N-formylkynurenine Research Articles

Novel inhibitor of brain indoleamine 2,3 dioxygenase, docking and experimental studies

Indoleamine 2,3-dioxygenase (IDO) is a haem-containing monomeric enzyme that catalyze the conversion of tryptophan (L-TRP) to N-formyl kynurenine. IDO activity is regulated by cytokines. Pro inflammatory cytokines are potent inducers of IDO, whereas anti-inflammatory cytokines are IDO inhibitors. Prostaglandin E2 induces IDO activity. In inflammation the relationship between immune system and the kynurenine pathway play an important role. IDO is an important therapeutic target for the treatment of inflammation. Present study evaluates the binding of Hypericum perforatum (HP) against IDO enzyme using MVD software acute and chronic effects of HP on IDO enzyme activity. Docking results show that HP fit well in the allosteric site of IDO. Energy scores for HP -158.687 Kcal/mol. Administration of HP (500mg/kg/3ml) shows that serum IDO activity was significantly increased (171%, P<0.01) and (114%, P<0.01) respectively after acute and chronic treatment. Brain IDO activity was decreased by 42%, (P<0.01) after acute and 43% (P<0.01) chronic treatment. It is concluded from the present study that HP is noncompetitive inhibitor of IDO as proofs by docking further its inhibitory effects on brain IDO reveals its anti-inflammatory effect.Dawood and Bano, International Current Pharmaceutical Journal, May 2017, 6(6): 34-39http://www.icpjonline.com/documents/Vol6Issue6/01.pdf

Characterization of 2-Oxindole Forming Heme Enzyme MarE, Expanding the Functional Diversity of the Tryptophan Dioxygenase Superfamily.

3-Substituted 2-oxindoles are important structural motifs found in many biologically active natural products and pharmaceutical lead compounds. Here, we report an enzymatic formation of the 3-substituted 2-oxindoles catalyzed by MarE in the maremycin biosynthetic pathway in Streptomyces sp. B9173. MarE is a homologue of FeII/heme-dependent tryptophan 2,3-dioxygenases (TDOs). Typical TDOs usually catalyze the insertion of two oxygen atoms from O2 into an indole ring to generate N-formylkynurenine (NFK)-like products. In contrast, MarE catalyzes the insertion of a single oxygen atom from O2 into an indole ring, to probably generate an epoxyindole intermediate that undergoes an unprecedented 2,3-hydride migration to form 2-oxindole structure. MarE shows substrate robustness to catalyze the conversion of a series of 3-substituted indoles into their corresponding 3-substituted 2-oxindoles. Although containing most key amino acid residues conserved in well-known TDO homologues, MarE falls into a separate new subgroup in the phylogenetic tree. The characterization of MarE and its homologue enriches the functional diversities of TDO superfamily and provides a new strategy for discovering novel natural products containing 3-substituted 2-oxindole pharmacophores by genome mining.

Kynurenine, a Tryptophan Metabolite That Accumulates With Age, Induces Bone Loss.

Age-dependent bone loss occurs in humans and in several animal species, including rodents. The underlying causal mechanisms are probably multifactorial, although an age-associated increase in the generation of reactive oxygen species has been frequently implicated. We previously reported that aromatic amino acids function as antioxidants, are anabolic for bone, and that they may potentially play a protective role in an aging environment. We hypothesized that upon oxidation the aromatic amino acids would not only lose their anabolic effects but also potentially become a catabolic byproduct. When measured in vivo in C57BL/6 mice, the tryptophan oxidation product and kynurenine precursor, N-formylkynurenine (NFK), was found to increase with age. We tested the direct effects of feeding kynurenine (kyn) on bone mass and also tested the short-term effects of intraperitoneal kyn injection on bone turnover in CD-1 mice. μCT analyses showed kyn-induced bone loss. Levels of serum markers of osteoclastic activity (pyridinoline [PYD] and RANKL) increased significantly with kyn treatment. In addition, histological and histomorphometric studies showed an increase in osteoclastic activity in the kyn-treated groups in both dietary and injection-based studies. Further, kyn treatment significantly increased bone marrow adiposity, and BMSCs isolated from the kyn-injected mice exhibited decreased mRNA expression of Hdac3 and its cofactor NCoR1 and increased expression of lipid storage genes Cidec and Plin1. A similar pattern of gene expression is observed with aging. In summary, our data show that increasing kyn levels results in accelerated skeletal aging by impairing osteoblastic differentiation and increasing osteoclastic resorption. These data would suggest that kyn could play a role in age-induced bone loss. © 2017 American Society for Bone and Mineral Research.

