Tryptophan In Solution Research Articles

Irradiation of Cells with Ultraviolet-A (320-400 nm) in the Presence of Cell Culture Medium Elicits Biological Effects Due to Extracellular Generation of Hydrogen Peroxide

Biological effects of ultraviolet A (UVA) irradiation have been ascribed to the photochemical generation of singlet oxygen. Not all effects described in the literature, however, are explicable solely by the generation of singlet oxygen, but rather resemble effects elicited by hydrogen peroxide (H 2 O 2 ). Here, we show that when cells are kept in cell culture media during exposure to UVA, stress kinases, including ERK 1 and ERK 2 as well as Akt (protein kinase B), are activated, whereas there is no or only minor activation when cells are kept in phosphate-buffered saline during irradiation. Indeed, the exposure of cell culture media to UVA (30 J/cm 2 ) results in the generation of significant amounts of H 2 O 2 , with concentrations of about 100 w M. H 2 O 2 concentrations are at least three-fold higher in HEPES-buffered culture media after UVA irradiation. From experiments with solutions of riboflavin, tryptophan or HEPES, as well as combinations thereof, it is concluded that riboflavin mediates the photooxidation of either tryptophan or HEPES, resulting in the generation of H 2 O 2 . Thus, if signaling effects of UVA radiation are to be investigated in cell culture systems, riboflavin and HEPES/tryptophan should be avoided during irradiation because of artificial H 2 O 2 generation. It should be taken into account, however, that in vivo tryptophan and riboflavin might play an important role in the generation of reactive oxygen species by UVA as both substances are abundant in living tissues.

Suitability of tryptophan radiation products as markers for the detection of gamma-irradiated protein rich food.

During gamma-irradiation (5 kGy) of aqueous tryptophan (Trp) solutions small amounts of 5-, 6-, and 7-hydroxytryptophan (OH-Trp) (0.04-0.08 Mol-%) are formed. Protein rich food like shrimps contain reasonable amounts of non-protein bound Trp (100 mg/kg). In order to detect the treatment of shrimps with gamma-irradiation a method for the determination of OH-Trp in gamma-irradiated shrimps was developed. After homogenization, squeezing of shrimp samples and protein precipitation, a two-step-SPE-clean up was performed using a C18-cartridge and a propylsulfonic acid cation-exchange SPE followed by HPLC analysis with electrochemical detection (750 mV). Results showed that 5-OH-Trp contents in shrimp samples increased with applied doses up to 3 kGy and then decreased with higher doses. Other OH-Trp isomers were not detectable in the irradiated shrimps. Similarly no formation of 4-, 6-, and 7-OH-Trp was detected in model solutions containing the same amino acid composition as in shrimps. This indicates a suppression of the reaction of OH-radicals with Trp by the 300 fold molar excess of other amino acids acting as well as radical scavengers. Therefore, non-physiological OH-Trp isomers formed from free Trp are not suitable as markers for the detection of gamma-irradiated protein-rich foodstuff.

Laser-induced fluorescence of estrogens

Spectra of the laser-induced fluorescence of estrogens—estradiol, estriol, and estrone—were measured under excitation by the fourth harmonic (λ=266 nm) of a Nd:YAG laser. Quantum yields of the fluorescence were evaluated by the relative technique to be 0.107 for estradiol, 0.116 for estriol, and 0.0052 for estrone. The water solution of tryptophan is used as the standard.

Autoxidation of tryptophan in aqueous solutions

Autoxidation of tryptophan was investigated in aqueous solutions by the gamma radiolytic technique. The oxygen uptake and formation of peroxide materials was followed as a function of pH, dose rate and concentration of tryptophan. The results obtained indicate that TrpH(OH)OO. radicals react with tryptophan by adduct formation thus propagating autoxidation. The chain propagation length (CPL) for a 2x10-2 mol dm-3 tryptophan solution at pH 9.5 and a dose rate 0.01Gy s-1 was estimated to be ? 5.8, which shows that the autoxidation of tryptophan is a short chain reaction.

