[Determination of penicillanic acid in solutions of penicillins using dithionitrobenzoic acid].
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Benzoates↗
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Reactivity of mitochondrial sulfhydryl groups toward dithionitrobenzoic acid and bromobimanes under oligomycin-inhibited and uncoupling conditions.
Thiol reactivity was determined in rat heart mitochondria using chromophores of differing polarities: monobromobimane (MB), dithionitrobenzoate (NbS2), and bromobimane-q (MQ). The purpose of this study is to correlate reaction rates of protein thiols in the mitochondrial membrane with the oligomycin-inhibited and uncoupled states: In all cases investigated the reactivity of -SH groups toward MB decreases under the above conditions. In parallel with an increase of their uncoupling activities the uncouplers reduce the reaction rate of thiol groups toward NbS2 and, progressively, toward MQ, indicating differences in sensitivity of thiol groups to uncouplers depending on the polarity of the environment. The pattern of -SH reactivity under inhibition by oligomycin resembles that of carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Functional changes of the mitochondrial membrane probably correlate with reactivity/polarity changes of membrane -SH groups. Masking of membrane thiol groups thus is not specific for uncouplers but is also observed under inhibition with oligomycin.
Animals↗
Modulation of activity of human alkaline phosphatases by Mg2+ and thiol compounds.
Interaction of purified human liver and placental alkaline phosphatases (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) with sulfhydryl groups, sulfhydryl reagents, and Mg2+ were studied. L-Cysteine (0.1 mmol/l) or Mg2+ activated the liver enzyme 4-5-fold and the placental enzyme 2-3-fold, with optimal pH 7.5-8.0; these activations were not additive. L-Cysteine (2 mmol/l) inhibited both enzymes maximally at pH greater than 9.0; phosphate protected the enzymes. S-Methylcysteine had little effect, with or without Mg2+. Inhibition by sulfur-containing compounds paralleled their ability to bind Zn2+. Fluoresceine mercury acetate (specific for sulfhydryl groups) inhibited the isoenzymes, whereas iodoacetic acid, iodoacetamide, dithionitrobenzoic acid, and p-chloromercuribenzoate had little effect. The inhibition was reversed by L-cysteine and only slightly protected by inorganic phosphate. Thus, there are two sites on human liver and placental alkaline phosphatase that interact with L-cysteine; a Mg2+-binding site, which results in activation, and a site that involves one or both of the bound Zn2+ ions and results in inactivation. Both enzymes have a protected essential thiol group.
Alkaline Phosphatase↗
Effects of cyclic nucleotides on the conformational states of the alpha core of the cyclic AMP receptor protein.
The alpha core gragment produced by limited proteolysis contains the cyclic AMP binding domain and the two buried sulfhydryl groups of the cyclic AMP receptor protein. The buried sulfhydryl groups of the alpha core react with 5,5'-dithio-bis(2-nitrobenzoic acid) after denaturation by 3 M urea or digestion with subtilisin. The rate of sulfhydryl modification in the presence of 3 M urea or subtilisin is markedly decreased in the presence of cyclic nucleotides which are proposed to tighten the conformation of the alpha core. Incubation of the alpha core in 3 M urea or dithionitrobenzoic acid does not affect cyclic AMP binding while dithionitrobenzoic acid plus 3 M urea inhibits cyclic AMP binding suggesting a role for the buried sulfhydryls in cyclic AMP binding or their proximity to the cyclic AMP binding domain of the alpha core. The data are consistent with a ligand-induced conformational change in the alpha region of the native cyclic AMP receptor protein that is required for DNA binding.
Binding Sites↗
Thiol groups of Escherichia coli citrate synthase and their influence on activity and regulation.