The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function

FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROO•) generated from AAPH (2,2′-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROO•. Quantification of the number of moles of amino acid consumed per mole of ROO• shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROO•-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples.

Inhibition of indoleamine 2,3-dioxygenase activity by fatty acids and prostaglandins: A structure function analysis

Indoleamine 2,3-dioxygenase-1 (IDO-1) catalyses the first and rate-limiting step in the metabolism of L-tryptophan. Degradation of L-Trp leads to the production of several immunosuppressive metabolites, including N-formyl kynurenine and kynurenine (Kyn). Apart from a normal physiological role, IDO-1 has also been identified to play a crucial role in immune suppression and tumour induced tolerance. Indeed, many primary tumours express high levels of IDO-1 compared to normal cells of the same stroma. IDO-1 is accepted as being an inducible negative regulator of T cell viability, proliferation and activation. As such, IDO-1 has become a target of intense interest for pharmacological inhibition, for the treatment of cancer. We have previously demonstrated that AA and the prostaglandin metabolite, PGD2, repressed the IFNγ mediated activity of IDO-1 in THP-1 cells and human monocytes. In this study, we characterise the structure-function relationship of fatty acids and eicosanoids towards inhibition of IDO-1 activity in THP-1 cells and human monocytes. Using a commercial library of fatty acids, 55% of fatty acids inhibited IDO-1 activity. The activity of individual FAs was affected by chain length, number of double bonds and bond configuration. Interrogation of an AA derived eicosanoid library identified 13 PGs with significant inhibitory activity. A structure-function analysis revealed that the γ position of the cyclopentenone ring, double bond in the α-β position of the cyclopentenone ring, the presence of multiple OH groups in the side arm and the addition of an ethanolamide group, significantly increased the inhibitory activity of the PGs. Based on this data we have identified the structure of two possible compounds that may be even more potent pharmacological repressors of IDO-1.

The efficacy of donepezil administration on acetylcholinesterase activity and altered redox homeostasis in Alzheimer’s disease

Alzheimer’s disease (AD) is a serious multifactorial disorder with progressive neurodegenerative outcomes related with impaired redox homeostasis. Inhibition of the enzyme acetylcholinesterase (AChE), as one of the major therapeutic strategies, is considered to be offering only symptomatic relief and moderate disease modifying effect. We intended to investigate the effects of acetylcholinesterase inhibition via donepezil on protein carbonyl (PCO), advanced protein oxidation products (AOPP) and ischemia modified albumin (IMA) as protein oxidation markers and ferric reducing antioxidant power (FRAP), prooxidant–antioxidant balance (PAB), total thiol (T-SH), protein thiol (P-SH) as antioxidant status markers and also kynurenine (KYN), N-formyl kynurenine (N-FKYN) and protein bound dityrosine (DT) levels all in one demonstrating the redox homeostasis in Alzheimer patients also correlated with AChE activity. The AChE activity and PCO, KYN, N-FKYN and DT levels were found to be significantly higher in the AD group than the control group. The FRAP, T-SH and P-SH levels were significantly lower in the AD group than in the control group. The AChE activity was significantly higher both in donepezil treated and untreated groups when compared with the control group. PCO levels were significantly higher in Alzheimer’s untreated group than the healthy control and donepezil treated groups. AChE activity was positively correlated with PCO, IMA, PAB, KYN and N-FKYN levels and negatively correlated with FRAP, T-SH and P-SH levels in all participants. Our data showed that treatment with donepezil had ameliorating effects on redox homeostasis in Alzheimer patients. AChE inhibition seems to be exhibiting a potent antioxidant role and may inhibit protein oxidation by decreasing AChE activity in AD, thus medicinal natural substances exhibiting the similar mechanism of action with their antioxidant behaviours can be recommended for the emphasis on new drug new drug development. Further clinical and experimental studies are needed to support our current findings and conclusions.

