Quantitation of the hydroxyl radical by reaction with dimethyl sulfoxide

This investigation was conducted to validate the use of dimethyl sulfoxide (DMSO) as a quantitative molecular probe for the generation of hydroxyl radicals (HO.) in aqueous systems. Reaction of HO. with DMSO produces methane sulfinic acid as a primary product, which can be detected by a simple colorimetric assay. To evaluate this method for estimating total HO. production, we studied three model systems, including the Fenton reaction, gamma irradiation of water, and ultraviolet photolysis of hydrogen peroxide, for which the theoretical maximum yield of HO. could be calculated and compared to measured DMSO oxidation. The results confirm that 0.05 to 1 M DMSO may be used to capture nearly all of the expected HO. radicals formed. Thus, methane sulfinic acid production from DMSO holds promise as an easily measured marker for HO. formation in aqueous systems pretreated with DMSO.     

α-[3H]Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding to Human Cerebral Cortical Membranes: Minimal Changes in Postmortem Brains of Chronic Schizophrenics

The binding of alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a selective ligand for the ion channel-linked quisqualate receptor, was evaluated in Triton X-100-treated membranes of human cerebral cortex. The presence of chaotropic ions produced divergent effects on specific [3H]AMPA binding: A twofold increase in the binding was observed with thiocyanide at 100 mM, although iodide (100 mM) and perchlorate (100 mM) reduced the binding. Chemical modifications of the sulfhydryl group with p-chloromercuriphenylsulfonic acid (PCMBS) produced threefold increases in specific [3H]-AMPA binding in the absence of KSCN as well as in the presence of KSCN. Treatment with dithiothreitol restored the enhanced specific [3H]AMPA binding by PCMBS to the basal level. Although specific [3H]AMPA binding in the absence of KSCN showed a single site (KD = 220 nM, Bmax = 235 fmol/mg of protein), curvilinear Scatchard plots of specific [3H]AMPA binding in the presence of 100 mM KSCN can be resolved into two binding sites with the following parameters: KD1 = 5.82 nM, Bmax1 = 247 fmol/mg of protein; KD2 = 214 nM, Bmax2 = 424 fmol/mg of protein. Quisqualate and AMPA were the most potent inhibitors of the [3H]AMPA binding in the presence of KSCN. Potent inhibitors of the binding included beta-N-oxalylamino-L-alanine (L-BOAA), cysteine-S-sulfate, L-glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione, and 6,7-dinitroquinoxaline-2,3-dione. Kainate, L-homocysteine sulfinic acid, and L-homocysteic acid were active with an IC50 value of a micromolar concentration, whereas L-cysteic acid and L-cysteine sulfinic acid were weakly active.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylene blue photosensitized oxidation of cysteine sulfinic acid and other sulfinates: the involvement of singlet oxygen and the azide paradox

The methylene blue photosensitized oxidation of cysteine sulfinic acid is investigated. Enhancement of the oxygen consumption rate in deuterium oxide suggests the involvement of singlet oxygen ((1)O(2)) in oxidation. Addition of the (1)O(2) quencher azide produced an unusual enhancement of the oxidation rate of all the sulfinates assayed. It is assumed that azide works as a one-electron carrier between (1)O(2) and the sulfur compounds. Analyses of the products indicate that the photochemical oxidation of cysteine sulfinic acid proceeds through two simultaneous mechanisms. The Type II (singlet oxygen) mechanism is responsible for oxidation of the sulfinic group to the sulfonic group with production of cysteic acid, stable to the photooxidation system, whereas the Type I (electron transfer) mechanism is involved in the degradation of cysteine sulfinic acid to acetaldehyde. Other products detected were ammonia, sulfate, and hydrogen peroxide which account for the degradation of cysteine sulfinic acid and for the excess of oxygen consumption detected during the oxidative reaction.     

In Vivo Elevation of Mouse Brain S-Adenosyl-L-Homocysteine after Treatment with L-Homocysteine

Intraperitoneal coadministration of adenosine and L-homocysteine markedly increased S-adenosyl-L-homocysteine in whole mouse brain, but further investigations showed that this elevation could also be produced following administration of L-homocysteine alone. The noted increase was maximal (+1325%) 10 min after treatment, remaining at about this level for 30-40 min before returning to control values after 180 min. Cerebral adenosine levels were decreased after treatment with L-homocysteine, adenosine, or these two substances in combination.     

Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B

Asparagine synthetase catalyzes the ATP-dependent formation of L-asparagine from L-aspartate and L-glutamine, via a beta-aspartyl-AMP intermediate. Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme. Prior work showed that L-cysteine sulfinic acid competitively inhibits this enzyme by interfering with L-aspartate binding. Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E. coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of beta-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle. While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor. A sulfoximine reported recently by Koizumi et al. as a competitive inhibitor of the ammonia-dependent E. coli asparagine synthetase A (AS-A) [Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J. (1999) J. Am. Chem. Soc. 121, 5799-5800] can be regarded as such a species. We found that this sulfoximine also inhibited AS-B, effectively irreversibly. Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine. Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B.     

Generation of a cysteine sulfinic acid analog for incorporation in peptides using solid phase peptide synthesis

The sulfinic acid analog of aspartic acid, cysteine sulfinic acid, introduces a sulfur atom that perturbs the acidity and oxidation properties of aspartic acid. Cysteine sulfinic acids are often introduced in peptides and proteins by oxidation of cysteine, but this method is limited as all cysteine residues are oxidized and cysteine residues are often oxidized to sulfonic acids. To provide the foundation for the specific incorporation of cysteine sulfinic acids in peptides and proteins, we synthesized a 9-fluorenylmethyloxycarbonyl (Fmoc) benzothiazole sulfone analog. Oxidation conditions to generate the sulfone were examined and oxidation of the Fmoc-protected sulfide (3) with NbC in hydrogen peroxide provided the corresponding sulfone (4) in the highest yield and purity. Reduction with sodium borohydride generated the cysteine sulfinic acid (5) suggesting this approach may be an efficient method to incorporate a cysteine sulfinic acid in biomolecules. A model tripeptide bearing a cysteine sulfinic acid was synthesized using this approach. Future studies are aimed at using this method to incorporate cysteine sulfinic acids in peptide hormones and proteins for use in the study of biological function.     

Measurement of Excitatory Sulfur Amino Acids, Cysteine Sulfinic Acid, Cysteic Acid, Homocysteine Sulfinic Acid, and Homocysteic Acid in Serum by Stable Isotope Dilution Gas Chromatography-Mass Spectrometry and Selected Ion Monitoring

Oxidized sulfur-containing amino acids are recognized as agonists of excitatory amino acid receptors in the mammalian nervous system. Homologues of glutamic acid (homocysteine sulfinic acid and homocysteic acid) and aspartic acid (cysteine sulfinic acid and cysteic acid) have been shown to be agonistic to N-methyl-D-aspartate receptors in animal brain and have been demonstrated in brain tissue. Considerable evidence exists for the role of homocysteic acid and cysteine sulfinic acid as endogenous ligands for excitatory amino acid receptors. We report, for the first time, the quantitation of these compounds in normal human serum, by a newly developed gas chromatography-mass spectrometry method that employs stable isotope-dilution selected ion monitoring using internal standards prepared in our laboratory. We also report new methods of synthesis of stable isotope-labeled internal standards used in measuring cysteine sulfinic acid, cysteic acid, homocysteine sulfinic acid, and homocysteic acid.     

Cysteine Sulfinic Acid in the Central Nervous System: Uptake and Release of Cysteine Sulfinic Acid by a Rat Brain Preparation

Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P₂) and slices of rat cerebral cortex were investigated. The P₂ fraction had a Na⁺-dependent high-affinity uptake system for cysteine sulfinic acid (K_m, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [¹⁴C]cysteine sulfinic acid from preloaded cortical slices or P₂ fractions was examined by a superfusion method, which avoided reuptake of released [¹⁴C]cysteine sulfinic acid. High K⁺ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P₂ fraction in a partly Ca²⁺-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P₂ fraction.     

Inhibition of protein carboxyl methylation by S-adenosyl-L-homocysteine in intact erythrocytes. Physiological consequences

S-Adenosyl-L-homocysteine was used to inhibit the methylation of carboxylic acid residues of membrane proteins in intact human erythrocytes. Incubation of erythrocytes for 24 h with 5 mM each of adenosine and L-homocysteine resulted in the intracellular accumulation of S-adenosyl-L-homocysteine and substantially inhibited membrane protein carboxyl methylation. From the degree of inhibition and from the observed turnover of methylated proteins, we estimate that the number of protein methyl esters in cells incubated with adenosine and L-homocysteine for 20 h is less than 20% that of cells incubated without these inhibitors. No significant differences in the physical deformability properties of the membrane of these hypomethylated cells were detected. However, there was a small but significant (p less than 0.001) increase in the amount of membrane protein D-aspartyl residues in these cells compared to control cells. These observations are consistent with the hypothesis that methylation of membrane proteins at D-aspartyl residues may result in the selective removal or repair of these uncommon residues.     

