cu(I) suggesting its potential application as an optical indicator for Cu(I) and Cu(II). In addition, miniGFPs showed the ability to selectively bind exogenous flavin mononucleotide demonstrating a potential for utilization as a selective fluorescent flavin indicator. Altogether, miniGFPs can serve as a multisensing platform for fluorescence biosensor development for in vitro and in-cell applications.

Heterologous Expression and Characterization of a Full-length Protozoan Nitroreductase from Leishmania orientalis isolate PCM2.

Leishmaniasis, a parasitic disease found in parts of the tropics and subtropics, is caused by Leishmania protozoa infection. Nitroreductases (NTRs), enzymes involved in nitroaromatic prodrug activation, are attractive targets for leishmaniasis treatment development. In this study, a full-length recombinant NTR from the Leishmania orientalis isolate PCM2 (LoNTR), which causes severe leishmaniasis in Thailand, was successfully expressed in soluble form using chaperone co-expression in Escherichia coli BL21(DE3). The purified histidine-tagged enzyme (His6-LoNTR) had a subunit molecular mass of 36kDa with no cofactor bound; however, the addition of exogenous flavin (either FMN or FAD) readily increased its enzyme activity. Bioinformatics analysis found that the unique N-terminal sequences of LoNTR is only present in Leishmania where the addition of this region might result in the loss of flavin binding. Either NADH or NADPH can serve as an electron donor to transfer electrons to nitrofurazone; however, NADPH was preferred. Molecular oxygen was identified as an additional electron acceptor resulting in wasteful electrons from NADPH for the main catalysis. Steady-state kinetic experiments revealed a ping-pong mechanism for His6-LoNTR with Km,NADPH, Km,NFZ, and kcat of 28µM, 68µM, and 0.84min-1, respectively. Besides nitroreductase activity, His6-LoNTR also has the ability to reduce quinone derivatives. The properties of full-length His6-LoNTR were different from previously reported protozoa and bacterial NTRs in many respects. This study provides information of NTR catalysis to be developed as a potential future therapeutic target to treat leishmaniasis.

A hybrid cyt c maturation system enhances the bioelectrical performance of engineered Escherichia coli by improving the rate-limiting step

Bioelectronic devices can use electron flux to enable communication between biotic components and abiotic electrodes. We have modified Escherichia coli to electrically interact with electrodes by expressing the cytochrome c from Shewanella oneidensis MR-1. However, we observe inefficient electrical performance, which we hypothesize is due to the limited compatibility of the E. coli cytochrome c maturation (Ccm) systems with MR-1 cytochrome c. Here we test whether the bioelectronic performance of E. coli can be improved by constructing hybrid Ccm systems containing protein domains from both E. coli and S. oneidensis MR-1. The hybrid CcmH increased cytochrome c expression by increasing the abundance of CymA 60%, while only slightly changing the abundance of the other cytochromes c. Electrochemical measurements showed that the overall current from the hybrid ccm strain increased 121% relative to the wildtype ccm strain, with an electron flux per cell of 12.3 ± 0.3 fA·cell−1. Additionally, the hybrid ccm strain doubled its electrical response with the addition of exogenous flavin, and quantitative analysis of this demonstrates CymA is the rate-limiting step in this electron conduit. These results demonstrate that this hybrid Ccm system can enhance the bioelectrical performance of the cyt c expressing E. coli, allowing the construction of more efficient bioelectronic devices.

Mosquito NADPH‐cytochrome P450 oxidoreductase: kinetics and role of phenylalanine amino acid substitutions at leu86 and leu219 in CYP6AA3‐mediated deltamethrin metabolism