Nanosecond UV laser photoionization of aqueous tryptophan: temperature dependence of quantum yield, mechanism, and kinetics of hydrated electron decay

The 266 nm photoionization of aqueous tryptophan (Trp) solution at natural pH has been studied by laser flash photolysis using 3–18 mJ, 7 ns, 266 nm pulses. The transient absorption method was employed to detect hydrated electrons. The photoionization mechanism is a combination of mono and biphotonic processes. Results from measurements of the power dependence of photoionization and of triplet (T 1) state production suggest that both processes involve the same one-photon and two-photon singlet intermediate state. The hydrated electron yield increases with temperature, and this can be attributed primarily to the monophotonic photoionization component. Hydrated electrons decay by a non-exponential rate law that is consistent with a decay mechanism involving their bimolecular scavenging by tryptophan radical cations with a rate constant of (7.2±0.6)×10 10 M −1 s −1.

Composite Ferric Oxyhydroxide-Containing Phases Formed in Neutral Aqueous Solutions of Tryptophan and Indole-3-Acetic Acid

Mossbauer, FTIR and XRD analyses showed that in aqueous medium in air in the presence of L-tryptophan (Trp) or indole-3-acetic acid (IAA) the ambient-temperature ageing of the precipitates formed from ferrous sulphate at pH∼7 gave composite phases with varying proportions of γ-FeOOH (a dominating crystalline phase), α-FeOOH (both fine-grained, showing superparamagnetic behaviour at 298 K, and relatively better crystallized) and amorphous ferric hydroxide. The experimental data suggested a competition for adsorption sites at the oxyhydroxide surface in the suspension during phase transformations, as well as the transformation of γ-FeOOH (and/or amorphous ferric hydroxide) to α-FeOOH via the dissolution-reprecipitation mechanism. The formation of certain ferric oxyhydroxide phases in the presence of Trp and IAA — released e.g., in the course of bacterial and plant metabolism — can contribute to the regulation of soil mineral composition.

Hydrogen abstraction by triplet flavins. I: time-resolved multi-channel absorption spectra of flash-irradiated riboflavin solutions in water

Multichannel detection with μs time resolution has been used for recording flash-induced changes in the absorbance of deaerated solutions of riboflavin in phosphate buffer at pH 6.8. By comparing the spectra of solutions containing, in addition to riboflavin (F v), different substrates (ascorbic acid, EDTA, indole acetic acid, tryptophan), and by recording the absorption spectrum of the neutral tryptophyl radical (produced by flash photolysis of aqueous solutions of tryptophan), the absorption spectrum and the extinction coefficient of F vH are ascertained. Under the experimental conditions pertaining to the present investigation (solute concentration (7.5±2.5)×10 −5 M, ca. 10% conversion into triplets), quenching of F v † (a riboflavin triplet) by F v 0 (an unexcited riboflavin molecule) was found to be insignificant; most triplets appear to decay through triplet–triplet annihilation, the loss of two triplets being accompanied by the gain of one neutral semiquinone radical (F vH ). It is proposed that (i) triplets deactivate mainly through triplet–triplet annihilation, (ii) the annihilation event leads to the formation of an ion pair, F v †+F v †→F v ++F v −, (iii) the anion rapidly converts to F vH , and (iv) the cation ejects a proton and splits into two neutral products, a molecule whose ground-state absorption spectrum resembles that of F v 0, and an odd-electronic species containing the remainder of the aliphatic side chain.

