Oxidation Of Tryptophan Residues Research Articles

P10. Detection and characterization of nitration and oxidation of tryptophan residues in proteins using mass spectrometry

P10. Detection and characterization of nitration and oxidation of tryptophan residues in proteins using mass spectrometry

Post-Translational Modifications in the Nuclear Region of Young, Aged, and Cataract Human Lenses

The urea-soluble proteins from the nucleus of two young, two aged, and two early-stage nuclear cataract lenses were subjected to tryptic digestion and analysis by 2D LC-MS/MS. Several novel post-translational modifications were identified. Deamidation was, by far, the most common modification. A number of differences were found in cataract compared to normal lenses, most notably an increase in the number of oxidized tryptophan residues. Semiquantitative analysis revealed that there appeared to be a trend toward increased levels of deamidation with age; however, there was no apparent increase upon the onset of nuclear cataract. This is in contrast to Trp oxidation, where an increase in the extent of modification was apparent in cataract lenses when compared to aged normal lenses. These findings suggest Trp oxidation may be involved in nuclear cataract development.

Basic research confirms coexistence of acquired Blaschkolinear Vitiligo and acrofacial Vitiligo

We report about a female patient with bilateral and unilateral blaschkolinear depigmentation on the extremities and coexistence of acrofacial vitiligo, who initially presented her first signs of depigmentation at the age of 32 years. The patient was otherwise healthy. The correct diagnosis was based on the latest up to date technology utilizing in vivo FT-Raman and Fluorescence spectroscopy, Wood's light examination of the depigmented skin and immunoreactivity of epidermal catalase expression in 3 mm punch biopsies from the linear depigmented area. The results yielded decreased catalase protein expression compared to healthy controls as well as complete absence of melanocytes. FT-Raman spectroscopy identified the presence of hydrogen peroxide (H(2)O(2)) in the mM range and Fluorescence spectroscopy demonstrated H(2)O(2)-mediated oxidation of tryptophan residues in the depigmented area. The results were in agreement with vitiligo. Repigmentation of the linear lesion was initiated after reduction/removal of epidermal H(2)O(2) with pseudocatalase PC-KUS further supporting the correct diagnosis. To the best of our knowledge this is the first case documented with vitiligo following Blaschko lines in coexistence with classical acrofacial vitiligo. This observation raises the question whether besides H(2)O(2)-mediated stress in association with genomic mosaicism could play a role in some cases with vitiligo.

Functioning Methionine-S-Sulfoxide Reductases A and B Are Present in Human Skin

Methionine residues in the structure of proteins and peptides are especially sensitive to oxidation by hydrogen peroxide (H(2)O(2)) yielding both the (R) and (S) diastereomers of methionine sulfoxide. This commentary shows that both diastereomers of methionine sulfoxide (R and S) can be repaired in the human epidermis by methionine sulfoxide reductases A and B, respectively.

Reversibility of Electron Transfer in Tryptophan−Tyrosine Peptide in Acidic Aqueous Solution Studied by Time-Resolved CIDNP

Time-resolved chemically induced dynamic nuclear polarization (CIDNP) has been used to study electron transfer reactions in tryptophan-tyrosine peptide under strongly acidic conditions. It is demonstrated that a decrease in pH from 2.4 to 1.6 reduces the overall efficiency of intramolecular electron transfer from the tyrosine residue to the oxidized tryptophan residue. A detailed analysis of the CIDNP kinetics revealed that the rate constant of this reaction k(f) stays unchanged upon pH variation, whereas the rate constant of electron transfer in the opposite direction k(r) increases with decreasing pH. The values of the rate constants extracted from model simulations are as follows: k(f) = (5.5 +/- 0.5) x 10(5) s(-1); k(r) = (5.5 +/- 1.0) x 10(4) s(-1) at pH 2.4, (1.2 +/- 0.2) x 10(5) s(-1) at pH 2.0, and (3.2 +/- 0.4) x 10(5) s(-1) at pH 1.6. The pH dependence of log K = log(k(f)/k(r)) is linear and allows for the determination of the difference between the one-electron reduction potentials of the tryptophanyl and tyrosyl radicals in the peptide. The efficiency of IET in acidic aqueous solution containing 10 M urea-d(4) was estimated.

A concise formal total synthesis of TMC-95A/B proteasome inhibitors.