The NADPH-cytochrome P450 oxidoreductase (CYPOR) enzyme is a membrane-bound protein and contains both FAD and FMN cofactors. The enzyme transfers two electrons, one at a time, from NADPH to cytochrome P450 enzymes to function in the enzymatic reactions. We previously expressed in Escherichia coli the membrane-bound CYPOR (flAnCYPOR) from Anopheles minimus mosquito. We demonstrated the ability of flAnCYPOR to support the An. minimus CYP6AA3 enzyme activity in deltamethrin degradation in vitro. The present study revealed that the flAnCYPOR purified enzyme, analyzed by a fluorometric method, readily lost its flavin cofactors. When supplemented with exogenous flavin cofactors, the activity of flAnCYPOR-mediated cytochrome c reduction was increased. Mutant enzymes containing phenylalanine substitutions at leucine residues 86 and 219 were constructed and found to increase retention of FMN cofactor in the flAnCYPOR enzymes. Kinetic study by measuring cytochrome c-reducing activity indicated that the wild-type and mutant flAnCYPORs followed a non-classical two-site Ping-Pong mechanism, similar to rat CYPOR. The single mutant (L86F or L219F) and double mutant (L86F/L219F) flAnCYPOR enzymes, upon reconstitution with the An. minimus cytochrome P450 CYP6AA3 and a NADPH-regenerating system, increased CYP6AA3-mediated deltamethrin degradation compared to the wild-type flAnCYPOR enzyme. The increased enzyme activity could illustrate a more efficient electron transfer of AnCYPOR to CYP6AA3 cytochrome P450 enzyme. Addition of extra flavin cofactors could increase CYP6AA3-mediated activity supported by wild-type and mutant flAnCYPOR enzymes. Thus, both leucine to phenylalanine substitutions are essential for flAnCYPOR enzyme in supporting CYP6AA3-mediated metabolism.

Riboflavin kinase couples TNF receptor 1 to NADPH oxidase

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22(phox), the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22(phox) is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase.

Redox-flexible NADH oxidase biosensor: A platform for various dehydrogenase bioassays and biosensors

A generic amperometric bioassay based on the enzymatic oxidation catalysed by the stable NADH oxidase (NAox) from Thermus thermophilus has been developed for NADH measurements. The NAox uses O 2 as its natural electron acceptor and produces H 2O 2 in a two-electron process. Electrochemical and spectrophotometric experiments showed that the NAox used in this work, presents a very good activity towards its substrate and, in contrary to previously mentioned NADH oxidases, does not require the addition of any exogenous flavin cofactor neither to promote nor to maintain its activity. In addition, the NAox used also works with artificial electron acceptors like ferrocene derivatives. O 2 was successfully replaced by redox mediators such as hydroxymethyl ferrocene (FcCH 2OH) for the regeneration of the active enzyme. Combining the NAox with the mediator and the horseradish peroxidase we developed an original, high sensitive “redox-flexible” NADH amperometric bioassay working in a large window of applied potentials in both oxidation and reduction modes. The biosensor has a continuous and complementary linearity range permitting to measure NADH concentrations starting from 5 × 10 −6 M in reduction until 2 × 10 3 M in oxidation. This redox-flexibility allows choosing the applied potential in order to avoid electrochemical interferences. The association of the “redox-flexible” concept with NADH dependent enzymes opens a novel strategy for dehydrogenases based bioassays and biosensors. The great number of dehydrogenases available makes the concept applicable for numerous substrates to analyse. Moreover it allows the development of a wide range of biosensors on the basis of a generic platform. This gives several advantages over the previous manufacturing techniques and offers a general and flexible scheme for the fabrication of biosensors presenting high sensitivities, wide calibration ranges and less affected by electrochemical interferences.

Properties of two hemolymp riboflavin-binding proteins from Heliothis virescens

Hemolymph from late final instar Heliothis virescens larvae contains substantial amounts of riboflavin which is bound to two abundant polypeptides, lipophorin and a hexameric protein comprised of 82,000 M r subunits (p82). The riboflavin binding properties of the two proteins as well as those of a well-characterized flavin-binding protein from chicken egg albumin are presented and compared here. The dissociation constants for riboflavin derived from fluorescence quench binding assays for lipophorin and the p82 protein (1.1 × 10 −7 M and 3.6 × 10 −7 M, respectively) were c 40–120-fold greater than that of the high affinity chicken flavin-binding protein (K d = 3 × 10 −9 M). These assays yielded binding capacities of 0.5 mol riboflavin bound/mole lipophorin and from 1 to 2 mol riboflavin bound/mole p82 protein hexamer, values which were consistent with those obtained by equilibrium dialysis. Determinations of hemolymph riboflavin titers through development revealed an almost 10-fold increase in this value (from 3 to 25 μg/ml hemolymph) in the final larval instar, a stage at which marked increases in the concentrations of both lipophorin and the p82 protein were shown to occur. Calculations using the derived binding data and the ligand/binding protein concentration values confirmed that all of the hemolymph flavin is protein-bound and further, that the concentration of binding sites was in excess of the apparent riboflavin concentration. This latter point was confirmed in binding assays in which the binding of exogenous flavin added to hemolymph samples was demonstrated.