UV-induced CYP1A1 gene expression in human cells is mediated by tryptophan

Induction of cytochrome P-4501A1 (CYP1A1) activity by UV has been observed earlier in animal studies via a mechanism that has not yet been resolved. Our previous data have indicated that formylated indolocarbazoles which are formed by UV irradiation of tryptophan solutions are very potent Ah-receptor agonists. To evaluate the effect of UV light on cytochrome P4501A1 gene expression, we studied the induction of CYP1A1 mRNA by UV irradiation of cultured human keratinocytes (HaCaT cell line), primary human blood lymphocytes and mouse Hepa-1 cells. The cells were exposed to UV light delivered by a bank of 6 Philips TL20/12RS sun lamps emitting primarily in the UVB range in the absence and presence of tryptophan. A semiquantitative reverse transcriptase-linked polymerase chain reaction (RT-PCR) was used for analysis of gene expression in the treated cells. The results show that the CYP1A1 mRNA level induced by UV in the presence of tryptophan was higher than that induced by UV alone in both HaCaT cells and lymphocytes after 3 h of incubation post-UV irradiation. To find out if the induction by UV light is caused by the formation of an Ah receptor ligand, Hepa-1 wild-type and Ah receptor deficient c12 cell lines were applied. Wild-type (wt) cells were inducible either by the tryptophan photoproduct 6-formylindolo[3,2- b]carbazole (FICZ) or by UV-irradiation but very low or undetectable levels were observed in the c12 cells. This shows that the induction of gene expression by FICZ and UV is Ah receptor dependent. Together, these results indicate that UV-induced CYP1A1 gene expression in mammalian cells is mediated by an Ah receptor ligand formed from tryptophan. Thus, the photoproducts of tryptophan are suggested to be mediators of light via binding to the Ah receptor and as such also could have a role in light-regulated biological rhythms.

Amphipathic alpha-helix bundle organization of lipid-free chicken apolipoprotein A-I.

Apolipoprotein A-I (apoA-I), the major protein component of plasma high-density lipoprotein (HDL), exists in alternate lipid-free and lipid-bound states. Among various species, chicken apoA-I possesses unique structural properties: it is a monomer in the lipid-free state and it is virtually the sole protein component of HDL. Near-UV circular dichroism (CD) spectroscopic studies provide evidence that chicken apoA-I undergoes a major conformational change upon binding to lipid, while far-UV CD data indicate its overall alpha-helix content is maintained during this transition. The fluorescence emission wavelength maximum (excitation 295 nm) of the tryptophans in apoA-I (W74 and W107) displayed a marked blue shift in both the lipid-free (331 nm) and HDL-bound (329 nm) states, compared to free tryptophan in solution. The effect of aqueous quenchers on tryptophan fluorescence was determined in lipid-free, dimyristoylphosphatidylcholine (DMPC)- and HDL-bound states. The most effective quencher in the lipid-free and HDL-bound states was acrylamide, giving rise to Ksv values of 1.6 +/- 0.1 and 1.2 +/- 0.1 M-1, respectively. Together, these data suggest that a hydrophobic environment around the two tryptophan residues (W74 and W107) is maintained in alternate conformations of the protein. To further probe the molecular organization of lipid-free apoA-I, its effect on the fluorescence properties of 8-anilino-1-naphthalenesulfonic acid (ANS) was determined. Human and chicken apoA-I induced a similar increase in ANS fluorescence quantum yield, in keeping with the hypothesis that these proteins adopt a similar global fold in the absence of lipid. When considered with near- and far-UV CD experiments, the data support a model in which lipid-free chicken apoA-I is organized as an amphipathic alpha-helix bundle. In other studies, lipid-soluble quenchers, 5-, 7-, 10-, and 12-DOXYL stearic acid (DSA), were employed to investigate the depth of penetration of apoA-I into the surface monolayer of spherical HDL particles. 5-DSA was the most effective quencher, suggesting that apoA-I tryptophan residues localize near the surface monolayer, providing a structural rationale for the reversibility of apoA-I-lipoprotein particle interactions.

Off-flavor compounds in wine and other food products formed by enzymatical, physical, and chemical degradation of tryptophan and its metabolites.