The modification of Escherichia coli citrate synthase (citrate oxaloacetatelyase(pro-3S-CH2.COO- leads to acetyl-CoA, EC 4.1.3.7) with 5,5'-dithiobis-(2-nitrobenzoic acid) has been investigated. (1) In low ionic strength (20 mM Tris.HCl, pH 8.0): (A) Eight thiol groups per tetramer of the native enzyme reacted with Nbs2. (b) Two of the eight accessible thiols were modified rapidly with the loss of 26% enzyme activity but with no change in the NADH inhibition. The remaining six were modified more slowly, resulting in a further 60% loss of activity and complete densensitization to NADH. (c) The 2nd-order rate constant for the modification of the rapidly reacting thiols is 2.5.10(4) M-1.min-1. At the reagent concentrations used (0.1 to 0.2 mM) the modification of the six thiols in the slow kinetic set appeared to be 1st-order; at 0.1 mM dithionitrobenzoic acid their rate of modification was approximately 30 times slower than the thiols in the fast kinetic set. (2) In high ionic strength (20 mM Tris.HCl, pH 8.0, 0.1 M KCl): (a) Four thiol groups were modified in a single kinetic set and it appeared that these thiols are four of the six slowly modified in the absence of KCl. (b) The modification resulted in 70% loss of enzyme activity and complete loss of NADH inhibition. (3) From the kinetic analysis it is proposed that the four thiol groups accessible to dithionitrobenzoic acid in the absence and presence of 0.1 M KCl are those involved in the response of NADH. Modification of any one of these four groups produced no reduction in the inhibition; instead, loss of NADH sensitivity was coincident with the appearance of tetrameric protein possessing three substituted thiols, whereas enzyme with one or two modified groups was still fully inhibited by NADH.
Chemical Phenomena↗
Redox-based control of the gamma heavy chain ATPase from Chlamydomonas outer arm dynein.
The outer dynein arm from Chlamydomonas flagella contains two redox-active thioredoxin-related light chains associated with the alpha and beta heavy chains; these proteins belong to a distinct subgroup within the thioredoxin family. This observation suggested that some aspect of dynein activity might be modulated through redox poise. To test this, we have examined the effect of sulfhydryl oxidation on the ATPase activity of isolated dynein and axonemes from wildtype and mutant strains lacking various heavy chain combinations. The outer, but not inner, dynein arm ATPase was stimulated significantly following treatment with low concentrations of dithionitrobenzoic acid; this effect was readily reversible by dithiol, and to a lesser extent, monothiol reductants. Mutational and biochemical dissection of the outer arm revealed that ATPase activation in response to DTNB was an exclusive property of the gamma heavy chain, and that enzymatic enhancement was modulated by the presence of other dynein components. Furthermore, we demonstrate that the LC5 thioredoxin-like light chain binds to the N-terminal stem domain of the alpha heavy chain and that the beta heavy chain-associated LC3 protein also interacts with the gamma heavy chain. These data suggest the possibility of a dynein-associated redox cascade and further support the idea that the gamma heavy chain plays a key regulatory role within the outer arm.
Amino Acid Sequence↗
Measurements of cysteine reactivity during protein unfolding suggest the presence of competing pathways.
Evidence that proteins may unfold utilizing complex competing pathways comes from a new pulse-labeling protocol in which the change in reactivity of a single cysteine residue in a protein during unfolding is measured, making use of its easily monitored reaction with the Ellman reagent, dithionitrobenzoic acid. The kinetics of unfolding of two single cysteine-containing mutant forms of the small protein barstar, C82A, which contains only Cys40, and C40A, which contains only Cys82, have been studied. The data suggest that unfolding occurs via two parallel pathways, each forming competing intermediates. In one of these early intermediates, Cys40 and Cys82 are already as reactive as they are in the fully unfolded protein, while in the other intermediate, the Cys thiol groups are unreactive. One more long-lived intermediate also needs to be included on the pathway defined by the early intermediate with unreactive Cys thiol groups to account for the difference in the rates of fluorescence change and of change in Cys40 reactivity. The demonstration of multiple intermediates and pathways for unfolding indicates that protein unfolding reactions can be as complex as protein folding reactions.
Amino Acid Substitution↗
Studies on acid soluble thiols in the human gastric juice.