Cysteine Sulfinic Acid in the Central Nervous System: Antagonistic Effect of Taurine on Cysteine Sulfinic Acid-Stimulated Formation of Cyclic AMP in Guinea Pig Hippocampal Slices

The stimulatory effect of cysteine sulfinic acid on cyclic AMP formation was examined in slices from three different regions of guinea pig brain. The inhibitory effect of taurine on the stimulated formation of cyclic AMP was also studied. Cysteine sulfinic acid (1--10 mM) greatly increased the cyclic AMP level in striatal, cortical, and especially hippocampal slices. In hippocampal slices, taurine (0.1--30 mM) markedly lowered the increase of cyclic AMP induced by cysteine sulfinic acid, but not that induced by glutamate or aspartate. In this region, taurine also reduced the stimulatory effects on cyclic AMP formation of adenosine, norepinephrine, and histamine, but not of depolarizing agents. It did not, however, inhibit the effects of any of these stimulants in cortical slices. These results suggest that sulfur-containing amino acids, such as cysteine sulfinic acid and taurine, regulate the cyclic AMP level in the hippocampus.     

Reduction of cysteine sulfinic acid by sulfiredoxin is specific to 2-cys peroxiredoxins

Cysteine residues of certain peroxiredoxins (Prxs) undergo reversible oxidation to sulfinic acid (Cys-SO2H) and the reduction reaction is catalyzed by sulfiredoxin (Srx). Specific Cys residues of various other proteins are also oxidized to sulfinic acid, suggesting that formation of Cys-SO2H might be a novel posttranslational modification that contributes to regulation of protein function. To examine the susceptibility of sulfinic forms of proteins to reduction by Srx, we prepared such forms of all six mammalian Prx isoforms and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Purified sulfiredoxin reduced the sulfinic forms of the four 2-Cys members (Prx I to Prx IV) of the Prx family in vitro, but it did not affect those of Prx V, Prx VI, or GAPDH. Furthermore, Srx bound specifically to the four 2-Cys Prxs in vitro and in cells. Sulfinic forms of Prx I and Prx II, but not of Prx VI or GAPDH, present in H2O2-treated A549 cells were gradually reduced after removal of H2O2; overexpression of Srx increased the rate of the reduction of Prx I and Prx II but did not induce that of Prx VI or GAPDH. These results suggest that reduction of Cys-SO2H by Srx is specific to 2-Cys Prx isoforms. For proteins such as Prx VI and GAPDH, sulfinic acid formation might be an irreversible process that causes protein damage.     

Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Identification as the metE gene product.

Methanobacterium thermoautotrophicum is a methane-forming archaeon that grows on H2 and CO2 as sole carbon and energy source. Cell extracts of the methanogen were found to contain methylcobalamin: homocysteine methyltransferase activity which was purified 3000-fold to a specific activity of approximately 500 U.mg-1 protein. SDS/PAGE revealed the presence of a polypeptide with an apparent molecular mass of 34 kDa. Via its N-terminal amino acid sequence, the 34-kDa polypeptide was identified as the metE gene product. The metE gene was heterologously expressed in Escherichia coli. The overproduced protein was recovered in the inclusion body fraction and was found to be inactive. The protein could be partially solubilized by unfolding in 8 M urea and then refolding. The solubilized protein had a specific activity of 450 U.mg-1. It exhibited first-order kinetics with respect to methylcobalamin concentration and Michaelis-Menten kinetics with respect to L-homocysteine concentration (apparent Km 0.1 mM). The enzyme was specific for L-homocysteine as methyl acceptor. Methylcobalamin could be substituted with methylcobinamide as methyl donor.     

Nature of papain products resulting from inactivation by a peptidyl O-acyl hydroxamate.

Mass spectrometry has been used to provide insights into the mechanism of inhibition of cysteine proteases by a hydroxylamine derivative, CBZ-Phe-Gly-NH-O-CO-(2,4,6-Me3)Ph. An oxidized form of papain resulting from the incubation of the enzyme with the peptidyl hydroxamate in the absence of a reducing agent has been identified as a sulfinic acid. The presence of a covalent enzyme-inhibitor complex of molecular mass consistent with a sulfenamide adduct of papain could also be detected by this method. Implications on the mechanism of inactivation of cysteine proteases by peptidyl hydroxamates are discussed.     

