Trp Oxidation Research Articles

Hollow-like three-dimensional structure of methyl orange-delaminated Ti3C2 MXene nanocomposite for high-performance electrochemical sensing of tryptophan.

Tryptophan(Trp) is being explored as a potential biomarker for various diseases associated with decreased tryptophan levels; however, metabolomic methods are expensive and time-consuming and require extensive sample analysis, making them urgently needed for trace detection. Toexploit the properties of Ti3C2 MXenes a rational porous methyl orange (MO)-delaminated Ti3C2 MXene was preparedvia a facile mixing process for the electrocatalytic oxidation of Trp. The hollow-like 3D structure with a more open structure and the synergistic effect of MO and conductive Ti3C2 MXene enhanced its electrochemical catalytic capability toward Trp biosensing. More importantly, MO can stabilize Ti3C2 MXene nanosheets through noncovalent π-π interactions and hydrogen bonding. Compared with covalent attachment, these non-covalent interactions preserve the electronic conductivity of the Ti3C2 MXene nanosheets. Finally, the addition of MO-derived nitrogen (N) and sulfur (S) atoms to Ti3C2 MXene enhanced the electronegativity and improved its affinity for specific molecules, resulting in high-performance electrocatalytic activity. The proposed biosensor exhibited a wide linear response in concentration ranges of 0.01-0.3 µM and 0.5-120 µM, with a low detection limit of 15 nM for tryptophan detection, and high anti-interference ability in complex media of human urine and egg white matrices. The exceptional abilities of the MO/Ti3C2 nanocatalyst make it a promising electrode material for the detection of important biomolecules.

Metabolic role of the amino acids in the adaptation of Holstein dairy cows to two contrasting challenging situations

This thesis investigated the role of the amino acids (AA) during the transition period and lactation under grazing conditions, and heat stress (HS) as two contrasting challenging situations for the dairy cow. In the experiment 1, the branched-chain AA (BCAA) metabolism was studied in North American (NAH) vs. New Zealand (NZH) Holstein cows between -45 and 180 days in milk (DIM). Higher plasma concentrations of BCAA were confirmed in NAH vs. NZH in early lactation. The temporal profile of hepatic gene expression of specific BCAA transporters, especially LAT1 and Eeg1, differed between the genetic strains in association with the level of somatotropic axis uncoupling and insulin resistance in the liver. In the experiments 2 and 3, we studied the role of the metabolism of the AA against HS and its amelioration through nutritional strategies under controlled conditions. The plasma and milk metabolomics indicated that the HS triggered skeletal muscle mobilization and enhanced AA catabolism. Dietary supply of vitamin E and Se above requirements seemed to have modulated the skeletal muscle mobilization, while D3 and Ca did not show significant effects (experiment 2). The results of experiment 3 suggested that the partial amelioration of HS effects on productive and physiological parameters through high dietary supply of Met (+70 %), Lys (+65 %) and His (+21 %) were mediated by a recovery of phosphatidylcholines and decreased oxidation of Val, Trp and Leu due to enhanced one-carbon metabolism and Lys oxidation, respectively.

Electrochemical detection of tryptophan in fish and pharmaceutical supplement at glassy carbon electrode modified with Fe doped ZnO nanoparticle

AbstractThe co‐precipitation approach was employed in this study to create Fe doped ZnO (Fe‐ZnO) nanoparticles (NPs). To characterize the synthesized NPs, Ultra violet‐Visible (UV‐Vis) spectroscopy, X‐ray diffraction (XRD), fourier transform infrared (FT‐IR), scanning electron microscopy (SEM), and Brunauer‐Emmett‐Teller (BET) were used. Here, a novel electrochemical approach for the detection of tryptophan (Trp) using a glassy carbon electrode modified with Fe‐ZnO NPs (Fe‐ZnO/GCE) is demonstrated. The research findings revealed that Fe‐ZnO/GCE enhanced the electron transfer rate of Trp oxidation. The sensitivity of Trp detection using Fe‐ZnO/GCE in phosphate buffer solution (PBS) at pH 64.0 was significantly improved compared to bare GCE due to the electrocatalytic activity of Fe‐ZnO NPs. Fe‐ZnO/GCE exhibited a linear response with Trp concentrations ranging from 0.1 to 150 μM with the limit of detection (LOD) is 0.089 μM (3σ/m) and limit of quantification (LOQ) about 0.3 μM (10σ/m) using linear sweep voltammetry (LSV). The Fe‐ZnO/GCE sensor was also effectively used to detect Trp in African catfish and multivitamin tablets. Trp analysis in real samples exhibited good recovery values of 89.60 to 99.15 %, demonstrating the accuracy and practicality of the method.