Human gastric juice was found to contain high levels of thiols as detected by reaction with dithionitrobenzoic acid (Ellman's reagent). Analysis of gastric juice for glutathione and cysteine, the known acid soluble thiols in biological system, by HPLC and calorimetric methods, indicated that they are absent in the gastric juice. Further studies on the nature of thiols in the gastric juice, by Sephadex G-25 gel filtration indicated the presence of both high and low molecular weight protein and peptide associated thiols. Dialysis of the gastric juice retained nearly 70% thiols in non-dialysable fraction. These studies suggest that biologically important low molecular weight thiols, glutathione and cysteine are absent in the human gastric juice and the thiols detected are mainly acid soluble proteins and possibly peptide associated thiols.
Chromatography, Gel↗
Cleavage of the S-S bond in 5,5'-dithiobis-(2-nitrobenzoic acid) in the presence of a cationic detergent: an approach to the cleavage of the S-S bond in bovine plasma albumin.
The hydrolysis of 5,5'-dithiobis-(2-nitrobenzoic acid) was studied at pH 9.20 and 25 degrees C in presence of tetradecyltrimethylammonium bromide or micellar tetradecyltrimethylammonium bromide. The reaction was pseudo-unimolecular reaction with regard to the concentration of dithionitrobenzoic acid. The rate constant k1 depended on the tetradecyltrimethylammonium bromide concentration and on the ionic strength of the buffer solution. At a constant ionic strength, the value of k1 increased with the increase in the tetradecyltrimethylammonium bromide concentration, attained maximum at a certain concentration above the critical micelle concentration, and then decreased. The value of k1 was larger when the ionic strength was lower. The rate of hydrolysis at the ionic strength 0.1 was the same as that without tetradecyltrimethylammonium bromide. On the contrary, sodium dodecyl sulfate or micellar sodium dodecyl sulfate had no effect on the rate of hydrolysis. These results lead to the conclusion that the S-S bond is cleaved easier, when it is surrounded by the cationic detergent. The SH/S-S exchange reaction of bovine plasma albumin in the presence of cationic detergent was slower when the ionic strength was higher. The fact could be explained by assuming that the exposed S-S bond is surrounded by the cationic detergent. Further, it was speculated that some S-S bonds in albumin are surrounded by the positively charged basic amino acid residues.
Detergents↗
Lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. Purification and characterization.
Lumazine protein, a novel protein containing 6,7-dimethyl-8-ribityllumazine as a bound prosthetic group, is one of the several major proteins produced by the bioluminescent bacteria, Photobacterium phosphoreum. purification to complete homogeneity from cell extracts is achieved in six steps. Lumazine protein is a near spherical, monomeric protein of average molecular weight 20,000; in amino acid composition it is acidic with two isoelectric isomers, pI 4.9 and 5.0, and is hydrophilic (974 cal/residue) with single methionine and tryptophan residues and two accessible cysteines. It contains no carbohydrate. Reaction of the cysteines with dithionitrobenzoic acid results in quenching of the bound lumazine fluorescence but is otherwise reversible. Estimates of protein by dry weight results in a mole ratio of one bound lumazine group per protein.
Amino Acids↗
Mechanisms of inhibitory effects of zinc and cadmium ions on agonist binding to adenosine A1 receptors in rat brain.