Simplified cysteine dioxygenase activity assay allows simultaneous quantitation of both substrate and product

A high-performance liquid chromatography (HPLC) method for enzyme activity assays using a hydrophilic interaction liquid chromatography (HILIC) column in combination with an evaporative light scattering detector was developed. The method was used to measure the activity of the non-heme mono-iron enzyme cysteine dioxygenase. The substrate cysteine and the product cysteine sulfinic acid are very weak chromophores, making direct ultraviolet (UV) detection without derivatization rather insensitive; moreover, derivatization of cysteine is often not efficient. Using the system described, underivatized substrate and product in samples from cysteine dioxygenase activity assays could be separated and analyzed. Furthermore, it was possible to quantify cysteic acid, the noncatalytic oxidation product of cysteine sulfinic acid. Acetone was used both to stop the enzymatic reaction by protein precipitation and as an organic mobile phase, making sample preparation very easy and the assay highly reproducible.     

Cysteine Sulfinic Acid in the Central Nervous System: Specific Binding of [35S]Cysteic Acid to Cortical Synaptic Membranes-An Investigation of Possible Binding Sites for Cysteine Sulfinic Acid

Abstract: Specific binding sites for cysteine sulfinic acid, an excitatory amino acid, in crude synaptic membrane fractions of rat cerebral cortex were examined, using L-[³⁵S]cysteic acid as a ligand. Two specific binding systems of [³⁵S] cystec acid were found, one Na⁺-dependent and the other Na⁺-independent. The Na⁺-independent specific binding of [³⁵S]Cysteic acid was saturable, with a K_d of 474 n M and B_max of 3.29 pmol/mg protein. The binding was optimal at pH 7.4 and at 37°C. Treatment of the membranes with proteases, concanavalin A, or Triton X-100 markedly reduced the binding. Of various compounds related to cysteic acid, L-cysteine sulfinic acid was the most effective competitor of this binding. These results indicate the existence of an Na⁺-independent specific binding site for cysteic acid in the synaptic membrane of rat cerebral cortex, which may be different from that for glutamate. Possible involvement of cysteine sulfinic acid as an endogenous ligand for this binding site is discussed.     

Antioxidant Properties of Sulfinates: Protective Effect of Hypotaurine on Peroxynitrite-Dependent Damage

It has been proposed that hypotaurine may function as an antioxidant in vivo. We investigated whether this compound can act as protective agent able to prevent damage from peroxynitrite, a strong oxidizing and nitrating agent that reacts with several biomolecules. The results showed that the compound efficiently protects tyrosine against nitration, alpha1-antiproteinase against inactivation, and human low-density lipoprotein against modification by peroxynitrite. Hypotaurine is also highly effective in inhibiting peroxynitrite-mediated nitration of tyrosine in the presence of added bicarbonate. This result suggests that hypotaurine could play an important role as protective agent under physiological conditions. Moreover, it was found that cysteine sulfinic acid, but not taurine, possesses protective properties against peroxynitrite-dependent damage similar to hypotaurine. These findings indicate that the protective effects exerted by these compounds may be attributable to the presence of the sulfinic group oxidizable into sulfonate by scavenging peroxynitrite and/or its derived species.     

S-Oxygenation of N-substituted thioureas catalyzed by the pig liver microsomal FAD-containing monooxygenase

The microsomal FAD-containing monooxygenase (EC 1.14.13.8, dimethylaniline monooxygenase) purified to homogeneity from hog liver catalyzes NADPH- and oxygen-dependent S-oxygenation of phenylthiourea, ethylenethiourea, thiocarbanilide, N-methylthiourea, and thiourea to their corresponding formamidine sulfinic acids. The sulfinic acids are formed by sequential enzymic oxidation of the thioureas through intermediate sulfenic acids. The reaction sequence was established by separating intermediate and final oxygenated metabolites of phenylthiourea and ethylenethiourea. The sulfenic and sulfinic acids of these two thioureas, produced enzymically, were chromatographically and spectrally identical with chemically synthesized reference compounds. Phenylformamidine and ethyleneformamidine sulfinic acids are slowly converted to their sulfonic acids upon prolonged incubation. While N-substituted formamidine sulfinic acids oxidize spontaneously to formamidine sulfonic acids at 37 °C, the further oxidation of ethyleneformamidine sulfinic acid may be, at least in part, enzyme catalyzed. The purified monooxygenase also catalyzes rapid oxygenation of mercaptoimidazoles to the corresponding imidazole sulfinic acids. The instability of S-oxygenated mercaptoimidazoles prevented their isolation and positive identification, but analysis of kinetic data obtained with sulfenic acid trapping agents suggests that these compounds are oxygenated by the same reaction sequence established for N-substituted thioureas. The NADPH- and oxygen-dependent oxidation of thiocarbamates and of 2-mercaptoimidazoles catalyzed by hog or hamster liver microsomes correlates with dimethylaniline N-oxidase activity and appears completely independent from cytochrome P-450. The S-oxidation of thiourea and its derivatives is not inhibited by n-octylamine, a known inhibitor of cytochrome P-450 dependent oxygenations. Furthermore, differential thermal inactivation of the flavin-containing monooxygenase totally abolishes phenylthiourea S-oxidase activity of hamster liver microsomes.     