Molecular characteristics of the cholesterol oxidase and factors influencing its activity

Abstract The purpose of this study was to characterize Pseudomonas cholesterol oxidase (cholesterol:oxygen oxidoreductase, EC 1.1.3.6) and define factors influencing its activity with a view to performing electrochemical determinations of cholesterol. The enzyme was purified by gel filtration and its molecular characteristics were determined. Its amino acid composition presents a high proportion of glutamic and aspartic acids. Its isoelectric point is between 5.0 and 5.5 unit/pH and its molecular mass is equal to 65.1 kDa, as determined by analytical ultracentrifugation. The influence of reaction products on enzyme activity was studied. The amount of hydrogen peroxide produced is not great enough to inhibit the enzyme. Adding exogenous flavin adenine dinucleotide increases enzyme activity. Bile salts stabilize cholesterol oxidase structure increasing its activity without changing its affinity for cholesterol.

Changes in lens and erythrocyte glutathione reductase in response to exogenous flavin adenine dinucleotide and liver riboflavin content of rat on riboflavin deficient diet

The effect of exogenous flavin adenine dinucleotide (FAD) and hepatic level of riboflavin on the activity of lenticular and erythrocyte glutathione reductase (GSH-R) was studied in rats fed a diet deficient in riboflavin. By 75 days of feeding, the GSH-R activity, under basal condition (in the absence of exogenous FAD) and the activation coefficient (AC, activity of the enzyme in response to exogenous FAD to that without it) values were significantly altered in lens as well as erythrocytes of rats fed riboflavin deficient diet. Significant reduction in riboflavin content of liver was also observed in the experimental animals. These findings suggest that the AC value for the lenticular GSH-R is as equally a reliable biochemical parameter for assessing riboflavin nutriture as that of the erythrocyte enzyme.

Kinetics of reduction of high redox potential ferredoxins by the semiquinones of Clostridium pasteurianum flavodoxin and exogenous flavin mononucleotide. Electrostatic and redox potential effects.

We have measured the ionic strength dependence of the rate constants for the electron-transfer reactions of flavin mononucleotide (FMN) and flavodoxin semiquinones with 10 high redox potential ferredoxins (HiPIP's). The rate constants were extrapolated to infinite ionic strength by using a theoretical model of electrostatic interactions developed in our laboratory. In all cases, the sign of the electrostatic interaction was the same as the protein net charge, but the magnitudes were much smaller. The results are consistent with a model in which the electrical charges are approximately uniformly distributed over the HiPIP surface and in which there are both short- and long-range electrostatic interactions. An electrostatic field calculation for Chromatium vinosum HiPIP is consistent with this. The presumed site of electron transfer includes that region of the protein surface to which the iron-sulfur cluster is nearest and appears to be relatively hydrophobic. The principal short-range electrostatic interaction would involve the negative charge on the iron-sulfur cluster. For some net negatively charged proteins, this effect is magnified, and for net positively charged HiPIP's, it is counterbalanced. The rate constants extrapolated to infinite ionic strength can be correlated with redox potential differences between the reactants, as has previously been shown for cytochrome-flavin semiquinone reactions. Both electrostatic and redox potential effects are magnified for the flavodoxin semiquinone as compared to the FMN semiquinone-HiPIP reactions. This was also observed previously for the flavin semiquinone-cytochrome reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Blue Light-Reducible Cytochromes in Membrane Fractions from Neurospora crassa

We have assayed absorbance changes generated by blue light in plasma membranes, endoplasmic reticulum, and mitochondrial membranes from Neurospora crassa. Light minus dark difference spectra, obtained anaerobically in the presence of ethylenediaminetetraacetate, indicated that b-type cytochromes could be photoreduced in all three membranes. In plasma membranes, a b-type cytochrome with a distinct difference spectrum was photoreducible without addition of exogenous flavin. Addition of riboflavin greatly stimulated the photoreduction of cytochromes in endoplasmic reticulum and mitochondrial membranes. In its spectral characteristics the cytochrome on the endoplasmic reticulum resembled cytochrome b(5) or nitrate reductase, while the cytochrome in mitochondrial membranes had the same spectrum as cytochrome b of the mitochondrial respiratory chain.Cytochromes in the three membrane fractions reacted differently to blue light in the presence of various inhibitors. Potassium azide inhibited reduction of plasma membrane cytochrome b, with 50% inhibition at 1.0 millimolar. The same concentration of azide stimulated photoreduction of cytochromes in both endoplasmic reticulum and mitochondria. Although photoreduction of cytochromes in all three membranes was inhibited by salicylhydroxamic acid, cytochromes in plasma membranes were more sensitive to this inhibitor than those in endoplasmic reticulum and mitochondria. Cells grown to induce nitrate reductase activity showed an elevated amount of blue light-reducible cytochrome b in the endoplasmic reticulum.