The dose-dependent inhibition of zinc and cadmium ions of agonist binding to A1 adenosine receptors in rat brain is prevented by histidine and cysteine, respectively. In the present study, the possible different mechanisms of Zn2+ and Cd2+ inhibitions were examined. The effects of Zn2+ and Cd2+ on equilibrium binding parameters of the agonists N6-cyclohexyl-[2,8-3H]-adenosine ([3H]CHA) or chloro-N6-cyclopentyl-adenosine ([3H]CCPA) and the antagonist cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) were compared with those effects of reagents or binding conditions which altered histidyl or cysteinyl residues of the A1 receptor. Zn2+ pretreatment did not change A1 agonist or antagonist affinity, but did reduce the Bmax. The inhibitory effects of Zn2+ pretreatments were also maintained after several membrane washings. Diethylpyrocarbonate, a histidine-specific alkylating reagent, behaved like zinc ions: pretreatment with A1 agonist protected the histidyl residues of the [3H]CHA binding site against modification by Zn2+, while the modification of the protonation state of the nitrogen of the imidazole group of histidines by changing pH indicated that the interactions of Zn2+ with the histidyl residues were feasible with their unprotonated form. These findings suggest the formation of coordination bonds between Zn2+ and histidines critical for [3H]CHA or [3H]DPCPX binding, which may prevent the ligand interaction with the specific sites without modifying the binding kinetics of radioligand to the non-chelated recognition sites. Cd2+ pretreatment reduced the [3H]CCPA affinity, but did not modify the affinity of the antagonist [3H]DPCPX, the Bmax remaining unaffected. As with cadmium effects, the oxidation of the thiol group of cysteine by dithionitrobenzoic acid (DTNB) reduced [3H]CCPA affinity without changing the number of binding sites. The reducing reagent dithiothreitol, which alone was unable to modify [3H]CCPA binding, overcame the inhibiting effects of both Cd2+ and DTNB. These findings suggest that cadmium ions may oxidize SH groups of cysteines localized on the A1 receptor molecule or a cysteine localized in the region of G(i)alpha subunit involved in the coupling with receptors. This mechanism can justify potential conformational modifications of the receptor molecule producing the decrease in affinity.
Adenosine↗
The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor.
The Rhodobacter sphaeroides extra cytoplasmic function sigma factor, sigma(E), directs transcription of promoters for the cycA gene (cycA P3) and the rpoEchrR operon (rpoE P1). These genes encode the periplasmic electron carrier cytochrome c(2) and sigma(E)/ChrR, respectively. Using in vitro transcription assays with purified R. sphaeroides core RNA polymerase and sigma(E), we show that ChrR is sufficient to inhibit sigma(E)-dependent transcription. Inhibition is proposed to proceed through a binding interaction, since sigma(E) and ChrR form a 1:1 complex that can be purified when expressed at high levels in Escherichia coli. Active preparations of ChrR and the sigma(E)/ChrR complex each contain stoichiometric zinc. Removal of zinc from ChrR or a single amino acid substitution that abolishes zinc binding, results in a protein that is incapable of inhibiting sigma(E) activity or forming a complex with the sigma factor, indicating that metal binding is important to ChrR activity. Treatment of ChrR with the thiol-modifying reagent p-hydroxymecuriphenylsulfonic acid results in the release of about one mole of zinc per mole of protein. Furthermore, two N-terminal cysteine residues are protected from reaction with the thiol-specific reagent dithionitrobenzoic acid until zinc is removed, suggesting that these residues may be involved in zinc binding. These data indicate that ChrR is a specific anti-sigma factor of sigma(E) that requires zinc for function. Based on amino acid sequence similarity, we propose that ChrR is part of a family of similar anti-sigma factors that are found in alpha and gamma proteobacteria.
Amino Acid Sequence↗
Measurement of oxidized glutathione by enzymatic recycling coupled to bioluminescent detection.
A new method is described for the quantification of oxidized glutathione (GSSG) in tissues by enzymatic recycling coupled to NADPH bioluminescent detection. Tissue samples are treated with metaphosphoric acid. In a first step, after derivatization of GSH with 4-chloro-7-trifluoromethyl-1-methylquinolinium (CFQ), GSSG is recycled in the presence of dithionitrobenzoic acid (DTNB) and NADPH by glutathione reductase. In a second step, the GSSG-dependent NADPH consumption is measured by luminescence with NADPH:FMN oxidoreductase-bacterial luciferase. The coefficient of variation for GSSG measurements on repeated assays (n = 5) is 2 and 3% for standards and tissue samples, respectively. The sensitivity of this method is at the picomole level and is convenient for determination of GSSG physiological concentrations in tissues: GSSG levels measured in rat liver and kidney ranged from 76 to 215 and 52 to 170 nmol/g wet weight, respectively.
