Reactions of tetranitromethane. Mechanism of the reaction of tetranitromethane with pseudo acids.
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Acetone↗
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Modification of bovine heart mitochondrial transhydrogenase with tetranitromethane.
Modification of pyridine dinucleotide transhydrogenase with tetranitromethane resulted in inhibition of its activity. Development of a membrane potential in submitochondrial particles during the reduction of 3-acetylpyridine adenine dinucleotide (AcPyAD+) by NADPH decreased to nearly the same extent as the transhydrogenase rate on tetranitromethane treatment of the membrane. Kinetics of the inactivation of homogeneous transhydrogenase and the enzyme reconstituted into phosphatidylcholine liposomes indicate that a single essential residue was modified per active monomer. NADP+, NADPH and NADH gave substantial protection against tetranitromethane inactivation of both the nonenergy-linked and energy-linked transhydrogenase reactions of submitochondrial particles and the NADPH leads to AcPyAD+ reaction of reconstituted enzyme. NAD+ had no effect on inactivation. Tetranitromethane modification of reconstituted transhydrogenase resulted in a decrease in the rate of coupled H+ translocation that was comparable to the decrease in the rate of NADPH leads to AcPyAD+ transhydrogenation. It is concluded that tetranitromethane modification controls the H+ translocation process solely through its effect on catalytic activity, rather than through alteration of a separate H+-binding domain. Nitrotyrosine was not found in tetranitromethane-treated transhydrogenase. Both 5,5'-dithiobis(2-nitrobenzoate)-accessible and buried sulfhydryl groups were modified with tetranitromethane. NADH and NADPH prevented sulfhydryl reactivity toward tetranitromethane. These data indicate that the inhibition seen with tetranitromethane results from the modification of a cysteine residue.
Animals↗
Identification of an essential tyrosine residue in nitroalkane oxidase by modification with tetranitromethane.
The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with production of nitrite and hydrogen peroxide. The enzyme is irreversibly inactivated by incubation with tetranitromethane, a tyrosine-directed reagent, at pH 7.3. The inactivation is time-dependent and shows first-order kinetics for two half-lives of inactivation. Further inactivation can be achieved upon a second addition of tetranitromethane. A saturation kinetic pattern is observed when the rate of inactivation is determined versus the concentration of tetranitromethane, indicating that a reversible enzyme-inhibitor complex is formed before irreversible inactivation occurs. Values of 0.096 +/- 0.013 min(-1) and 12.9 +/- 3.8 mM were determined for the first-order rate constant for inactivation and the dissociation constant for the reversibly formed complex, respectively. The competitive inhibitor valerate protects the enzyme from inactivation by tetranitromethane, suggesting an active-site-directed inactivation. The UV-visible absorbance spectrum of the inactivated enzyme is perturbed with respect to that of the native enzyme, suggesting that treatment with tetranitromethane resulted in nitration of the enzyme. Comparison of tryptic maps of nitroalkane oxidase treated with tetranitromethane in the presence and absence of valerate shows a single peptide differentially labeled in the inactivated enzyme. The spectral properties of the modified peptide are consistent with nitration of a tyrosine residue. The amino acid sequence of the nitrated peptide is L-L-N-E-V-M-C-(NO(2)-Y)-P-L-F-D-G-G-N-I-G-L-R. The possible role of this tyrosine in substrate binding is discussed.
Amino Acid Sequence↗
Reaction of tetranitromethane with lutropin, oxytocin, and vasopressin.