Tryptophan (TRP) and its metabolites are considered as potential precursors of 2-aminoacetophenone (AAP) in different food products causing different off-flavors. AAP is also responsible for the "untypical aging flavor (UTA)" in wine, developing a floor polish-like flavor in white wines within a few months of storage. In this study the formation of AAP was elucidated by GC-MS analysis of volatile components in model systems, grape musts and wines, spiked with TRP and different TRP metabolites like indole-3-acetic acid (IAA) and sulfite. In sulfurized wines and model solutions which were stored at different temperatures (20 degrees C, 45 degrees C) formylaminoacetophenone (FAP) and AAP were formed mainly from IAA with formation rates up to 20 mole%. Minor formation rates of AAP (< 1 mole%) were found in sulfurized solutions of TRP, indole-3-lactic acid, and indole-3-pyruvic acid. The results showed that the formation of AAP in wine can be referred to an oxidative degradation of IAA by superoxide- and hydroxyl-radicals, which can be formed in wine after the sulfuration by cooxidation of sulfite to sulfate. After decarboxylation, pyrrole oxidation, and ring cleavage, FAP was the main volatile compound of the nonenzymatic degradation of IAA by sulfite which was quantitatively hydrolyzed to AAP. The formation of AAP and FAP was significantly lower in white wines than in ethanolic solutions spiked with IAA. However AAP formation rates of up to 5 mole% were still enough for an UTA. Due to the fact that the AAP- and UTA-formation by cooxidation of sulfite and IAA was completely blocked in red wines, it could be deduced that polyphenolic compounds, typical for red wines, have a scavenger effect on the radical oxidation of sulfite. Possibilities for an inhibition of the IAA degradation during winemaking to avoid the UTA in white wines by addition of radical scavengers like grape marc or ascorbic acid are discussed.

Binding of dl-tryptophan to BSA adsorbed in multilayers by polymer chains grafted onto a porous hollow-fiber membrane in a permeation mode

An anion-exchange-group-containing porous membrane in the form of a hollow fiber was prepared to immobilize bovine serum albumin (BSA) as a chiral selector. First, an epoxy-group-containing polymer chain was grafted onto the pore surface of the polyethylene porous hollow-fiber membrane pre-irradiated with an electron beam. Second, the epoxy group was converted to diethylamino and 2-hydroxyethylamino groups. Third, a BSA solution was forced to permeate through the pores of the membrane. As a result, 190 mg BSA per gram of membrane, which amounted to a degree of multilayer binding of about four, were immobilized. Subsequently, a racemic solution of tryptophan (0.02 mM) was forced to permeate through the BSA-multilayered porous membrane at a flow rate ranging from 10 to 80 ml/h. A two-stage stepwise concentration change of tryptophan in the effluent was observed due to independent chiral recognition of d- and l-tryptophan by BSA adsorbed in multilayers within the graft chains.

Influence of some aromatic amino acids on the swelling behavior of acrylamide/maleic acid hydrogel

Influence of some aromatic amino acids (histidine, phenylalanine and tryptophan) on the swelling behavior of acrylamide/maleic acid hydrogel (AAm/MA) prepared by γ-radiation was investigated. Swelling tests of AAm/MA hydrogel were made in buffer solutions and amino acid solutions at various pH at 37°C. The pH values are ionization of α-carboxyl groups (pK'1), α-amino groups (pK'2) and, isoelectric points (pI) of amino acids. The swelling of AAm/MA hydrogel increased when pH values of solutions were increased. The value of equilibrium swelling of AAm/MA hydrogel was 1035% at pH 10 buffer, while it was 880% at pH 2 buffer. The values of equilibrium swelling of AAm/MA hydrogel in phenylalanine, tryptophan and histidine solutions varied among 1130–1245% at pH 10, while they were among 790–975% at pH 2. The rate constant of swelling, diffusional exponent, network parameter and, diffusion and intrinsic diffusion coefficient were calculated by swelling kinetics. Diffusion of the penetrants into the hydrogel was found to be non-Fickian character. The diffusion coefficients of the hydrogel varied between 3.33×10−6– 7.71×10−6 cm 2s−1, while the intrinsic diffusion coefficients waried between 4.03×10−6– 8.48×10−6 cm 2s−1.