Animals↗
Sulfhydryl reagent susceptibility in proteins with high sequence similarity--triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi and Leishmania mexicana.
The amino acid sequence of triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68%. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei triosephosphate isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100-fold more sensitive. The sensitivity of wild type triosephosphate isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana triosephosphate isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accessibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more susceptible for perturbation.
Animals↗
Reactions of phenoxyl radicals with NADPH-cytochrome P-450 oxidoreductase and NADPH: reduction of the radicals and inhibition of the enzyme.
Phenoxyl radicals are intermediates of one-electron oxidation of phenolic compounds by various peroxidases. This report describes reactions of phenoxyl radicals with human NADPH-cytochrome P-450 oxidoreductase (OR) and NADPH. Purified truncated OR catalyzed quenching of EPR signal of the phenoxyl radical of a vitamin E homolog, 2,2,5,7,8-pentamethyl-6-hydroxychromane. The quenching required both reductase and NADPH and was not supported by NADH. NADPH quenched directly the EPR signal of phenoxyl radical of a phenolic antitumor drug, etoposide, in the absence of the OR. Quenching of the EPR signal was accompanied by increased rate of NADPH oxidation and decreased rate of etoposide oxidation. Phenoxyl radicals of etoposide did not inactivate the OR. In the absence of NADPH, OR was inhibited irreversibly when exposed to phenoxyl radicals of phenol. The activity of the flavoprotein could not be recovered by dithiothreitol (DTT) but the inhibition was prevented by saturation of OR with NADP+ prior to the exposure to phenoxyl radicals. The OR was also inhibited by 5,5'-dithionitrobenzoic acid (DTNB). The inhibition was reversible by subsequent addition of DTT. OR pretreated with DTNB was protected from inhibition by phenoxyl radicals of phenol. The results indicate that phenoxyl radical of 2,2,5,7,8-pentamethyl-6-hydroxychromane is likely reduced enzymatically by transfer of electrons from NADPH via the FAD/FMN of the OR. Phenoxyl radicals with higher redox potential, e.g., phenoxyl radicals of etoposide, oxidize NADPH directly. Phenoxyl radicals of phenol can also inactivate OR likely by oxidation of cysteine 565 in the NADPH binding region of the enzyme.
Animals↗
The effects of thiol reduction and oxidation on the inhibition of NMDA-stimulated neurotransmitter release by ethanol.
The N-methyl-D-aspartate (NMDA) receptor is sensitive to inhibition by pharmacologically relevant concentrations of ethanol and may be an important target for the actions of ethanol in the brain. The mechanisms responsible for ethanol's action are unknown but may involve perturbation of one of the multiple regulatory sites that have been identified on the receptor. Previous studies have shown that thiol reducing and oxidizing agents selectively after the activity of the NMDA receptor via a redox modulatory site. In this study, these agents were tested for their ability to modify the inhibitory actions of ethanol on NMDA-stimulated neurotransmitter release from rat brain slices and the potentiation of NMDA-stimulated release by the glycine agonist D-serine. Treatment of hippocampal slices with the thiol reducing agent dithiothreitol (DTT) significantly potentiated (> 2-fold) the NMDA-stimulated release of tritiated norepinephrine ([3H]NE) from rat hippocampal slices. Slices treated with the thiol oxidizing agent dithionitrobenzoic acid (DTNB) had slightly lower (10-15%) levels of NMDA-stimulated neurotransmitter release. Ethanol (25-200 mM) dose-dependently inhibited the NMDA-stimulated release of [3H]NE from control slices with a calculated IC50 value of 73.1 mM. The inhibitory potency for ethanol was increased following DTNB treatment (IC50 of 40.9 mM) while DTT treatment slightly reduced ethanol's potency (IC50 of 85.6 mM). The ability of D-serine to augment NMDA-stimulated neurotransmitter release was tested in the presence and absence of ethanol following treatment of slices with either DTT or DTNB to examine any potential interaction between these modulatory sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effects of reducing agents and oxidants on excitation-contraction coupling in skeletal muscle fibres of rat and toad.