Tetranitromethane reaction with intact ovine lutropin and its isolated subunits was studied using spectrophotometric measurements, amino acid analysis, and isolation of tyrosyl peptides. Tyrosyl residues in the beta subunit (beta37, beta59) did not react with tetranitromethane in the intact hormone, but were nitrated in the isolated subunit. The sequence and extent of reaction of tetranitromethane with the tyrosyl residues in the alpha subunit was alpha21 = alpha92 = alpha93 (in intact hormone or isolated subunit) greater than alpha 41 (reacted in isolated subunit only) greater than alpha 30 (reacted in isolated subunit in 8 M urea only). Polymerization was observed as a side reaction in agreement with previous studies. The degree of polymerization appeared to be related to both primary sequence and tertiary structure, and for lutropin had the relation: alpha subunit (93% polymerized) greater than intact hormone greater than beta subunit (less than 40%). Polymerization observed with vasopressin was significantly greater than with oxytocin; for these peptides the tyrosine residues in the monomeric product were converted to 3-nitrotyrosine. Neither 3-nitrotyrosine nor tyrosine was detected in the polymerized by-products. In the tetranitromethane reaction with intact ovine lutropin, other reaction products charcterized by absorption spectra were found. Peptides isolated from these products lacked the characteristic 428 nm abosrption maxima of 3-nitrotyrosyl peptides and showed instead absorption in the 310 to 350 nm region. Similar products from tetranitromethane reactions with di- and tripeptides containing tyrosine have been observed previously (Boyd, N.D., and Smith, D.B. (1971) Can. J. Biochem, 49, 154-161), but they have not been studied in proteins. A possible relationship to the polymerization side reaction is suggested.
Amino Acid Sequence↗
The reaction of bovine alpha-thrombin with tetranitromethane. Characterization of the modified protein.
Previous studies from several laboratories have shown that thrombin is inactivated by tetranitromethane with the formation of nitrotyrosine. The inactivation is characterized by an apparently greater loss of fibrinogen-clotting activity than activity toward synthetic ester substrates, suggesting that the residues modified by tetranitromethane are involved in the interaction of thrombin with fibrinogen. This study was designed 1) to determine the effect of solvent conditions on the rate of modification and the stoichiometry of the reaction of tetranitromethane with bovine alpha-thrombin; 2) to identify the residue(s) modified; and 3) to characterize the modified enzyme with respect to its interaction with peptide nitroanilide substrates and fibrinogen. The inactivation of thrombin by tetranitromethane proceeded more rapidly in 50 mM Tris, pH 8.0, than in 50 mM sodium phosphate, 100 mM NaCl, pH 8.0. Approximately 10% fibrinogen-clotting activity remained at maximal inactivation. A study of the effect of tetranitromethane concentration on the rate of inactivation suggested that the loss of activity was the result of the modification of 1 mol of tyrosine/mol of thrombin. A similar result was obtained from the analysis of the extent of inactivation as a function of the extent of protein modification. Structural analysis of the modified protein showed substantial modification at both Tyr71 and Tyr85. Enzyme kinetic studies were performed with the modified protein and a control thrombin with N2-tosylglycylprolylarginine p-nitroanilide. H-D-phenylalanylpipecolylarginine p-nitronailide, and purified bovine fibrinogen. With all three substrates, a substantial decrease in kcat was observed, whereas there was essentially no change in Km. These results suggest that, contrary to previous suggestions, the modification of Tyr71 and Tyr85 in thrombin does not influence the binding of substrates, but rather influences active site reactivity.
Animals↗
Tetranitromethane modification of the tyrosine residues of the lactose repressor.
Repressor protein modified with N-ethylmaleimide has been used to determine the exclusive effects of tyrosine nitration by tetranitromethane. Since modification of proteins with tetranitromethane generally results in both cysteine oxidation and tyrosine nitration, N-ethylmaleimide has been used to protect the cysteines in the repressor against oxidation in subsequent tetranitromethane reactions. Nitration of tyrosine residues in repressor previously reacted with N-ethylmaleimide results in loss of both specific and nonspecific DNA-binding activities. Na2S2O4 reduction of tetranitromethane-modified protein restores partial operator DNA-binding and complete nonspecific DNA-binding capability. Residues primarily affected are tyrosines 7 and 17, which are both in the NH2 terminus. Inter- and intramolecular cross-links which are observed in the modified protein can be minimized by altering reaction conditions; the cross-links present occur between sites located in the NH2 termini. Modification of the core protein also results in loss of the operator DNA-binding capacity, and subsequent reduction restores partial operator-binding activity. Both operator and nonspecific DNA-binding capabilities of the repressor protein are protected by the presence of nonspecific DNA during the tetranitromethane modification, and simultaneously the extent of nitration is decreased.