Flavocytochromes c: transient kinetics of photoreduction by flavin analogues.

Kinetics of reduction of phototrophic bacterial flavocytochromes c by exogenous flavin semiquinones and fully reduced flavins generated by laser flash photolysis have been studied. The mechanisms of reduction of Chromatium and Chlorobium flavocytochromes c are more similar to one another than previously thought. Neither protein is very reactive with neutral flavin semiquinones (k less than 10(7) M-1 s-1), and the reactions with fully reduced flavins are slower than expected on the basis of comparison with other electron-transfer proteins of similar redox potentials. Deazaflavin radical is reactive with the flavocytochromes c by virtue of its low redox potential, but this reaction is also slower than expected on the basis of comparison with other electron-transfer proteins. These experiments indicate that the active site for reduction of flavocytochrome c is relatively buried and probably inaccessible to solvent. Fully reduced FMN does not show an ionic strength effect in its reaction with flavocytochrome c, which demonstrates that the active site is uncharged. Sulfite, which forms an adduct with protein-bound FAD, partially blocks heme reduction. This shows that heme is reduced via the FAD. The rate constant for intramolecular electron transfer between FAD and heme must be on the order of 10(4) s-1 or larger.

Chromatium flavocytochrome c: Kinetics of reduction of the heme subunit, and the flavocytochrome c-mitochondrial cytochrome c complex

The kinetics of reduction of Chromatium vinosum flavocytochrome c heme subunit by exogenous flavin neutral semiquinones generated by laser flash photolysis have been investigated. Unlike the holoprotein, the isolated heme subunit was appreciably reactive with lumiflavin neutral semiquinone. The measured rate constant for the reaction (2.7 × 10 7 m −1 s −1) was comparable to those of c-type cytochromes having similar redox potentials. The ionic strength dependence of the reaction with FMN neutral radical indicated that the heme subunit had a small negative charge at the site of reduction. Taken together, these results suggest that the active site of the heme subunit is buried on complexation with the flavin subunit in the holoprotein. Horse cytochrome c formed a strong complex with Chromatium, but not Chlorobium, flavocytochrome c. Possible physiological electron acceptors such as HiPIP, cytochrome c′, and cytochrome c-555 apparently did not bind to the flavocytochromes c. The rate constant for reduction by lumiflavin radical of horse cytochrome c complexed to flavocytochrome c was about twofold smaller than for reduction of horse cytochrome c alone. Flavocytochrome c was itself unreactive with exogenous flavin semiquinones. The ionic strength dependence of the reduction of the complex by FMN radical was also smaller than for horse cytochrome c in the absence of flavocytochrome c. Sulfite, which forms an adduct with the protein-bound FAD (FAD is bound in an 8-α- S-cysteinyl linkage), did not affect the reduction of horse cytochrome c in its complex with flavocytochrome c. We conclude that horse cytochrome c is reduced directly by exogenous flavins in its complex with flavocytochrome c, although the kinetics are slightly modified. These results are not unlike observations made with complexes of mitochondrial cytochrome c with cytochrome oxidase or cytochrome b 5.

PHOTOREACTIVATION BY TRIPLET FLAVIN and PHOTOINACTIVATION BY SINGLET OXYGEN OF Neurospora crassa NITRATE REDUCTASE*

Abstract— In the present paper the mechanism of photoreactivation and photodestruction of partially purified nitrate reductase from Neurospora crassa was investigated. For both photoreactions exogenous flavin was needed; the rates of the photoreactions depended on the concentration of free flavin in the irradiated sample. Their efficiency as photoreceptors increased in the sequence FAD–FMN–riboflavin. No effect of enzyme‐bound flavin as photoreceptor was found in these photoreactions. Photoreactivation of nitrate reductase inactivated by reduction in the presence of KCN is due to its reaction with flavins in their triplet state. Photodestruction is due to singlet oxygen generated from triplet flavin in the presence of oxygen. Rate constants are estimated to be k =1–5 × 10‐1M‐1 s‐1 for the reaction of nitrate reductase with the flavin triplet (photoreactivation) and k= 5 × 10‐1M‐1 s‐1 for the reaction with singlet oxygen (photodestruction).