Oxidation Products of Tryptophan and Proline in Adipokinetic Hormones-Artifacts or Post-Translational Modifications?

Adipokinetic hormones (AKHs) regulate important physiological processes in insects. AKHs are short peptides with blocked termini and Trp in position 8. Often, proline occupies position 6. Few post-translational modifications have been found, including hydroxyproline ([Hyp6]) and kynurenine. Our recent data suggest that the Hyp- and Kyn-containing AKHs occur more often than originally thought and we here investigate if they are natural or artifactual. From crude extracts of the corpora cardiaca (CC) of various insect species, AKHs were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS). Synthetic [Hyp6]-AKHs were tested in an in vivo metabolic assay. Freshly dissected Periplaneta americana and Blaberus atropos CCs (with precautions taken against oxidation) were analyzed. B. atropos CC were placed into a depolarizing saline and the released AKHs were measured. Hyp was detected in several decapeptides from cockroaches. The modified form accompanied the AKH at concentrations below 7%. The [Hyp6]-AKHs of B. atropos were present in fresh CC preparations and were shown to be releasable from the CC ex vivo. Synthetic [Hyp6]-containing peptides tested positively in a hypertrehalosemic bioassay. Hydroxyprolination was also detected for Manto-CC from the termite Kalotermes flavicollis and for Tetsu-AKH of the grasshopper, Tetrix subulata. Oxidized Trp-containing forms of Nicve-AKH were found in species of the burying beetle genus Nicrophorus. Trp oxidation is known to occur easily during sample handling and is likely the reason for the present findings. For hydroxyprolination, however, the experimental evidence suggests endogenous processes.

Nitrogen Defect-Rich Graphitic Carbon Nitride for Highly Sensitive Voltammetric Determination of Tryptophan.

Here, a highly sensitive electrochemical sensor for detection of tryptophan (Trp) using a nitrogen defect graphitic carbon nitride-modified glassy carbon electrode (ND-CN/GCE) was introduced. ND-CN/GCE showed a higher oxidation current for Trp than the graphitic carbon nitride-modified glassy carbon electrode (g-CN/GCE) and bare glassy carbon electrode (BGCE). The synthesized nitrogen defect-rich graphitic carbon nitride (ND-CN) was characterized using X-ray photoelectron spectroscopy, X-ray diffraction spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy, and transmission electron microscopy. Electrochemical impedance spectroscopy and cyclic voltammetry were used to further analyze the electrochemical properties of BGCE, g-CN/GCE, and ND-CN/GCE. The oxidation of Trp at ND-CN/GCE is a diffusion-controlled process at pH 3.0. It was calculated that the transfer coefficient, rate constant, and diffusion coefficient of Trp were 0.53, 2.24 × 103 M-1 s-1, and 8.3 × 10-3 cm2 s-1, respectively, at ND-CN/GCE. Trp was detected using square wave voltammetry, which had a linear range from 0.01 to 40 μM at pH 3.0 and a limit of detection of about 0.0034 μM (3σ/m). Analyzing the presence of Trp in a milk and multivitamin tablet sample with a percentage recovery in the range of 97.0-108% satisfactorily demonstrated the practical usability of the electrochemical sensor. The ND-CN/GCE additionally displays good repeatability and reproducibility and satisfactory selectivity.