[reaction: see text] A formal total synthesis of proteasome inhibitors TMC-95A/B is described. The synthesis features a stereoselective modified Julia olefination and a diastereoselective dihydroxylation to construct the highly oxidized tryptophan residue.

Oxidative modification of proteins: oxidation of tryptophan and production of dityrosine in purified proteins using Fenton's system.

Specific features of metal-catalyzed oxidation (MCO) of purified proteins (human serum albumin and human erythrocyte superoxide dismutase) were analyzed by the oxidation level of tryptophan and tyrosine. The production of dityrosine cross-links and the oxidation of tryptophan residues were recorded by fluorescence. The degree of oxidative modification of the amino acid residues of the proteins depended on the concentration of the Fenton's medium components and on the incubation time. These changes were different in different proteins. By electrophoresis and gel-permeation chromatography, changes in the superoxide dismutase structure are shown to be caused by oxidative modification of the enzyme and to be accompanied by a decrease in its activity. Findings with OH* scavengers (mannitol and ethanol) suggest that oxidative modification of the proteins in Fenton's medium should be associated not only with hydroxyl radical but also with ferryl and perferryl ions and with the radical CO(-.)(3).

Hypochlorous acid damages erythrocyte membrane proteins and alters lipid bilayer structure and fluidity

Treatment of human erythrocyte membranes with active forms of chlorine (hypochlorous acid and chloramine T) resulted in a concentration-dependent inhibition of the membrane Na +, K +- and Mg 2+-ATPases. Membrane protein thiol group oxidation was consistent with inactivation of enzymes and preceded oxidation of tryptophan residues and chloramine formation. Erythrocyte exposure to hypochlorous acid led to complex changes of cell membrane rigidity and cell morphological transformations: cell swelling, echinocyte formation, and haemolysis. The inhibition of ion pump ATPases of human erythrocyte membranes may be due to direct oxidation of essential residues of enzyme (thiol groups) and structural rearrangement of the membrane.

Gastroenteropancreatic Hormones (Insulin, Glucagon, Somatostatin, and Multiple Forms of PYY) from the Pallid Sturgeon, Scaphirhynchus albus (Acipenseriformes)

Insulin, glucagon, somatostatin-14, and three structurally related molecular forms of peptide tyrosine–tyrosine (PYY) were isolated from an extract of the combined pancreas and gastrointestinal tract of the pallid sturgeon, Scaphirhynchus albus. Pallid sturgeon insulin was identical to insulin from the Russian sturgeon, Acipenser guldenstaedti, and to insulin-2 from the paddlefish, Polyodon spathula, and was approximately twofold less potent than human insulin in inhibiting the binding of [3-[125I] iodotyrosine-A14] human insulin to the soluble human insulin receptor. The sturgeon glucagon (HSQGMFTNDY10-SKYLEEKLAQ20 EFVEWLKNGK30S), like the two paddlefish glucagons, contains 31 rather than 29 amino acid residues, indicative of an anomalous pathway of posttranslational processing of proglucagon. Pallid sturgeon somatostatin, identical to human somatostatin-14, was also isolated in a second molecular form containing an oxidized tryptophan residue, but [Pro2]somatostatin-14, previously isolated from the pituitary of A. guldenstaedti, was not identified. Sturgeon PYY (FPPKPEHPGD10DAPAEDVAKY20YTALRHYINL30 ITRQRY.HN2) was also isolated in variant forms containing the substitutions (Phe1 → Ala) and (Ala18 → Val), indicative of at least two gene duplications occurring within the Acipenseriformes lineage. The amino acid sequences of the pallidsturgeon PYY peptides are appreciably different from the proposed “ancestral” PYY sequence that has otherwise been very strongly conserved among the actinopterygian and elasmobranch fish.

