Tryptophane nitration

It was recently reported that the tryptophan residues of proteins could be nitrated by the action of peroxynitrite (67). This reactive nitrogen species (RNS) is generated from the reaction of nitric oxide with superoxide at a rate that is ten times greater than the destruction of superoxide by dismutases. The authors propose that the nitration of tryptophan, although less common than tyrosine nitration, could serve to modulate the function of some proteins. However, at this time the in vivo evidence for tryptophan nitration by RNS has yet to be reported. 

Barry SM, Kers JA, Johnson EG, Song L, Aston PR, Patel B, Krasnoff SB, Crane BR, Gibson DM, Loria R et al (2012) Cytoehrome P450-eatalyzed /.-tryptophan nitration in tfaaxtomin phytotoxin biosynthesis. Nat Chem Biol 8 814-816... 

What kind of business This depends largely on what kind of dope you plan to manufacture. Amine-type precursors go hand in hand with perfumery or dyes. Nitrating-type compounds (nitromethane, nitroethane) are also used as solvents, and rocket fuels. Indole has been used in perfumery. Tyrosine and tryptophan are nutrients added to feeds. Urea is a plant fertilizer and a feed additive. 

Triazoles have been used extensively in analytical chemistry. Nitron (182) has been used for the determination of boron, rhenium, and tungsten. Nitron has been bonded to a polymer and used for the removal of nitrate from water <79Mi 402-oi >. Other triazoles have been used for the spectrophoto-metric assay of cobalt, rhodium, and platinum <68Mi 402-01,72ZAK2209). Hydroxyphenylazotriazoles form brilliant lakes with a number of metal ions <73AJC1585>. 5-" C-3-Diazotriazole couples only to tryptophane below pH 6.8 and can used for the specific determination of tryptophane in proteins <84M1 402-02). 

Figure 6.8 Experimental variation of the retention of 23 phenylthiohydantoin (PTH) derivatives of amino acids with mobile phase composition in RPLC. Mobile phase mixtures of acetonitrile and 0.05M aqueous sodium nitrate buffer (pH — 5.81). All mobile phases contain 3% THF. Stationary phase ODS silica. Solutes D = aspartic acid C-OH = cysteic acid E = glutamic acid N = asparagine S = serine T = threonine G = glycine H = histidine Q = glutamine R = arginine A = alanine METS = methionine sulphone ABA = a-aminobutyric acid Y = tyrosine P = proline V = valine M = methionine NV = norvaline I = isoleucine F = phenylalanine L = leucine W = tryptophan K = lysine. Figure taken from ref. [610]. Reprinted with permission.

Tyrosine is more fluorescent than tryptophan in solution, but when present in proteins, its fluorescence is weaker. This can be explained by the fact that the protein tertiary structure inhibits tryosine fluorescence. Also, energy transfer from tyrosines to tryptophan residues occurs in proteins inducing a total or important quenching of tyrosine fluorescence. This tyrosine — tryptophan energy transfer can be evidenced by nitration of tyrosine residues with tetranitromethane (TNM), a highly potent pulmonary carcinogen. Because TNM specifically nitrates tyrosine residues on proteins, the effects of TNM on the phosphorylation and dephosphorylation of tyrosine, and the subsequent effects on cell proliferation, can be investigated. 

CGTases (EC 2.4.1.19) are bacterial enzymes that facilitate the biosynthesis of cyclodextrins from starch through intramolecular transglucosylation. The primary structures of most of these enzymes have been published, and the three-dimensional structure of Bacillus circulans CGTase has been established. Studies of transglucosylation molecular mechanism have indicated that amino acids such as histidine and tryptophan are implicated in such mechanisms. Nitration of CGTase with TNM induces a loss of enzyme activity, a decrease in enzyme affinity towards the (i-CD copolymer, and a loss of tryptophan fluorescence (Villette etal. 1993). 

The fluorescence intensity of CGTase tryptophan residues decreases (Figure 7.13) after treatment of the enzyme with increasing concentrations of TNM and purification of the modified enzyme on co-polymer. Tryptophan fluorescence intensity of the 8 mM-TNM-modified CGTase (0.03 /uM 1 tryptophan) is similar to that of free L-tryptophan (0.034 /xM 1). The loss of tryptophan fluorescence observed during nitration may then be related to elimination of this energy transfer. 

CGTase nitration induces an 11.5 nm shift in the fluorescence maximum to shorter wavelengths (A.max = 326.5 nm instead of 338 nm for 8 mM TNM), suggesting a relative increase in the buried tryptophan fluorescence. Tyr -> Trp energy transfer may then involve solvent-exposed residue(s). 