Characterization of tryptophan oxidation affecting D1 degradation by FtsH in the photosystem II quality control of chloroplasts.

Photosynthesis is one of the most important reactions for sustaining our environment. Photosystem II (PSII) is the initial site of photosynthetic electron transfer by water oxidation. Light in excess, however, causes the simultaneous production of reactive oxygen species (ROS), leading to photo-oxidative damage in PSII. To maintain photosynthetic activity, the PSII reaction center protein D1, which is the primary target of unavoidable photo-oxidative damage, is efficiently degraded by FtsH protease. In PSII subunits, photo-oxidative modifications of several amino acids such as Trp have been indeed documented, whereas the linkage between such modifications and D1 degradation remains elusive. Here, we show that an oxidative post-translational modification of Trp residue at the N-terminal tail of D1 is correlated with D1 degradation by FtsH during high-light stress. We revealed that Arabidopsis mutant lacking FtsH2 had increased levels of oxidative Trp residues in D1, among which an N-terminal Trp-14 was distinctively localized in the stromal side. Further characterization of Trp-14 using chloroplast transformation in Chlamydomonas indicated that substitution of D1 Trp-14 to Phe, mimicking Trp oxidation enhanced FtsH-mediated D1 degradation under high light, although the substitution did not affect protein stability and PSII activity. Molecular dynamics simulation of PSII implies that both Trp-14 oxidation and Phe substitution cause fluctuation of D1 N-terminal tail. Furthermore, Trp-14 to Phe modification appeared to have an additive effect in the interaction between FtsH and PSII core in vivo. Together, our results suggest that the Trp oxidation at its N-terminus of D1 may be one of the key oxidations in the PSII repair, leading to processive degradation by FtsH.

Characterization of tryptophan oxidation affecting D1 degradation by FtsH in the photosystem II quality control of chloroplasts

Photosynthesis is one of the most important reactions for sustaining our environment. Photosystem II (PSII) is the initial site of photosynthetic electron transfer by water oxidation. Light in excess, however, causes the simultaneous production of reactive oxygen species (ROS), leading to photo-oxidative damage in PSII. To maintain photosynthetic activity, the PSII reaction center protein D1, which is the primary target of unavoidable photo-oxidative damage, is efficiently degraded by FtsH protease. In PSII subunits, photo-oxidative modifications of several amino acids such as Trp have been indeed documented, whereas the linkage between such modifications and D1 degradation remains elusive. Here, we show that an oxidative post-translational modification of Trp residue at the N-terminal tail of D1 is correlated with D1 degradation by FtsH during high-light stress. We revealed that Arabidopsis mutant lacking FtsH2 had increased levels of oxidative Trp residues in D1, among which an N-terminal Trp-14 was distinctively localized in the stromal side. Further characterization of Trp-14 using chloroplast transformation in Chlamydomonas indicated that substitution of D1 Trp-14 to Phe, mimicking Trp oxidation enhanced FtsH-mediated D1 degradation under high light, although the substitution did not affect protein stability and PSII activity. Molecular dynamics simulation of PSII implies that both Trp-14 oxidation and Phe substitution cause fluctuation of D1 N-terminal tail. Furthermore, Trp-14 to Phe modification appeared to have an additive effect in the interaction between FtsH and PSII core in vivo. Together, our results suggest that the Trp oxidation at its N-terminus of D1 may be one of the key oxidations in the PSII repair, leading to processive degradation by FtsH.

Neuroanatomical zones of human traumatic brain injury reveal significant differences in protein profile and protein oxidation: Implications for secondary injury events.