Binding Sites↗
Tetranitromethane as a surface antiviral disinfectant.
Tetranitromethane, a protein nitrating agent, was tested for its ability to disinfect surfaces from viruses. Different surfaces on commercially available pocket calculators were pretreated with either the Indiana strain of vesicular stomatitis virus or the Herts' strain of Newcastle disease virus. The calculators surfaces were then sprayed with either tetranitromethane or control solutions. The calculators were incubated for 30 min at ambient temperature, and then the surfaces were wiped with sterile swabs. The swabs were placed into test tubes containing phosphate-buffered saline. Samples of the phosphate-buffered saline were then titered on appropriate cell lines by plaque assay. The results indicated that the amount of vesicular stomatitis virus and Newcastle disease virus recovered from the tetranitromethane-treated surfaces was dramatically decreased compared to the amount of virus recovered from control-treated surfaces. These data suggest that tetranitromethane may be useful to disinfect surfaces from both enveloped and non-enveloped RNA viruses.
Antiviral Agents↗
The cross-linking of tyrosine by treatment with tetranitromethane.
1. Tyrosine was treated with tetranitromethane. 2. Approx. 10% of the tyrosine was converted into 3-nitrotyrosine. 3. Three fluorescent compounds were also formed. They appear to be a dimer, trimer and tetramer in which tyrosine units are linked by biphenyl bonds. 4. The dimer and trimer have also been isolated from some proteins after treatment with tetranitromethane. 5. The yield of 3-nitrotyrosine from ovotransferrin after treatment with tetranitromethane was much smaller than the loss of tyrosine. 6. Several unidentified compounds were also formed by the reaction between tyrosine and tetranitromethane.
Electrophoresis↗
Effect of tetranitromethane on the biological activities of botulinum neurotoxin types A, B and E.
Botulinum neurotoxin serotypes A, B and E were modified at pH 7.9 with tetranitromethane, a reagent highly specific for tyrosine residues. The type B and E neurotoxins were completely detoxified without significant damage to their serological activities. Under similar modification conditions, the type A neurotoxin was incompletely detoxified with some alteration in its serological reactivity. Modification of only tyrosine residues to nitrotyrosine was evident from amino acid analysis of the acid hydrolysates of the modified proteins. The completely detoxified type B and E neurotoxins, used as toxoid, elicited antibodies in rabbits. The antisera precipitated and neutralized the homologous neurotoxin. The two toxoids, type B and E, were prepared with greater than 99% pure neurotoxins as tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis whereas the traditional toxoids produced with formaldehyde are very crude preparations of the neurotoxin (approximately 90% impure). Chemical modification using tetranitromethane is more specific than products that form during approximately 7 days of reaction between a protein and formaldehyde. The toxoids produced with tetranitromethane may be considered second-generation toxoids, compared with the first-generation toxoids (crude preparation of neurotoxins detoxified with formaldehyde).
Amino Acids↗
Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives.
The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49-64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118-123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the pKa of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (pKa = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine pKa shift in the colipase micelle complex may explain why nitrated colipase can reactivate lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5.
Animals↗
The modification with tetranitromethane of an essential tyrosine in the active site of pig fumarase.