Trisomy 8 in the bone marrow associated with high red cell glutathione reductase activity

In a series of 841 patients with hematologic disorders, 10 individuals were found to have an extra C group chromosome in their bone marrow cells. In two the extra chromosome was not identified, but in the remaining eight it was No. 8. Four of these ten patients had leukemia, and the others had cytopenias or other probably preleukemic conditions. The mean value for glutathione reductase activity in the red cells of four patients with trisomy 8 was significantly higher (2980 +/- 940 mumoles/min/liter of erythrocytes) than in normal controls (1930 +/- 360) or in any of five different control groups of patients with hematologic disorders. The extent of enzyme activation as a result of preincubation with exogenous flavin adenine dinucleotide was similar in the erythrocytes of all groups. The reasons for the high values of red cell glutathione reductase activity in patients with trisomy 8 are discussed in the light of the proposed assignment of the gene for that enzyme to chromosome 8.

Trisomy 8 in the Bone Marrow Associated With High Red Cell Glutathione Reductase Activity

Abstract In a series of 841 patients with hematologic disorders, 10 individuals were found to have an extra C group chromosome in their bone marrow cells. In two the extra chromosome was not identified, but in the remaining eight it was No. 8. Four of these ten patients had leukemia, and the others had cytopenias or other probably preleukemic conditions. The mean value for glutathione reductase activity in the red cells of four patients with trisomy 8 was significantly higher (2980 +/- 940 mumoles/min/liter of erythrocytes) than in normal controls (1930 +/- 360) or in any of five different control groups of patients with hematologic disorders. The extent of enzyme activation as a result of preincubation with exogenous flavin adenine dinucleotide was similar in the erythrocytes of all groups. The reasons for the high values of red cell glutathione reductase activity in patients with trisomy 8 are discussed in the light of the proposed assignment of the gene for that enzyme to chromosome 8.

Dechlorination of DDT by membranes isolated from Escherichia coli.

The capacity of cellular components of Escherichia coli to metabolize DDT-14C was investigated. Washed membrane fractions were obtained by lysozyme treatment followed by osomotic shock. Gas chromatography with electron capture detection and thin-layer chromatography were used to measure the effect of exogenous Kreb’s cycle cofactors and intermediates on the metabolism of DDT by particulate components of the bacteria. Reductive dechlorination of DDT to TDE occurred in the membranous fraction of the bacterial cell and was stimulated by a factor (s) present in the cytoplasmic fraction. Isolated membranes produced substantial amounts of TDE only in the presence of exogenous flavin adenine dinucleotide (FAD), and the prior reduction of FAD is essential to the production of TDE. The presence of Kreb’s cycle energy inhibited TDE production, indicating that DDT does not accept electrons produced by the oxidation of Kreb’s cycle intermediates directly from the cytochrome system.

Toxicity of Air-Oxidized Soybean Oil

The toxicity of air-oxidized soybean oil to weanling rats was investigated in several aspects. Growth studies demonstrated that the concentration of the toxic principle corresponded closely to the peroxide concentration of the oil. Moreover, separation of the oxidized oil into high-, medium- and low-peroxide fractions revealed that toxicity again followed peroxide concentration. Histopathological examination of rats fed oxidized soybean oil gave generally negative findings but indicated that the intestine might be involved. Moreover, absorption of fatty acid peroxides in mature rats, studied by means of thoracic duct cannulation, indicated that although the reduced products of the peroxides were absorbed, the peroxides themselves were destroyed in the intestine and probably had their action at that site. Recovery from the effects of whole-body irradiation, a condition also affecting the intestine, was delayed by diets containing fat with peroxide numbers as low as 100. Inhibition of intestinal xanthine oxidase by air-oxidized soybean oil and its reversal by exogenous flavin adenine dinucleotide suggest that the specific toxicity of the lipid peroxides may be at the level of the intestinal enzymes.