Traumatic brain injury (TBI) causes significant neurological deficits and long-term degenerative changes. Primary injury in TBI entails distinct neuroanatomical zones, i.e., contusion (Ct) and pericontusion (PC). Their dynamic expansion could contribute to unpredictable neurological deterioration in patients. Molecular characterization of these zones compared with away from contusion (AC) zone is invaluable for TBI management. Using proteomics-based approach, we were able to distinguish Ct, PC and AC zones in human TBI brains. Ct was associated with structural changes (blood-brain barrier (BBB) disruption, neuroinflammation, axonal injury, demyelination and ferroptosis), while PC was associated with initial events of secondary injury (glutamate excitotoxicity, glial activation, accumulation of cytoskeleton proteins, oxidative stress, endocytosis) and AC displayed mitochondrial dysfunction that could contribute to secondary injury events and trigger long-term degenerative changes. Phosphoproteome analysis in these zones revealed that certain differentially phosphorylated proteins synergistically contribute to the injury events along with the differentially expressed proteins. Non-synaptic mitochondria (ns-mito) was associated with relatively more differentially expressed proteins (DEPs) compared to synaptosomes (Syn), while the latter displayed increased protein oxidation including tryptophan (Trp) oxidation. Proteomic analysis of immunocaptured complex I (CI) from Syn revealed increased Trp oxidation in Ct > PC > AC (vs. control). Oxidized W272 in the ND1 subunit of CI, revealed local conformational changes in ND1 and the neighboring subunits, as indicated by molecular dynamics simulation (MDS). Taken together, neuroanatomical zones in TBI show distinct protein profile and protein oxidation representing different primary and secondary injury events with potential implications for TBI pathology and neurological status of the patients.

The inflammatory oxidant peroxynitrous acid modulates the structure and function of the recombinant human V3 isoform of the extracellular matrix proteoglycan versican

Continued oxidant production during chronic inflammation generates host tissue damage, with this being associated with pathologies including atherosclerosis. Atherosclerotic plaques contain modified proteins that may contribute to disease development, including plaque rupture, the major cause of heart attacks and strokes. Versican, a large extracellular matrix (ECM) chondroitin-sulfate proteoglycan, accumulates during atherogenesis, where it interacts with other ECM proteins, receptors and hyaluronan, and promotes inflammation. As activated leukocytes produce oxidants including peroxynitrite/peroxynitrous acid (ONOO−/ONOOH) at sites of inflammation, we hypothesized that versican is an oxidant target, with this resulting in structural and functional changes that may exacerbate plaque development. The recombinant human V3 isoform of versican becomes aggregated on exposure to ONOO−/ONOOH. Both reagent ONOO−/ONOOH and SIN-1 (a thermal source of ONOO−/ONOOH) modified Tyr, Trp and Met residues. ONOO−/ONOOH mainly favors nitration of Tyr, whereas SIN-1 mostly induced hydroxylation of Tyr, and oxidation of Trp and Met. Peptide mass mapping indicated 26 sites with modifications (15 Tyr, 5 Trp, 6 Met), with the extent of modification quantified at 16. Multiple modifications, including the most extensively nitrated residue (Tyr161), are within the hyaluronan-binding region, and associated with decreased hyaluronan binding. ONOO−/ONOOH modification also resulted in decreased cell adhesion and increased proliferation of human coronary artery smooth muscle cells. Evidence is also presented for colocalization of versican and 3-nitrotyrosine epitopes in advanced (type II-III) human atherosclerotic plaques. In conclusion, versican is readily modified by ONOO−/ONOOH, resulting in chemical and structural modifications that affect protein function, including hyaluronan binding and cell interactions.

Mitochondrial Complex I Inhibition in Dopaminergic Neurons Causes Altered Protein Profile and Protein Oxidation: Implications for Parkinson's disease.