Photodegradation of tryptophan in wool

The amino acid tryptophan is degraded upon light exposure or during acid and oxidative treatments. After irradiation of tryptophan solutions, the photodegradation products N′‐formylkynurenine, kynurenine, tryptamine, and oxindolylalanine were identified by means of HPLC. These photoproducts of tryptophan contribute to the photoyellowing of wool. Oxygen plays a major role in tryptophan photodegradation. An attack of oxygen at the indole residue initiates the photodegradation reaction. The main photodegradation of tryptophan in proteins (e. g. in polytryptophan or in wool) is the cleavage of the indole ring and the formation of kynurenines. In irradiated wool, only N′‐formylkynurenine was detected. N′‐Formylkynurenine is known to be a photosensitiser.

Energetics of photoinduced electron transfer in the indole.O2 cluster in gas phase: possible consequences for photoexcited tryptophan in solution.

A direct process for an activationless electron transfer from photoexcited tryptophan to molecular oxygen is proposed. By photodetachment of mass-selected indole+.O2- clusters in gas phase a neutral indole+.O2- charge-separated exciplex state is found at 2.25 +/- 0.2 eV above the neutral ground state. By theory also, the existence of an excited charge separated state at 3.05 +/- 0.2 eV is postulated. In gas phase both charge-separated cluster states are energetically below the first singlet states 1Lb and 1La and the lower even below the first triplet state T1 of indole. In gas-phase clusters these energetics imply a very efficient quenching of photoexcited indole by fast electron transfer to oxygen. We discuss a similar mechanism for tryptophan.O2 in aqueous environment and find it without activation barrier and presumably extremely fast. In the collisional tryptophan*-O2 complex the efficiency and the time scale of the charge transfer process should be mostly solvent independent. In polar solvent a complete charge separation and free superoxide formation are expected. We correlate this model with previous fluorescence and phosphorescence quenching data of excited tryptophan by O2 and propose electron transfer to be the relevant process.

Determination of γ-radiation induced proudcts in aqueous solutions of tryptophan and synthesis of 4-, 6- and 7-hydroxytryptophan

A HPLC method with UV and fluorescence detection was developed to determine γ-radiation induced products in aqueous solutions of tryptophan. Therefore 4-, 6- and 7-hydroxytryptophan were synthesized in purities which allow their use as reference substances for analytical studies. Oxindolylalanine, N-formylkynurenine, 4-, 5, 6- and 7-hydroxytryptophan were determined as the main radiation induced products.

Classical and Quantum Simulations of Tryptophan in Solution

We use path integral molecular dynamics to study the effect of nuclear tunneling on the structure, fluctuations, and energetics of tryptophan in water. Tryptophan is important as a spectroscopic probe; it can also serve as a miniature model of a protein, possessing charged, polar, hydrophobic, and aromatic groups, hard and soft degrees of freedom, and conformational heterogeneity around its dihedral angles. At room temperature, nuclear tunneling increases the fluctuations of hard internal degrees of freedom by a factor of approximately 2; dihedral fluctuations are not affected. Water structure is weakened, and the solvation energies of the amino, carboxyl, and indole groups change by 1−2 kcal/mol. Solvation energies and free energies of the 1La and 1Lb excited states were analyzed, using semiempirical and ab initio atomic charges, and a free energy perturbation approach. The dielectric relaxation free energy, from Marcus theory, is 1/2 of the dielectric relaxation energy, for a wide class of systems; this...

Reactivity of singlet oxygen toward proteins: the effect of structure in basic pancreatic trypsin inhibitor and in ribonuclease A.

The reactions of singlet oxygen, 1O2, with large peptides have been described previously. It was found that even in these systems, which in their native form are generally not supposed to possess a stable structure in solution, the polypeptide does impede the access of 1O2 to the amino acids that react readily with 1O2. Here we describe the 1O2 reaction with two proteins of well-defined structure. The quenching of 1O2 by bovine pancreatic trypsin inhibitor (BPTI) and by ribonuclease A (RNase A) was compared to that of a solution at the same concentration as those of its constituent amino acids that react readily with 1O2. The proteins were studied in their native form, when partly denatured by splitting their S-S bonds and when fully denatured. It was found that while in the native form the quenching rate constant was seven times lower in BPTI (2.2 vs 15.2 x 10(7) M-1 s-1) and three times lower in RNase A (11.0 vs 32 x 10(7) M-1 s-1) than in a mixture of its constituent amino acid residues, it increased upon denaturation reaching in the fully denatured state the value of the corresponding amino acid mixture. More striking is the effect of the protein structure when comparing the fraction of the encounters between 1O2 and protein, which cause damage to the protein, as reflected in the decrease of its biological activity. This decrease is assumed to be due to the chemical (oxidative) reactions of 1O2 in the protein. In the exceptionally stable BPTI the fraction of such encounters was 0.05 and in RNase A it was 0.2, whereas for the amino acid tryptophan in solution, 0.7 of the collisions with 1O2 led to a chemical reaction.