The role of tryptophan residues in the hemolytic activity of stonustoxin,a lethal factor from stonefish ( Synanceja horrida) venom

Stonustoxin (SNTX) is a pore-forming cytolytic lethal factor, isolated from the venom of the stonefish Synanceja horrida, that has potent hemolytic activity. The role of tryptophan residues in the hemolytic activity of SNTX was investigated. Oxidation of tryptophan residues of SNTX with N-bromosuccinimide (NBS) resulted in loss of hemolytic activity. Binding of 8-anilino-1-naphthalenesulphonate (ANS) to SNTX resulted in occlusion of tryptophan residues that resulted in loss of hemolytic activity. Circular dichroism and fluorescence studies indicated that ANS binding resulted in a conformational change of SNTX, in particular, a relocation of surface tryptophan residues to the hydrophobic interior. NBS-modification resulted in oxidised surface tryptophan residues that did not relocate to the hydrophobic interior. These results suggest that native surface tryptophan residues play a pivotal role in the hemolytic activity of STNX, possibly by being an essential component of a hydrophobic surface necessary for pore-formation. This study is the first report on the essentiality of tryptophan residues in the activity of a lytic and lethal factor from a fish venom.

Delineation of key amino acid side chains and peptide domains for antimicrobial properties of divercin V41, a pediocin-like bacteriocin secreted by Carnobacterium divergens V41.

Divercin V41 (DV41) is a class IIa bacteriocin produced by Carnobacterium divergens V41. This antilisterial peptide is homologous to pediocin PA-1 and contains two disulfide bonds. To establish the structure-activity relationships of this specific family of bacteriocin, chemical modifications and enzymatic hydrolysis were performed on DV41. Alteration of the net charge of this cationic bacteriocin by succinylation and acetylation revealed that, in a certain range, the electrostatic interactions were surprisingly not necessary for the activity of DV41. Cleavage of DV41 by endoproteinase Asp-N released two fragments N1[1-17] and N2[18-43] corresponding to the conserved hydrophilic N-terminal and the variable hydrophobic C-terminal sequences, respectively. Inhibitory assays showed that only the C-terminal fragment was active, and after trypsin cleavage at Lys42 or disulfide reduction it lost its inhibitory activity. These results suggested that both hydrophobicity and folding imposed by the Cys25-Cys43 disulfide bond were essential for antilisterial activity of the C-terminal hydrophobic peptide. Chemical oxidation of tryptophan residues by N-bromosuccinimide demonstrated that these residues were crucial for inhibitory activity since modification of any one of them rendered DV41 inactive. On the contrary, only the modification of all the three tyrosine residues caused a total loss of antilisterial activity. These latter results strengthened previous results suggesting that the N-terminal domain containing the YGNGV consensus sequence was not involved in the binding of DV41 to a potential specific receptor on listerial cells.

Protective effects of β-blockers against 2,2′-azobis(2-amidinopropane)-dihydrochlorideinduced damage

The protective effects of β-blockers against 2,2′-azobis(2-amidinopropane)-dihydrochloride (AAPH)-induced damage were investigated. With the exception of pindolol, none of the β-blockers tested inhibited arachidonate peroxidation induced by AAPH in the absence of iron. In contrast, ADP-Fe 3+- and NADPH-dependent microsomal lipid peroxidation was inhibited by all the β-blockers tested, although the inhibitory effects of atenolol and metoprolol were very slight. Oxidation of tryptophan residues in bovine serum albumin (BSA) induced by AAPH was strongly inhibited by pindolol and propranolol but not by atenolol or metoprolol. All the β-blockers tested, however, inhibited AAPH-induced carbonyl formation of BSA. Furthermore, all the β-blockers tested also strongly inhibited the deoxyribose degradation induced by AAPH, suggesting that these agents act as hydroxyl radical scavengers to inhibit carbonyl formation. DNA strand scission was induced by AAPH in the absence or presence of O 2. Only pindolol strongly inhibited the DNA damage in the absence of O 2. In the presence of O 2, however, all the β-blockers tested effectively prevented the DNA damage. These results suggested that the hydroxyl radicals produced from AAPH damaged DNA and, that β-blockers might act as hydroxyl radical scavengers to protect DNA against the AAPH-induced oxidative damage.

A kinetic model for the competitive reactions of ozone with amino acid residues in proteins in reverse micelles.