Mitochondrial dysfunction and oxidative stress are critical to neurodegeneration in Parkinson's disease (PD). Mitochondrial dysfunction in PD entails inhibition of the mitochondrial complex I (CI) in the dopaminergic neurons of substantia nigra. The events contributing to CI inhibition and downstream pathways are not completely elucidated. We conducted proteomic analysis inadopaminergic neuronal cell line exposed individually to neurotoxic CI inhibitors: rotenone (Rot), paraquat (Pq) and 1-methyl-4-phenylpyridinium (MPP+). Mass spectrometry (MS) revealed the involvement of biological processes including cell death pathways, structural changes and metabolic processes among others, most of which were common across all models. The proteomic changes induced by Pq were significantly higher than those induced by Rot and MPP+. Altered metabolic processes included downregulated mitochondrial proteins such as CI subunits. MS of CI isolated from the models revealed oxidative post-translational modifications with Tryptophan (Trp) oxidation as the predominant modification. Further, 62 peptides in 22 subunits of CI revealed Trp oxidation with 16 subunits common across toxins. NDUFV1 subunit had the greatest number of oxidized Trp and Rot model displayed the highest number of Trp oxidation events compared to the other models. Molecular dynamics simulation (MDS) of NDUFV1 revealed that oxidized Trp 433 altered the local conformation thereby changing the distance between the Fe-S clusters, Fe-S 301(N1a) to Fe-S 502 (N3) and Fe-S 802 (N4) to Fe-S 801 (N5), potentially affecting the efficiency of electron transfer. The events triggered by the neurotoxins represent CI damage, mitochondrial dysfunction and neurodegeneration in PD.

Implications of differential peroxyl radical-induced inactivation of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase for the pentose phosphate pathway

Escherichia coli glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are key enzymes of the pentose phosphate pathway, responsible for the NADPH production in cells. We investigated modification of both enzymes mediated by peroxyl radicals (ROO·) to determine their respective susceptibilities to and mechanisms of oxidation. G6PDH and 6PGDH were incubated with AAPH (2,2′-azobis(2-methylpropionamidine)dihydrochloride), which was employed as ROO· source. The enzymatic activities of both enzymes were determined by NADPH release, with oxidative modifications examined by electrophoresis and liquid chromatography (LC) with fluorescence and mass (MS) detection. The activity of G6PDH decreased up to 62.0 ± 15.0% after 180 min incubation with 100 mM AAPH, whilst almost total inactivation of 6PGDH was determined under the same conditions. Although both proteins contain abundant Tyr (particularly 6PGDH), these residues were minimally affected by ROO·, with Trp and Met being major targets. LC–MS and in silico analysis showed that the modification sites of G6PDH are distant to the active site, consistent with a dispersed distribution of modifications, and inactivation resulting from oxidation of multiple Trp and Met residues. In contrast, the sites of oxidation detected on 6PGDH are located close to its catalytic site indicating a more localized oxidation, and a consequent high susceptibility to ROO·-mediated inactivation.

Label-free tumor cell screening based on IDO1-mediated tryptophan metabolism at single cell level

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a critical role in inflammatory and immunometabolism programming through catalyzing the oxidation of tryptophan (Trp) into downstream N-formylkynurenine. IDO1 is typically up-regulated in malignant tumors, making it a potential biomarker for cancer diagnosis. Here we show an effective strategy for tumor cell detection by integrating IDO1 activity assay with single cell-encapsulated droplets on a microfluidic platform for high-throughput bioanalysis. Mixed cells, as well as other cofactors, are encapsulated in individual droplets, which act as dynamic microreactors for IDO1-catalyzed oxidation of Trp. After pico-injection of a biosensing ensemble consisting of the macrocycle cucurbit [8]uril (Q8) and a fluorescent guest, rapid and robust screening of tumor cells by fluorescence signal is achieved in a few minutes reporting to Trp depletion, expanding the scope of conventional antibody-based detection of protein biomarkers. The results represent the first example of quantifying IDO1 enzymatic activity at the single cell level with a high-throughput performance, therefore promising warning signs and early diagnosis of tumor cells.