Tryptophan and Non-Tryptophan Fluorescence of the Eye Lens Proteins Provides Diagnostics of Cataract at the Molecular Level

The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of “free” Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their “free” counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age.

Molecular basis for catalysis and substrate-mediated cellular stabilization of human tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) play a central role in tryptophan metabolism and are involved in many cellular and disease processes. Here we report the crystal structure of human TDO (hTDO) in a ternary complex with the substrates L-Trp and O2 and in a binary complex with the product N-formylkynurenine (NFK), defining for the first time the binding modes of both substrates and the product of this enzyme. The structure indicates that the dioxygenation reaction is initiated by a direct attack of O2 on the C2 atom of the L-Trp indole ring. The structure also reveals an exo binding site for L-Trp, located ~42 Å from the active site and formed by residues conserved among tryptophan-auxotrophic TDOs. Biochemical and cellular studies indicate that Trp binding at this exo site does not affect enzyme catalysis but instead it retards the degradation of hTDO through the ubiquitin-dependent proteasomal pathway. This exo site may therefore provide a novel L-Trp-mediated regulation mechanism for cellular degradation of hTDO, which may have important implications in human diseases.

Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.

Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loop(C)) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop(N)) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop(N) suggests that it may function as a target for post-translational modifications and/or a mediator for protein-protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved "GTGG" motif of JK loop(C), thereby anchoring JK loop(C) in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loop(C) conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loop(C) in controlling substrate and product dynamics.

Tryptophan oxidation catabolite, N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance.

Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC-MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N-formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance.

Formation of fluorophores from the kynurenine pathway metabolite N-formylkynurenine and cyclic amines involves transamidation and carbon–carbon bond formation at the 2-position of the amine

BackgroundTryptophan catabolism along the kynurenine pathway is associated with a number of pathologies including cataract formation and cancer. Whilst the chemical reactions of kynurenine are well studied, less is known about the reactivity of its precursor N-formylkynurenine (NFK). We previously reported the generation of a strong fluorophore in an aqueous reaction of NFK with piperidine, and herein we describe its structure and mechanism of formation. MethodsCompounds were identified using NMR, mass and UV spectroscopic techniques. The products from the reaction of amines with amino acids were quantified using HPLC-MS. ResultsThe novel fluorophore was identified as a tetrahydroquinolone adduct (PIP-THQ), where piperidine is N-formylated and attached at its 2-position to the quinolone. NFK is initially deaminated to generate an unsaturated enone, which forms an adduct with piperidine and is subsequently converted into the fluorophore. Testing of a variety of other secondary amines showed that only cyclic amines unsubstituted at both positions adjacent to nitrogen could form fluorophores efficiently. The amino acids tryptophan and kynurenine, which lack the formamide group do not form such fluorophores. ConclusionsNFK forms fluorophores in a not previously published reaction with cyclic amines. General significanceOur study is the first to provide evidence for concurrent transamidation and substitution at the 2-position of a cyclic amine occurring under moderately-heated aqueous conditions with no added catalysts. The high reactivity of NFK demonstrated here could result in formation of biologically relevant metabolites yet to be characterised.

EGCG prevents tryptophan oxidation of cataractous ocular lens human γ-crystallin in presence of H2O2

Disruption of the short range order of proteins present in the ocular lens leads to cataract resulting in a loss of transparency. Human γ-crystallin (HGC), a water soluble protein present in the lens is known to aggregate with aging. A modified form of HGC (HGCc) was isolated from cataractous human ocular lens extract and the number of Trp residues that undergo oxidation was determined. The extent of oxidized Trp (N-formyl kynurenine) in HGC due to cataract formation was determined, primarily using fluorescence spectroscopy. The ability of (−)-epigallocatechin gallate (EGCG) to retain its antioxidant effect even in the presence of H2O2 was investigated. This was monitored by its ability to prevent the modification of intact Trp residues in HGCc isolated from cataractous human eye lens. Significant Trp fluorescence quenching occurs on interaction of the green tea component, EGCG with HGCc accompanied by a red shift. Docking studies were employed to substantiate the experimental results. As eye lens proteins are prone to oxidative stress it is essential that a clear understanding of the effects of the components generated in vivo vis-à-vis the antioxidant effects of natural polyphenols be obtained.