New lignan metabolites in rat urine

Ten potential lignan metabolites were quantified in rat urine extracts using liquid chromatography-tandem mass spectrometry. The rats were orally administered with the plant lignans 7-hydroxymatairesinol, matairesinol, lariciresinol or secoisolariciresinol, or with the mammalian lignan enterolactone. The samples were enzymatically hydrolysed and solid-phase extracted before analysis. Of the analysed compounds, only trace amounts of 7-oxoenterolactone could be detected in the urine extracts before administration, but after administration of any of the lignans, the excretion of 7-oxoenterolactone increased and monodemethylated matairesinol and 4,4'-dihydroxyenterolactone could be detected. In addition, other novel lignan metabolites were detected, i.e., 7-oxomatairesinol, alpha-conidendrin, and alpha- and beta-conidendric acid.     

Characterization of mammalian sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine

Peroxiredoxins (Prxs) are a family of peroxidases that reduce hydroperoxides. The cysteine residue in the active site of certain eukaryotic Prx enzymes undergoes reversible oxidation to sulfinic acid (Cys-SO2H) during catalysis, and sulfiredoxin (Srx) has been identified as responsible for reversal of the resulting enzyme inactivation in yeast. We have now characterized mammalian orthologs of yeast Srx with an assay based on monitoring of the reduction of sulfinic Prx by immunoblot analysis with antibodies specific for the sulfinic state. Sulfinic reduction by mammalian Srx was found to be a slow process (kcat = 0.18/min) that requires ATP hydrolysis. ATP could be efficiently replaced by GTP, dATP, or dGTP but not by CTP, UTP, dCTP, or dTTP. Both glutathione and thioredoxin are potential physiological electron donors for the Srx reaction, given that their Km values (1.8 mM and 1.2 microM, respectively) are in the range of their intracellular concentrations, and the Vmax values obtained with the two reductants were similar. Although its pKa is relatively low (approximately 7.3), the active site cysteine of Srx remained reduced even when the active site cysteine of most Prx molecules became oxidized. Finally, depletion of human Srx by RNA interference suggested that Srx is largely responsible for reduction of the Cys-SO2H of Prx in A549 human cells.     

Bacterial overgrowth affects urinary proteome analysis: recommendation for centrifugation, temperature, duration, and the use of preservatives during sample collection

Bacterial overgrowth is one of the major concerns in collection and storage of biofluids, particularly 24-h urine. However, there is no previous systematic analysis of effects of bacterial overgrowth on urinary proteome analysis, and necessity, type, and appropriate concentration of preservatives to prevent bacterial overgrowth in the urine remain unclear. We, therefore, performed such systematic evaluation. Pooled normal urine was either centrifuged at 1500 g (to remove cell debris) or uncentrifuged. The samples were then added with either sodium azide (NaN3) or boric acid with various concentrations, and kept at room temperature (RT) or at 4 degrees C. Bacterial overgrowth was determined by UV-visible spectrophotometry (lambda620 nm) and Gram staining. At both temperatures, centrifugation to remove cell debris could effectively delay the bacterial overgrowth. At RT, both centrifuged and uncentrifuged samples without any preservative had the detectable overgrowth of Gram-positive and Gram-negative cocci and bacilli as early as 12 and 8 h, respectively, whereas 0.1-1 mM NaN3 and 2-20 mM boric acid could delay bacterial overgrowth, which started at 16-20 h in the centrifuged urine and 12-16 h in the uncentrifuged urine. Greater delay (for at least 48 h) was achieved with 10 mM NaN3 and 200 mM boric acid. At 4 degrees C, no bacterial overgrowth was detected in all centrifuged samples. However, it was observed at 20 h in the uncentrifuged urine without preservative, and at 48 h for the uncentrifuged urine with 0.1 mM NaN3 or 2 mM boric acid. There was no bacterial overgrowth detectable in the uncentrifuged urine preserved with higher concentrations of NaN3 or boric acid. 2-DE showed obvious changes in the urinary proteome profile of the sample with bacterial contamination, and the bacterial proteins could be identified by MALDI-TOF MS. Our data suggest that the urine should be centrifuged to remove cell debris and kept at 4 degrees C, rather than at RT, during the collection interval prior to long-term storage in the freezer. Moreover, the addition of 200 mM boric acid or 10 mM NaN3 is highly recommended for the prevention of bacterial overgrowth in the urine.