Modification of pig heart fumarase (L-malate hydro-lyase, EC 4.2.1.2) with tetranitromethane results in loss of enzymatic activity. The inactivation is slowed down in the presence of substrates, indicating that the modification reaction takes place at the level of the substrate binding sites. From these inactivation kinetics, a value Kd = 78 microM is calculated for the mixture of substrates (L-malate + fumarate). This is in fairly good agreement with the Michaelis constant Km = 31 microM. Spectrophotometric data indicate that modification of one tyrosine residue per fumarase subunit is responsible for the inactivation; one or more additional residues, which do not participate in the binding sites, are modified at much lower rates. Amino acid analyses confirm the presence of nitrotyrosine and exclude the possibility of tetranitromethane-mediated polymerization side-reactions. It is concluded from the pH-dependence of the nitration reaction that the inactivation of fumarase is not caused by cysteine modification. Additional studies of nitration of melittin, a tryptophan-containing model peptide, are described. From the absorption spectra of modified melittin, in comparison with the spectra of nitrofumarase, it is concluded that the tryptophan residues of the latter enzyme remain intact during the reaction with tetranitromethane. Finally, evidence is given for an independent action of the four fumarase subunits, i.e., inactivation of one subunit does not influence the catalysis by the other three subunits. Moreover, it is shown that only fumarase tetramers with all four subunits nitrated are unable to bind to a Sepharose-pyromellitic acid affinity column.
Amino Acids↗
Chemical modification of adrenocortical cytochrome P-450scc with tetranitromethane.
Selective chemical modification of adrenocortical cytochrome P-450scc, responsible for key stages of steroid biogenesis, with tetranitromethane has been carried out. Nitration of the cytochrome P-450scc tyrosine residues results in heme protein inactivation with syncatalytic loss of enzyme activity. Analysis of the cytochrome P-450scc inactivation kinetics indicates that there are several pools of tyrosine residues, differing in their accessibility to tetranitromethane. The modification of cytochrome P-450scc results in changes in the hemeprotein spectral properties and its conformation which indicates to the involvement of essential tyrosine residue(s) in the heme-protein interaction. Cholesterol and adrenodoxin (high-spin effectors) prevent the inactivation of cytochrome P-450scc with tetranitromethane, i.e., protect the essential tyrosine residue(s) from modification. Possible functions of the tyrosine residues in the cytochrome P-450scc molecule are discussed.
Adrenal Cortex↗
Inhalation of tetranitromethane causes nasal passage irritation and pulmonary carcinogenesis in rodents.
Fischer 344 rats and B6C3F1 mice were exposed for 2 years to vapors of tetranitromethane at concentrations below (0.5 ppm) and slightly above (2 or 5 ppm) the current U.S. recommended occupational exposure limit. Under the conditions of exposure of 6 h/day, 5 days/week, tetranitromethane was found to cause mild irritation and hyperplastic lesions in the nasal passages, but not nasal cavity neoplasms were observed. In contrast, nearly all animals exposed to the higher TNM concentrations, and the majority of animals exposed to the lower concentrations developed alveolar/bronchiolar adenoma or carcinoma; squamous cell neoplasms of the lung also occurred in exposed rats. The extent of the lung tumor response, and the low concentrations of tetranitromethane required for this response, are unprecedented in National Toxicology Program (NTP) studies.
Adenocarcinoma, Bronchiolo-Alveolar↗
Chemical modification of the brown-fat-mitochondrial uncoupling protein with tetranitromethane and N-ethylmaleimide. A cysteine residue is implicated in the nucleotide regulation of anion permeability.
Treatment of brown adipose tissue mitochondria with tetranitromethane or N-ethylmaleimide decreases the affinity with which inhibitory nucleotide GDP binds to the tissue-specific uncoupling protein. Both reagents modify cysteine residues which are 'accessible' and 'buried' to 5,5'-dithio-bis(2-nitrobenzoic acid) (Nbs2). Modification of the single Nbs2-accessible residue correlates with the loss of high-affinity binding sites for GDP. Tetranitromethane does not affect the Cl- or H+ permeability of the protein in the absence of nucleotide, while N-ethylmaleimide increases both by 70-80%. Bound GDP is a less effective inhibitor of Cl- permeability after N-ethylmaleimide or tetranitromethane treatment, but retains much of the ability to inhibit H+ permeation.
Adipose Tissue, Brown↗
[Modification of micrococcal histidine decarboxylase with tetranitromethane].