Lysozyme and 10 other proteins are solubilized in reverse micelles formed by 0.1 M sodium di-2-ethyl-hexylsulfosuccinate and 2.0-2.5 M water (pH 7.4) in isooctane solvent. Exposure of the protein-containing reverse micellar solutions to ozone causes oxidative damage to the proteins, as assessed by the oxidation of tryptophan residues. The oxidation product of the protein-bound tryptophan has a molar absorption coefficient of 3275 +/- 81 M-1 cm-1 (mean +/- S.D., n = 6) at 320 nm. The product is suggested to be a Criegee ozonide or a tautomer of the Criegee ozonide and not N-formylkynurenine. Ozonation of lysozyme in reverse micelles results in the formation of hydrogen peroxide in yields of only approximately 0.07 mol/mol of tryptophan residues oxidized. The recovery of hydrogen peroxide added as an internal standard to the lysozyme-containing reverse micellar solutions ranges from 84 to 88%, whether or not the samples are subjected to ozonation. This suggests that hydrogen peroxide is neither destroyed during the process of ozonation nor consumed by the protein to a significant extent in an adventitious reaction. A kinetic model for the overall reaction of ozone with the proteins is developed, taking into account the concentrations and the reactivities of individual amino acid residues toward ozone. The model predicts the fractional reaction of ozone with tryptophan residues in the proteins, despite differences in amino acid composition, molecular weight, and tertiary structures. The lack of influence of protein structure is confirmed further by the observation that the native lysozyme (with and without external S-carboxymethylcysteine) and S-carboxymethylated lysozyme give identical values of the fractional reaction of ozone with tryptophan residues. The kinetic equations for the competitive reactions of ozone with amino acid residues in proteins, with some minor modification, are applicable to ozonations on complex mixtures of lipids, proteins, and antioxidants.

Ozonation of lysozyme in the presence of oleate in reverse micelles of sodium DI-2-ethylhexylsulfosuccinate

Ozone is shown to react with lysozyme in reverse micelles formed by 0.1 M sodium di-2-ethylhexylsulfosuccinate and 1.2-3 M water (pH 7.4) in isooctane solvent. The reaction of ozone is assessed by the oxidation of tryptophan residues in the protein to N-formylkynurenine. Cosolubilization of oleate in lysozyme-containing reverse micellar solutions at concentrations of 0.5-10 mM results in a progressive inhibition (19% to 82%) of the oxidation of tryptophan residues with a concentration for 50% inhibition around 2 mM. At this concentration of oleate, the magnitude of inhibition is independent of the micelle size and concentration, the overall interfacial area of reverse micelles, and the amount of ozone employed. These findings are discussed in terms of competitive reactions of ozone with unsaturated fatty acids and proteins in the lung lining fluid and in biological membranes.

Identification of the tryptophan residue present in the low affinity saccharide-binding site of ricin E.

The tryptophan residue located at the low affinity saccharide-binding site (LA-site) of ricin E was identified using two derivatives of ricin E, in which tryptophan residues were oxidized with N-bromosuccinimide (NBS) in the absence and presence of lactose. From the lysyl endopeptidase and tryptic digests of the B-chains of the respective derivatives, peptides containing oxidized tryptophan residue(s) were isolated by gel filtration on Bio Gel P-2 and HPLC. Analysis of the peptides containing oxidized tryptophan showed that tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative, in which the saccharide-binding ability at the LA-site was abrogated by modification of tryptophan residues with NBS in the absence of lactose. On the other hand, two tryptophan residues at positions 93 and 160 in the B-chain were found to be oxidized in the active derivative that retained the saccharide-binding ability at the LA-site after NBS-oxidation in the presence of lactose,...

Proton NMR resonance assignments and surface accessibility of tryptophan residues of a dimeric phospholipase A 2 from Trimeresurus flavoviridis

Proton NMR spectra of a dimeric phospholipase A 2 from Trimeresurus flavoviridis have been recorded. N-1 proton resonances of the tryptophan indole rings have been detected and assigned to specific positions, Trp-3/Trp-30, Trp-68 and Trp-108, by comparing the spectra of the enzyme derivatives with tryptophans oxidized to differing extents. Photo-CIDNP experiments have revealed that Trp-68 and Trp-108 are exposed while Trp-3 and Trp-30 are buried in the molecule. This is consistent with the X-ray crystal structure of a homologous phospholipase A 2 from Crotalus atrox where residues 3 and 30 are located at a dimer interface, but inconsistent with the results of stepwise oxidation of tryptophan residues.

Tryptophan residue essential for activity of Naja naja atra phospholipase A2.