Tryptophan oxidation in young children with environmental enteric dysfunction classified by the lactulose rhamnose ratio

ABSTRACTBackgroundIn young children, associations between linear growth faltering, environmental enteric dysfunction (EED), and the plasma kynurenine (Kyn)/tryptophan (Trp) ratio (KTR) have led to the proposal that higher Trp catabolism in response to intestinal/systemic inflammation limits Trp availability for protein synthesis, resulting in impaired growth.ObjectivesWe sought to estimate the Trp oxidation rate and the Trp conversion rate to Kyn in young children with and without EED.MethodsChildren aged 18–24 mo, from urban slums, were assigned to EED (n = 19) or no-EED (n = 26) groups on the basis of a urinary lactulose/rhamnose ratio (LRR) cutoff based on mean + 2 SDs of LRR (≥0.068) in normal age- and sex-matched, high–socioeconomic status children. Plasma KTR and fecal biomarkers of EED were measured. Trp oxidation in the fed state was measured using 13C1-Trp in an oral plateau feeding protocol.ResultsThe median (quartile 1, quartile 3) fasted KTR was 0.089 (0.066, 0.110) in children with EED compared with 0.070 (0.050, 0.093) in children with no EED (P = 0.077). However, there was no difference in fed-state Trp oxidation [median (quartile 1, quartile 3) 3.1 (1.3, 5.8) compared with 3.9 (1.8, 6.0) µmol/kg FFM/h, respectively, P = 0.617] or Trp availability for protein synthesis [42.6 (36.5, 45.7) compared with 42.5 (37.9, 46.9) µmol/kg FFM/h, respectively, P = 0.868] between the groups. In contrast, the median (quartile 1, quartile 3) fractional synthesis rates of Kyn [12.5 (5.4, 20.0) compared with 21.3 (16.1, 24.7) %pool/h, P = 0.005] and the fraction of Ala derived from Trp [0.007 (0.005, 0.015) compared with 0.012 (0.008, 0.018), P = 0.037], respectively, in the plasma compartment were significantly slower in the EED group. Fecal biomarkers of EED did not differ between the groups.ConclusionsThe static plasma KTR value is not a good indicator of the dynamic Trp flux down its oxidative pathway. In a poor sanitary environment, children without EED actually have a faster Kyn synthesis rate, which might be beneficial, because of the cytoprotective and anti-inflammatory functions of downstream metabolites. This study was registered in the Clinical Trials Registry of India as CTRI/2017/02/007921.

Large-scale top-down proteomics of the Arabidopsis thaliana leaf and chloroplast proteomes.

We present a large-scale top-down proteomics (TDP) study of plant leaf and chloroplast proteins, achieving the identification of over 4700 unique proteoforms. Using capillary zone electrophoresis coupled with tandem mass spectrometry analysis of offline size-exclusion chromatography fractions, we identify 3198 proteoforms for total leaf and 1836 proteoforms for chloroplast, with 1024 and 363 proteoforms having post-translational modifications, respectively. The electrophoretic mobility prediction of capillary zone electrophoresis allowed us to validate post-translational modifications that impact the charge state such as acetylation and phosphorylation. Identified modifications included Trp (di)oxidation events on six chloroplast proteins that may represent novel targets of singlet oxygen sensing. Furthermore, our TDP data provides direct experimental evidence of the N- and C-terminal residues of numerous mature proteoforms from chloroplast, mitochondria, endoplasmic reticulum, and other sub-cellular localizations. With this information, we suggest true transit peptide cleavage sites and correct sub-cellular localization signal predictions. This large-scale analysis illustrates the power of top-down proteoform identification of post-translational modifications and intact sequences that can benefit our understanding of both the structure and function of hundreds of plant proteins.

A New Electrochemical Method to Determine Tryptophan in Fruit Juices: Development and Validation.