Tetranitromethane inhibited distinctly the histidine decarboxylase activity at pH above 7.0. Spectral and fluorescence properties as well as amino acid composition of the transformed enzyme were studied. Nitration of tyrosine residues occurred simultaneously with oxidation of cysteine in a molecule of histidine decarboxylase treated with tetranitromethane, while the other amino acids such as tryptophane, histidine and methionine were not altered. The histidine decarboxylase pretreated with dithiothreitol, became insensitive to the tetranitromethane effect but complete inactivation of the enzyme was observed under these conditions. Possible mechanisms of the histidine decarboxylase inactivation are discussed.
Amino Acids↗
Chemical modification of tyrosine residues in active-site of human placental estradiol 17 beta-dehydrogenase by tetranitromethane.
The native estradiol 17 beta-dehydrogenase purified from human placenta was rapidly inactivated by tetranitromethane, which is a reagent for chemical modification of tyrosine and cysteine residues. The inactivation followed pseudo-first-order reaction kinetics, and the activity was partially recovered by addition of dithiothreitol. On the other hand, the enzyme sulfhydryl-blocked by 5,5'-dithiobis(2-nitrobenzoic acid) was treated with tetranitromethane, and the activity was assayed in the presence of dithiothreitol. Tetranitromethane inactivated the enzyme in a time-dependent manner, following pseudo-first-order kinetics. The rate of inactivation was significantly decreased by addition of NADP(H), 2'-AMP, 5'-ATP, 2',5'-ADP, 3-pyridine aldehyde-DPN and 3-acetylpyridine-DPN(reduced form). These results suggest that the tyrosyl residues are located at or near the cofactor-binding site of the estradiol 17 beta-dehydrogenase and play an essential role in the catalytic function of the enzyme.
17-Hydroxysteroid Dehydrogenases↗
Spectrophotometric pH titrations and nitration with tetranitromethane of the tyrosyl residues in yeast phosphoglycerate kinase.
Spectrophotometric pH titrations of phosphoglycerate kinase (EC 2.7.2.3) reveal seven tyrosyl residues. In the native state one tyrosyl residue has pKapp equal to 9.3, another has pKapp of about 12.9, and five have pKapp values close to 11.0. Titration above pH 10 causes concomitant reduction of the catalytic activity. Reactivation of the enzyme occurs during storage at pH 7.8. In 6 M guanidine - HCl seven tyrosyl residues with pKapp values equal to 10.0 appear. Nitration of three tyrosyl residues occurs easily when tetranitromethane is used in excess. Four tyrosyl residues appear to be masked or buried. The tyrosyl residue having pKapp equal to 9.3 can be selectively nitrated. Simultaneously the enzyme loses 40% of its catalytic activity. No change in the Km value for one or the other of the two substrates, MgATP or 3-phospho-D-glycerate, was observed in the mononitrated enzyme. On the other hand MgATP protects the tyrosyl residue from nitration whereas 3-phospho-D-glycerate at corresponding condition appears harmless. These results suggest the low ionizing tyrosyl residue to be situated close to the binding site of MgATP, possibly in a pocket just behind. Circular dichroism measurements indicated that minor successive changes occur in the secondary structure, mainly the beta-structure, when the enzyme is being nitrated. It is reasonable to think that these structural changes, possible in combination with steric hindrance, are responsible for the decrease in catalytic activity. Dimerization of the enzyme occurs if the single thiol group is not masked before the tetranitromethane treatment.
Adenosine Triphosphate↗
The inactivation of Clostridium perfringens epsilon toxin by treatment with tetranitromethane and N-acetylimidazole.
When one residue of tyrosine per molecule of epsilon toxin was nitrated by tetranitromethane, the modified toxin lost more than 90% of its original activity. The toxin also was inactivated by treatment with N-acetylimidazole. The lethality was restored when the inactive N-acetylimidazole-treated toxin was treated with hydroxylamine. The inactive N-acetylimidazole-treated toxin was not nitrated by tetranitromethane under conditions where one residue of Tyr per molecule of the untreated toxin is nitrated. These data suggest that only one Tyr residue in the toxin is important for the lethal activity.
Acetylation↗