1. The mechanically skinned fibre technique was used to examine the role of oxidation-reduction in the control of Ca2+ release and contraction in rat and toad skeletal muscle fibres under physiological conditions of myoplasmic [Mg2+] and [ATP] and sarcoplasmic reticulum (SR) Ca2+ load. 2. None of the reducing agents, dithiothreitol (DTT, 10 mM), glutathione (GSH, 10 mM) or cysteine (1 and 5 mM), had any detectable effect on the peak force, duration or the total number of depolarization-induced responses that could be elicited in skinned fibres from either toad or rat muscle, except for a slight alteration in one case (GSH on the duration of the response in rat fibres) caused by an effect of the agent of the Ca2+ sensitivity of the contractile apparatus. 3. Application of the reactive disulphide, 2,2'-dithiodipyridine (DTDP, 100 microM), a potent oxidizing agent, never induced any measurable force response or noticeable depletion of SR Ca2+ in any fibre under the conditions used. When all Ca2+ uptake was prevented, DTDP treatment of rat fibres was found to cause a 2- to 3-fold increase in the low rate of Ca2+ "leak' from the SR. DTDP treatment also increased the responsiveness of toad muscle fibres to 1 or 2 mM caffeine. These effects could be largely reversed by treatment with DTT. These results indicate that oxidation of the Ca2+ release channel does not cause substantial channel opening under physiological conditions. 4. Depolarization-induced force responses in both rat and toad fibres were rapidly abolished in the presence of DTDP (10 or 100 microM), in a manner favoured by inactivation of the voltage sensors. The relatively impermeant oxidant, 5,5'-dithionitrobenzoic acid (DTNB, 100 microM), had an effect very similar to DTDP if applied intracellularly, but unlike DTDP, had little or no effect if applied extracellularly (at 5 mM) before skinning. Depolarization-induced responses could be restored by treatment with DTT (10 mM). Intracellular application of the sulfhydryl-alkylating agent, N-ethylmaleimide (NEM, 100 microM), had effects very similar to DTDP and DTNB. 5. These results are not consistent with the proposal that excitation-contraction coupling in skeletal muscle primarily involves the oxidative linkage of the voltage sensors to the Ca2+ release channels, but do show that oxidation of an intracellularly accessible site can interfere with the coupling, in a process made more sensitive by voltage sensor inactivation.
2,2'-Dipyridyl↗
Accumulation of genistein and lipophilic genistein derivatives in lipoproteins during incubation with human plasma in vitro.
Atherosclerosis is initiated by the uptake and retention of oxidized low-density lipoprotein (LDL) into the arterial intima. We have previously shown that dietary isoflavone phytoestrogens inhibit LDL oxidation in vitro. The inhibition could have been caused by undetected isoflavone metabolites associated with lipoproteins. In the present study, we incubated human plasma with [3H]genistein, both with and without the lecithin:cholesterol acyltransferase (LCAT) inhibitor dithionitrobenzoic acid (DTNB). Our results indicated that the 3H-label was attached to both high-density lipoprotein (HDL) and LDL, and that it represented both underivatized genistein and lipophilic derivatives of genistein, part of which were identified as fatty acid monoesters. The latter was demonstrated by the findings that DTNB decreased the HDL and LDL associated radioactivity in the lipophilic fraction isolated by hydrophobic chromatography and that saponification hydrolysis liberated a corresponding part of the 3H-label. Two-dimensional reversed-phase thin-layer chromatography (TLC) demonstrated that a corresponding part of the radioactivity comigrated with genistein monoester standards in the absence of DTNB but was abolished if DTNB had been present in the incubation. In summary, incubation of plasma with [3H]genistein resulted in accumulation of underivatized genistein as well as lipophilic genistein derivatives in lipoproteins. A smaller part of the latter were genistein monoesters, while part remained unidentified. Our results suggest an explanation for the increased oxidation resistance of isolated LDL during intake of soybean isoflavones.
Dithionitrobenzoic Acid↗