Induction of indoleamine 2, 3-dioxygenase in human dendritic cells by a cholera toxin B subunit-proinsulin vaccine.

Dendritic cells (DC) interact with naïve T cells to regulate the delicate balance between immunity and tolerance required to maintain immunological homeostasis. In this study, immature human dendritic cells (iDC) were inoculated with a chimeric fusion protein vaccine containing the pancreatic β-cell auto-antigen proinsulin linked to a mucosal adjuvant the cholera toxin B subunit (CTB-INS). Proteomic analysis of vaccine inoculated DCs revealed strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1). Increased biosynthesis of the immunosuppressive enzyme was detected in DCs inoculated with the CTB-INS fusion protein but not in DCs inoculated with proinsulin, CTB, or an unlinked combination of the two proteins. Immunoblot and PCR analyses of vaccine treated DCs detected IDO1mRNA by 3 hours and IDO1 protein synthesis by 6 hours after vaccine inoculation. Determination of IDO1 activity in vaccinated DCs by measurement of tryptophan degradation products (kynurenines) showed increased tryptophan cleavage into N-formyl kynurenine. Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system. Treatment of vaccinated DCs with pharmacological NF-κB inhibitors ACHP or DHMEQ significantly inhibited IDO1 biosynthesis, suggesting a role for NF-κB signaling in vaccine up-regulation of dendritic cell IDO1. Heat map analysis of the proteomic data revealed an overall down-regulation of vaccinated DC functions, suggesting vaccine suppression of DC maturation. Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance. Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention of type 1 diabetes autoimmunity.

Ameliorative Effects of Testosterone Administration on Renal Redox Homeostasis in Naturally Aged Rats.

Testosterone biosynthesis gradually decreases with age. Impaired redox homeostasis-related oxidative damage in cellular macromolecules has a high risk for the development of renal insufficiency. Our aim was to study the effects of testosterone replacement therapy on redox homeostasis. We investigated various oxidative damage biomarkers in kidney. Experimental animals were separated into three groups-naturally aged rats, testosterone-administered naturally aged rats (single dose of 25 mg/kg testosterone enanthate), and their respective young controls. Our results showed that the testosterone-administered naturally aged group shared significant similarities with the young rats with respect to their redox status. In testosterone-administered naturally aged rats, kynurenine, protein carbonyl, advanced oxidation protein products, lipid peroxidation markers, and xanthine oxidase activities were significantly lower and Cu-Zn superoxide dismutase activities and testosterone levels were higher than naturally aged rats. In testosterone-administered naturally aged rats, catalase activities, ferric reducing anti-oxidant power, and testosterone levels were significantly lower and dityrosine, N-formyl kynurenine, protein carbonyl, and protein hydroperoxides were significantly higher than in young rats. On the other hand, in naturally aged rats, Cu-Zn superoxide dismutase, catalase activities, ferric reducing anti-oxidant power, and testosterone levels were lower and dityrosine, kynurenine, protein carbonyl, protein hydroperoxide, advanced oxidation protein products, lipid peroxidation markers, advanced glycation end products, and xanthine oxidase activities were higher than controls. Our results showed that a single dose of testosterone administration has a positive effect on the redox status of the aged kidney. Future studies are needed to clarify the exact molecular mechanism(s) involved in the action of testosterone in maintaining kidney redox homeostasis.