The optical resolution of amino acids by plasma polymerized terpene membranes

Four kinds of terpenes, i.e. l-menthol, S-(−)- β-citronellol, [(1 S)-endo]-borneol, and R-limonene, were fixed on a cellulose acetate membrane by plasma polymerization method at various periods of plasma irradiation. The fractional fixations of terpenes increased with increasing time of plasma irradiation. The optical resolutions of racemic tryptophan ( dl-Trp) solutions were performed by the dialysis and the ultrafiltration using the plasma polymerized membranes. All the membranes were permeable with respect to the d-isomer tryptophan in in preference to the l-isomer. The larger value of separation factor was obtained at lower volume flux. The maximum value of separation factor obtained for dl-Trp was 9.5 at volume flux J v=4.7 × 10 −8 m s −1. The solute permeabilities and apparent viscous parameters were evaluated from the dialysis and ultrafiltration data, respectively. A linear relation between the solute permeability and the ratio of apparent viscous parameters was obtained. It is indicated that the strength of chemical interaction between the amino acid solute and the terpene fixed in the membrane has a high influence on the solute flow inside the membrane pore.

Development of monoclonal antibodies against a riboflavin-tryptophan photoinduced adduct: reactivity to eye lens proteins.

We describe here the development of monoclonal antibodies to the hapten tryptophan-riboflavin, generated by irradiation of a solution of bovine serum albumin in the presence of riboflavin. The specificity of the three obtained monoclonal antibodies, named 1E6, 5H5, 5A8 all belonging to the IgG1 isotype, was assessed by a competitive enzyme-linked immunosorbent assay in the presence of an increasing concentration of the tryptophan-riboflavin adduct, obtained from an irradiated riboflavin-sensitized tryptophan solution. It was demonstrated that the tryptophan-riboflavin antibodies react with the soluble proteins of the eye lens; this reaction was more intense in the old rat lenses as compared to the young ones, and a maximum binding of the antibodies was obtained with the soluble protein fraction from the human cataractous lens. By indirect immunofluorescence, a reactivity associated with the protein matrix, localized in the lens central zone, was observed. In the peripheral zone of the lens, where the younger cells are found, a marked immunofluorescent emission was observed on structures preferentially localized in the nuclei.

Emission spectra and decay kinetics of pulse irradiated polycrystalline tryptophans

Pulse radiolysis of solid, polycrystalline, d, l and dl-tryptophan has been performed with 13 MeV electron pulses of 2.5 or 5.5 μs duration. In addition to the expected short-lived UV emission identical to the emission frequently described in irradiated aqueous solutions of tryptophan, a long-lived ( T 1 2 = 2.2 μ s ) emission in the visible spectral range (maximum at 580 nm) has been recorded. The long-lived emission in visible range is supposed to originate from species formed in at least two ionization/excitation spurs located in one molecule. This phenomenon cannot occur in solution, but is unique to the solid state in which tryptophan is the main component interacting with radiation. The long-lived emission is efficient in the case of d and l-enantiomers but is very low for dl-racemate. The results are presumed to depend on differences of structures of enantiomers on the one hand and on the dl-racemate structure on the other. The primary excitation effect of a molecule does not depend on the arrangement of molecules in the crystal. However, in more tightly packed racemate and different geometry of indole moieties arrangement, the phenomenon of radiationless deactivation occurs.