Figure 7.13 Tryptophan fluorescence spectra of CGTase after 18 h of nitration with 0, 0.25, 0.5, 1, 2, 4, and 8 mM TNM. Xex = 295 nm. Source Villette J.R., Helbecque, N., Albani, J.R., Sicard, RJ. and Bouquelet, S.J. (1993). Biotechnology and Applied Biochemistry, 17, 205-216. Reprinted with permission from Portland Press.

These results support a conformational impairment of the enzyme during nitration and would account for the loss of tryptophan fluorescence by the elimination of (Tyr -> Trp) energy transfer (removal of the chromophores) and hence enzyme inactivation. Table 7.2 lists the principal absorption and fluorescence characteristics of the three aromatic amino acids within the proteins and of the Y-base. 

Tetranitromethane (TNM) reacts with tyrosyl residues in proteins to give 3-nitrotyrosine derivatives. Nitrating tyrosyl residues in rat liver microsomal stearyl coenzyme A desaturase with TNM leads to inactivation with concomitant loss of the iron prosthetic group (29). TNM also is known to react with sulfhydryls in proteins and, under certain conditions, with tryptophan, histidine, and methionine (30). 

Fig. 6.9 Activation of nitrite by peroxydases (a) and (b) nitration of phenol derivatives (c) nitration of Y-acetyl-tryptophan amide (d) nitration of dopamine...

Sala A, Nicolis S, Roncone R, Monzani E, Casella L (2004) Peroxidase catalyzed nitration of tryptophan derivatives. Mechanism, products and comparison with chemical nitrating agents. Eur J Biochem 271 2841-2852... 

Besides amide formation and partial removal of acid-labile protecting groups, nitration of sensitive amino acid residues such as tyrosine and tryptophan has been reported.h Sulfoxide formation in 5-benzylcysteine and methionine residues was also detected.h l... 

The awareness of the many advantages of this modification reaction should be matched by the knowledge of the numerous side-reactions observed to result from the reaction of TNM with proteins. In addition to nitration of tyrosine, the following side-reactions have been reported in several (but not all) proteins studied (1) inter- and intramolecular cross-linking, (2) oxidation of sulfhydryl groups to a variety of products, (3) oxidation of methionine, (4) modification of tryptophan and histidine, (5) modification of prosthetic groups. It is apparent therefore that a successful application of TNM to the selective modification of tyrosine is achieved in those cases where an unusually rapid reaction... 

The visible absorption spectrum of a solution containing a known concentration of nitrated protein is measured in a solution buffered at pH 9.0, and the absorbance at the maximum (near 428 nm) used to calculate the nitrotyrosine content ( 428nm for the nitrophenoxide ion is 4200). The tyrosine and nitrotyrosine content of the modified protein should also be determined by amino acid analysis. If the sum of these values does not add up to the tyrosine content of the unmodified protein, intra- or intermolecular cross-linking may have occurred. The amino acid analysis may also reveal whether other side-reactions have taken place. Particular attention should be paid to the half-cystine, cysteine, methionine, histidine and tryptophan contents of the modified proteins. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate offers a rapid and highly sensitive way of detecting products of intermolecular cross-linking. Such products are readily removed by gel filtration. 

Proteins respond to the following color tests (a) biuret, pink to purple with an excess of alkali and a small amount of copper sulfate (b) ninhydrin. a blue color when boiled with ninhydrin (triketohydrindene hydrate), which is intensified by the presence of pyridine (c) Millon s test for tyrosine, a brick-F color or precipitate when boiled with mercuric nitrate in an excess of nitric acid (d) Hopkins-Cole test for tryptophan, a violet zone with a salt of glyoxylic acid and stratified over sulfuric acid and (e) xanthoproteic test, a brilliant orange zone when a solution in concentrated nitric acid is stratified under ammonia. Almost all so-called alka-loidal reagents will precipitate proteins in slightly acid solution. 

It was shown that tryptophan is also nitrated by peroxynitrite in the absence of transition metals to one predominant isomer of nitrotryptophan, as determined from spectral characteristics and liquid chromatography-mass spectrometry analysis. Typical hydroxyl radical scavengers partially inhibited the nitration" . The yields of the nitration of tyrosine and salicylate by peroxynitrite are significantly improved by the Fe(III)-EDTA complex " ". ... 

A recent study has described the inhibition of tyrosine nitration but not oxidation by tryptophan and tryptamine, indicating that nitration of the indole ring proceeds more favourably than nitration of the phenoxyl ring [118]. Methionine oxidation can be elicited by peroxynitrite and proceeds via two-step single electron reduction reactions yielding methionine sulphoxide as described previously [119]. 

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