Unraveling the intramolecular cyclization mechanism of oxidized tryptophan in aqueous solution as a function of pH

The aqueous intramolecular cyclization of 3a-hydroperoxitryptophan, Trp-OOH, (an intermediate in the photodynamic treatment of cancer) is studied at the PCM-MP2/aug-cc-pVDZ//PCM-B3LYP/aug-cc-pVDZ computational level with and without explicit water molecules. The three-cycle product may evolve to the metabolite N-formyl kynurenine in living beings or can be a building block in the formation of indole alkaloids in organic synthesis. When the pH is close to the isoelectric point of tryptophan, we have found two cyclization mechanisms, one passing along zwitterion intermediates, I-route (beginning with a proton transfer from the ammonium to the N-indole atom) and the other involving neutral isomers, C-route (starting with the attack of N-amino to C2-indole). At this pH, the discrete-continuum model with six explicit water molecules predicts a Gibbs energy barrier of 14.6 kcal mol(-1) for the I-route and of 11.6 kcal mol(-1) for the C-route. It is possible to experimentally tune the operating mechanism since in acidic environments only the I-route is available (Gibbs energy barrier of 8.4 kcal mol(-1)) whereas in basic media only the C-route can operate (Gibbs energy barrier of 6.7 kcal mol(-1)). These data explain the trend of Trp-OOH to easily decompose under basification.

High-Throughput Fluorescence-Based Screening Assays for Tryptophan-Catabolizing Enzymes

Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the preclinical IDO1 inhibitor Compound 5l shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384- or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors.

P51 - Consumption of peptide-included and free tryptophan induced by peroxyl radicals: A kinetic study

It is well-known that tryptophan residues are efficiently oxidized by peroxyl radicals, generating kynurenine, and N-formyl kynurenine as well as hydroperoxide derivatives as products. In the present work we studied the kinetic of such reaction employing free and peptide-included tryptophan. Two azocompounds were used to produce peroxyl radicals: AAPH (2,2'-Azobis(2-methylpropionamidine) dihydrochloride) and ABCVA (4,4'-Azobis(4-cyanovaleric acid)), which generate cationic and anionic peroxyl radicals, respectively. Tryptophan consumption was assessed by fluorescence spectroscopy and the reactions were carried out in phosphate buffer (75mM, pH 7.4) at 45°C. Only a slight effect of the peroxyl radical charge was evidenced on the consumption of free tryptophan and the dipeptide Gly-Trp. Employing AAPH as peroxyl radical source, at low free tryptophan concentrations (1-10µM) near 0.3 mol of tryptophan were consumed per each mol of peroxyl radicals introduced into the system. However, at high free tryptophan concentrations (100µM–1mM) such stoichiometry increased in a tryptophan concentration-way. At 1mM three moles of tryptophan were consumed per mol of AAPH-derived peroxyl radicals, evidencing the presence of chain reactions. A similar behavior was observed when di and tri-peptides (Gly-Trp, Trp-Gly, Gly-Trp-Gly, Trp-Ala, Ala-Trp-Ala) were studied. Nonetheless, at low initial concentration (5µM), the initial consumption rate of tryptophan included in the peptides was two times higher than free tryptophan. In contrast, at high concentration (1mM) free and peptide-included tryptophan showed similar initial consumption rates. These results could be explained considering a disproportionation process of tryptophanyl radicals at low free tryptophan concentrations, a process that would be inhibited when tryptophan is included in peptides.

Characterization of the Degradation Products of a Color-Changed Monoclonal Antibody: Tryptophan-Derived Chromophores