Tryptophan (Trp) is an essential amino acid usually found in fruit juices. Its determination is necessary for food companies because of its relation to human health. In this work, a new electrochemical method based on sonogel–carbon electrodes (SNGCEs) was developed and validated using an ultra performance liquid chromatography (UPLC) method as a reference method for the determination of Trp in fruit juices. Cyclic voltammetry (CV), chronoamperometry, and differential pulse voltammetry (DPV) techniques were applied to investigate the oxidation of Trp on a previously polarized SNGCE surface in a Britton–Robinson (BR) buffer solution at pH 3.6. The operating conditions for electroanalysis were optimized using a Box–Behnken design (BBD), obtaining an oxidation peak for Trp at 0.749 V. The linear range for this method was from 0.1 to 5 mg/L. The intraday and interday precision, expressed as a relative standard deviation (RSD), were 3.1% and 2.7%, respectively. The average recovery was 99.01%, and the limit of detection and quantitation were 0.33 and 1.09 mg/L, respectively. Therefore, from the quality analytical parameters obtained, it can be concluded that the new electrochemical method can be successfully used for the routine analysis of Trp in fruit juices. As far as we are concerned, this is the first time that a methodology for Trp determination was performed in this kind of real food matrices.

3D Co-doped Ni-based conductive MOFs modified electrochemical sensor for highly sensitive detection of l-tryptophan

L-tryptophan (Trp) is an essential amino acid for humans and plays crucial roles in many metabolic functions. Trp levels can be used for diagnosing different kinds of metabolic disorders and the symptoms associated with those diseases. Herein, a novel, simple and sensitive sensor based on 3D peony-like bimetallic conductive MOFs (Co–Ni-MOFs) was fabricated for the electrochemical determination of Trp. The bimetallic conductive MOFs were synthesized by a facile one-pot hydrothermal process. On account of the synergy between the Ni2+ and Co2+ ions, the bimetallic Co–Ni-MOFs showed excellent electrochemical performance, including good conductivity, large effective surface areas, and high electrocatalytic reactivity toward the oxidation of Trp. Consequently, the Co–Ni-MOFs-modified electrodes obtained a wide linear range from 10 nmol L−1 to 300 μmol L−1 and a low detection limit of 8.7 nmol L−1 (S/N = 3) for Trp. Additionally, the prepared sensor also displayed high selectivity, long-term stability and reproducibility. Moreover, the proposed sensor was successfully applied to determine the levels of Trp in the plasma of mice after cadmium intoxication.

Oxidation of specific tryptophan residues inhibits high-affinity binding of cocaine and its metabolites to a humanized anticocaine mAb

Cocaine addiction remains a serious problem lacking an effective pharmacological treatment. Thus, we have developed a high-affinity anti-cocaine monoclonal antibody (mAb), h2E2, for the treatment of cocaine use disorders. We show that selective tryptophan (Trp) oxidation by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) resulted in a loss of high-affinity binding of cocaine to this mAb. The newly developed use of excess methionine (Met) to protect mAb met residues from AAPH oxidation did not substantially attenuate the effects of oxidation on cocaine binding but greatly decreased the modification of met residues in the mAb. Similar large decreases in ligand affinity (5000–10,000-fold) upon oxidation were observed using cocaine and two cocaine metabolites, cocaethylene and benzoylecgonine, which also bind with nanomolar affinity to this h2E2 mAb. The decrease in binding affinity was accompanied by a decrease of approximately 50% in Trp fluorescence, and increases in mAb 310 to 370 nm absorbance were consistent with the presence of oxidized forms of Trp. Finally, mass spectral analysis of peptides derived from control and AAPH-oxidized mAb indicated that excess free met did effectively protect mAb met residues from oxidation, and that AAPH-oxidized mAb heavy-chain Trp33 and light-chain Trp91 residues are important for cocaine binding, consistent with a recently derived h2E2 Fab fragment crystal structure containing bound benzoylecgonine. Thus, protection of the anti-cocaine h2E2 mAb from Trp oxidation prior to its clinical administration is critical for its proposed therapeutic use in the treatment of cocaine use disorders.