When Naja naja atra phospholipase A2, which contains three tryptophan residues at the 18th, 19th, and 61st positions, was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased in a convex manner with increase in the extent of oxidation of tryptophan residues. The curve shape showed that the tryptophan residue oxidized last is most responsible for the activity. The order of accessibilities of the three tryptophan residues, which was analyzed according to the method reported previously (Mohri et al. (1876) J. Biochem. 100, 883-893), was Trp-61 greater than Trp-19 greater than Trp-18. Thus, Trp-18 was evaluated to be essential for activity. Difference spectra of phospholipase A2 produced by titrating with laurylphosphorylcholine in the presence of Ca2+, which are due in large part to perturbation of the tryptophan residue(s), were retained with phospholipase A2 derivatives containing 1.2 and 2.0 mol of tryptophan residues oxidized but not with the derivative containing 3.0 mol of tryptophan residues oxidized. Such observations led us to assume that Trp-18 is involved in the specific site that interacts with phospholipid.

Studies on tryptophan residues of Abrus agglutinin. Stopped-flow kinetics of modification and fluorescence-quenching studies.

The presence of two essential tryptophan residues/molecule was implicated in the binding site of Abrus agglutinin [Patanjali, Swamy, Anantharam, Khan & Surolia (1984) Biochem. J. 217, 773-781]. A detailed study of the stopped-flow kinetics of the oxidation of tryptophan residues revealed three classes of tryptophan residues in the native protein. A discrete reorganization of tryptophan residues revealed three classes of tryptophan residues in the native protein. A discrete reorganization of tryptophan residues into two phases was observed upon ligand binding. The heterogeneity of tryptophan exposure was substantiated by quenching studies with acrylamide, succinimide and Cs+. Our study revealed the microenvironment of tryptophan residues to be hydrophobic, and also the presence of acidic amino acid residues in the vicinity of surface-localized tryptophan residues.

Free radicals in hypothermic rat heart preservation--prevention of damage by mannitol and desferrioxamine.

The free radical mediated oxidation of proteins in stored hypothermic rat hearts has been investigated by the estimation of fluorescence (Ex 360, Em 440-460 nm) believed to result from the oxidation of tryptophan residues. Damage occurring during ischaemia results in the release of oxidised proteins. This damage is significantly attenuated by mannitol and desferrioxamine although the mechanism of action of the two compounds may be different.

Identification of the tryptophan residue located in the calcium binding site of Trimeresurus flavoviridis phospholipase A2.

When phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased linearly with increase in the extent of oxidation of tryptophan residues. Oxidation of two of the four tryptophan residues caused an apparent loss of activity. The accessibilities of the tryptophan residues were analyzed with differently oxidized phospholipase A2 preparations and were determined to be in the following order: Trp-3 approximately Trp-30 greater than Trp-68 greater than Trp-108. The magnitude of the difference spectrum with a negative peak at 292 nm which is produced upon the binding of Ca2+ in the vicinity of tryptophan residue(s) decreased in a concave manner with increase in the extent of oxidation of tryptophan residues and was greatly diminished when 2 mol of tryptophan residues were oxidized. The activity and Ca2+-induced difference spectrum are thus related to either Trp-3 or Trp-30 or both. Des-octapeptide(1-8)-phospholipase A2 (L-fragment) is 14% as active as phospholipase A2 and is able to give a Ca2+-induced difference spectrum which is smaller than, but similar to, that of phospholipase A2. Its activity and the magnitude of the Ca2+-induced difference spectrum decreased along similar paths with increase in the amount of tryptophan residues oxidized, but in a manner indicating that two tryptophan residues are apparently responsible for the activity and the Ca2+-induced difference spectrum. The order of accessibility of the tryptophan residues of L-fragment was Trp-30 approximately Trp-108 greater than Trp-68. Trp-108, however, could be excluded from the residues located in the active site by reference to the tertiary structure of homologous Crotalus atrox phospholipase A2. Thus, Trp-30 is located in the Ca2+ binding site and is responsible for the activity of L-fragment. It is thus concluded that in phospholipase A2 Trp-30 is located in the Ca2+ binding site. From the concave decrease of relative magnitude of the Ca2+-induced difference spectrum and the linear decrease of relative activity upon oxidation of phospholipase A2, it may be assumed that both Trp-3 and Trp-30 are required to produce the Ca2+-induced difference spectrum, while only Trp-30 need be intact for activity. Anomalous binding of Ca2+ was observed for oxidized phospholipase A2.