We describe the characterization of degradation products responsible for color change in near UV-visible light-irradiated and heat-stressed monoclonal antibody (mAb) drug product in liquid formulation. The treated samples were characterized using reversed-phase HPLC and size-exclusion HPLC with absorption spectroscopy. Both methods showed color change was due to chromophores formed on the mAb but not associated with the formulation excipients in both light-irradiated and heat-stressed mAb samples. These chromophores were further located by a new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy. Mass spectrometry identified the major tryptophan oxidation products as kynurenine (Kyn), N-formylkynurenine (NFK), and hydroxytryptophan (OH-Trp). The absorption spectra showed that each of the tryptophan oxidation products exhibited a distinct absorption band above 280 nm shifted to the longer wavelengths in the order of OH-Trp < NFK < Kyn. The Kyn-containing peptide was detected by absorption at 420 nm. No new absorption bands were observed for either methionine or histidine oxidation products. This confirmed that tryptophan oxidation products, but not methionine and histidine oxidation products, were responsible for the color change. It is worth noting that a new oxidation product with the loss of hydrogen (2 Da mass decrease) for Trp-107 of the heavy chain was identified in the heat-stressed mAb sample. This oxidized tryptophan residue exhibited a distinct absorption band at the maximum absorbance wavelength 335 nm, which is responsible for the color change to yellow. This study showed that the new peptide mapping methodology with a combination of mass spectrometry and absorption spectroscopy is useful to identify tryptophan oxidation products as chromophores responsible for color change in stressed mAb drug product.

Effects of ozone and relative humidity on fluorescence spectra of octapeptide bioaerosol particles

The effects of ozone and relative humidity (RH) at common atmospheric levels on the properties of single octapeptide bioaerosol particles were studied using an improved rotating reaction chamber, an aerosol generator, an ultraviolet aerodynamic particle sizer (UVAPS), an improved single particle fluorescence spectrometer (SPFS), and equipments to generate, monitor and control the ozone and RH. Aerosol particles (mean diameter ~2μm) were generated from a slurry of octapeptide in phosphate buffered saline, injected into the rotating chamber, and kept airborne for hours. Bioaerosols were sampled from the chamber hourly for the measurements of particle-size distribution, concentration, total fluorescence excited at 355-nm, and single particle fluorescence spectra excited at 266-nm and 351-nm under different controlled RH (20%, 50%, or 80%) and ozone concentration (0 or 150ppb). The results show that: (1) Particle size, concentration, and the 263-nm-excited fluorescence intensity decrease at different rates under different combinations of the RH and ozone concentrations used. (2) The 263-nm-excited UV fluorescence (280–400nm) decreased more rapidly than the 263-nm-excited visible fluorescence (400–560nm), and decreased most rapidly when ozone is present and RH is high. (3) The UV fluorescence peak near 340nm slightly shifts to the shorter wavelength (blue-shift), consistent with a more rapid oxidation of tryptophan than tyrosine. (4) The 351/355-nm-excited fluorescence (430–580nm/380–700nm) increases when ozone is present, especially when the RH is high. (5) The 351/355-nm-excited fluorescence increase that occurs as the tryptophan emission in the UV decreases, and the observation that these changes occur more rapidly at higher RH with the present of ozone, are consistent with the oxidation of tryptophan by ozone and the conversion of the resulting ozonides to N-formyl kynurenine and kynurenine.

Kinetics and Mechanism of Oxidation of Tryptophan by Ferrate(VI)

Kinetics of the oxidation of tryptophan (Trp) and kynurenine (Kyn), precursors of nitrogenous disinfection byproducts (N-DBP), by ferrate(VI) (Fe(VI)O4(2-), Fe(VI)) were investigated over the acidic to basic pH range. The second-order rate constants decreased with increase in pH, which could be described by the speciation of Fe(VI) and Trp (or Kyn). The trend of pH dependence of rates for Trp (i.e., aromatic α-amino acid) differs from that for glycine (i.e., aliphatic α-amino acid). A nonlinear relationship between transformation of Trp and the added amount of Fe(VI) was found. This suggests that the formed intermediate oxidized products (OPs), identified by LC-PDA and LC-MS techniques, could possibly compete with Trp to react with Fe(VI). N-Formylkynurenine (NFK) at pH 7.0 and 4-hydroxyquinoline (4-OH Q) and kynurenic acid (Kyn-A) at pH 9.0 were the major OPs. Tryptophan radical formation during the reaction was confirmed by the rapid-freeze quench EPR experiments. The oxygen atom transfer from Fe(VI) to NFK was demonstrated by reacting Fe(18)O4(2-) ion with Trp. A proposed mechanism explains the identified OPs at both neutral and alkaline pH. Kinetics and OPs by Fe(VI) were compared with other oxidants (chlorine, ClO2(•), O3, and (•)OH).