Effect of macromolecular crowding on protein oxidation: Consequences on the rate, extent and oxidation pathways

Biological systems are heterogeneous and crowded environments. Such packed milieus are expected to modulate reactions both inside and outside the cell, including protein oxidation. In this work, we explored the effect of macromolecular crowding on the rate and extent of oxidation of Trp and Tyr, in free amino acids, peptides and proteins. These species were chosen as they are readily oxidized and contribute to damage propagation. Dextran was employed as an inert crowding agent, as this polymer decreases the fraction of volume available to other (macro)molecules. Kinetic analysis demonstrated that dextran enhanced the rate of oxidation of free Trp, and peptide Trp, elicited by AAPH-derived peroxyl radicals. For free Trp, the rates of oxidation were 15.0 ± 2.1 and 30.5 ± 3.4 μM min−1 without and with dextran (60 mg mL−1) respectively. Significant increases were also detected for peptide-incorporated Trp. Dextran increased the extent of Trp consumption (up to 2-fold) and induced short chain reactions. In contrast, Tyr oxidation was not affected by the presence of dextran. Studies on proteins, using SDS-PAGE and LC-MS, indicated that oxidation was also affected by crowding, with enhanced amino acid loss (45% for casein), chain reactions and altered extents of oligomer formation. The overall effects of dextran-mediated crowding were however dependent on the protein structure. Overall, these data indicate that molecular crowding, as commonly encountered in biological systems affect the rates, and extents of oxidation, and particularly of Trp residues, illustrating the importance of appropriate choice of in vitro systems to study biological oxidations.

Pathogenesis of Age-Related Cataract: A Systematic Review of Proteomic Studies

Aims: The aim of this systematic review is to identify all the available data on human lens proteomics with a critical role in age-related cataract formation in order to elucidate the physiopathology of the aging lens. Methods:: We searched on Medline and Cochrane databases. The search generated 328 manuscripts. We included nine original proteomic studies that investigated human cataractous lenses. Results: Deamidation was the major age-related post-translational modification. There was a significant increase in the amount of αA-crystallin D-isoAsp58 present at all ages, while an increase in the extent of Trp oxidation was apparent in cataract lenses when compared to aged normal lense. During aging, enzymes with oxidized cysteine at critical sites included GAPDH, glutathione synthase, aldehyde dehydrogenase, sorbitol dehydrogenase, and PARK7. Conclusion: D-isoAsp in αA crystallin could be associated with the development of age-related cataract in humans by contributing to the denaturation of a crystallin and decreasing its ability to act as a chaperone. Oxidation of Trp may be associated with nuclear cataract formation in humans, while the role of oxidant stress in age-related cataract formation is dominant.

Enhancement of electrocatalytic activity in tungsten trioxide nanoparticles by UV-light irradiation: Application for simultaneous detection of tyrosine and tryptophan

Electromagnetic radiation is widely used as a tool for grafting or partially modifying materials for specific applications. Here, we demonstrate that the UV-rays irradiation on tungsten trioxide (WO3) nanoparticles, reduces its crystallite size and induces oxygen vacancy, thereby enhancing the catalytic ability significantly. This unique feature of WO3 NPs has been utilised for the development of a novel electrochemical sensor for the simultaneous determination of two important amino acids l-Tyrosine (Tyr) and l-Tryptophan (Trp). Both the Tyr and Trp act as precursors for catecholamine and indolamine neurotransmitters which help neural communication and regulate the psychological, physiological, and behavioural responses in human beings. Therefore, it is important to know the precise amount of these amino acids in the human body fluids, food, and pharmaceutical supplements. Here, we report the fabrication of a simple method for the determination of Tyr and Trp using UV-rays irradiated WO3 nanoparticles modified glassy carbon electrode (GCE) without using any additional surface mediator. Among various exposition times, 4 h irradiated WO3 modified GCE showed an excellent electrocatalytic activity towards the oxidation of Tyr and Trp over wider concentration ranges of 0.01–1100 μM and 0.02–2000 μM for Tyr and Trp with the lowest detection limits of 2.3 nM and 4.4 nM respectively. A good separation in the oxidation potentials was realized during the simultaneous detection of Tyr and Trp from their binary mixture in phosphate buffer saline at pH 6.0. The developed sensor displayed good reproducibility, high sensitivity, and good selectivity towards the determination of the amino acids, making it suitable for the precise determination of Tyr and Trp in farm foods such as milk, curd, and egg.