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Purpose: To evaluate the effect of excipients, including sugars and amino acids, on photo-degradation reactions in pharmaceutical buffers induced by near UV and visible light., Methods: Solutions of citrate or acetate buffers, containing 1 or 50 μM Fe 3+ , the model peptides methionine enkephalin (MEn), leucine enkephalin (LEn) or proctolin peptide (ProP), in the presence of commonly used amino acids or sugars, were photo-irradiated with near UV or visible light. The oxidation products were analyzed by reverse-phase HPLC and HPLC-MS/MS., Results: The sugars mannitol, sucrose and trehalose, and the amino acids Arg, Lys, and His significantly promote the oxidation of peptide Met to peptide Met sulfoxide. These excipients do not increase the yields of hydrogen peroxide, suggesting that other oxidants such as peroxyl radicals are responsible for the oxidation of peptide Met. The addition of free Met reduces the oxidation of peptide Met, but, in citrate buffer, causes the addition of Met oxidation products to Tyr residues of the target peptides., Conclusions: Commonly used excipients enhance the light-induced oxidation of amino acids in model peptides.

Inteins change the structure and function of their host protein in a unique way and the Gp41-1 split intein is the fastest protein trans-splicing intein known to date. To design a photo-activatable variant, we have incorporated ortho-nitrobenzyl-tyrosine (ONBY) at the position of a structurally conserved phenylalanine in the Gp41-1-N fragment. Using irradiation at 365 nm, the splicing reaction was triggered with virtually unchanged rates. The partial cellular reduction of the nitro group in ONBY, previously observed during bacterial protein expression for several photo-caged amino acids, was overcome by periplasmatic expression and by using an E. coli K12(DE3) strain instead of BL21(DE3). Together, our findings provide new tools for the artificial photo-control of proteins.

In this study, the combined toxicity of UV/chlorinated products on Escherichia coli (E. coli) was investigated when ibuprofen (IBP) and tyrosine (Tyr) were used as two precursors. The median-effect equation and combined index (CI)-isobologram equation were used to evaluate the combined toxicity of UV/chlorinated products. Results revealed that the UV/chlorinated products originated from binary Tyr and IBP showed a synergism in toxicity on Escherichia coli at low concentration level while it turned into a clear antagonism effect above a f a value of 0.2 in the toxicity trial. The combined toxic effects on E. coli were determined by both the potential toxicity mode of specific disinfection byproducts (DBPs) and the complicated interaction caused by Tyr and IBP. The addition of IBP decreased the yield of N-DBPs generated from Tyr, which dominated the effect of combined toxicity. Even though the antagonism predominated in toxicity effect on E. coli, the synergistic toxicity at low dose levels should be getting attention, which was more close to the natural concentration of N-DBPs in waters., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The self-assembly of tyrosyl bolaamphiphiles is exploited to create a colloidal protein-like host matrix, upon which sacrificial electron-donor molecules associate to create a photosystem II (PSII) mimetic electron-relay system. This system harnesses the tyrosine phenol groups abundant on the surface of the assemblies to mediate photoinduced intermolecular electron transfer. Compared with the l-tyrosine molecules, the tyrosyl bolaamphiphile assembly facilitates electron transfer from the sacrificial electron donor to the oxidized photosensitizer. The enhanced electron relay is likely to be driven by the host function of the assembly associated with the sacrificial electron donor and by the suppression of the oxidative cross-linking of phenoxyl radicals. The tyrosyl bolaamphiphile assembly is advantageous in the construction of a PSII mimetic system with a protein-like nature and displaying biochemical functions., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We genetically encoded three new caged tyrosine analogues with improved photochemical properties by using an engineered pyrrolysyl-tRNA synthetase/tRNA CUA pair in bacterial and mammalian cells. We applied the new tyrosine analogues to the photoregulation of firefly luciferase by caging its key tyrosine residue, Tyr340, and observed excellent off-to-on light switching. This reporter was then used to evaluate the activation rates of the different light-removable protecting groups in live cells. We identified the nitropiperonyl caging group as an excellent compromise between incorporation efficiency and photoactivation properties. To demonstrate applicability of the new caged tyrosines, an important proteolytic enzyme, tobacco etch virus (TEV) protease, was engineered for optical control. The ability to incorporate differently caged tyrosine analogues into proteins in live cells further expands the unnatural amino acid and optogenetic toolbox., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Blue light using flavin adenine dinucleotide (BLUF) proteins are essential for the light regulation of a variety of physiologically important processes and serve as a prototype for photoinduced proton-coupled electron transfer (PCET). Free-energy simulations elucidate the active site conformations in the AppA (activation of photopigment and puc expression) BLUF domain before and following photoexcitation. The free-energy profile for interconversion between conformations with either Trp104 or Met106 closer to the flavin, denoted Trp in /Met out and Trp out /Met in , reveals that both conformations are sampled on the ground state, with the former thermodynamically favorable by ∼3 kcal/mol. These results are consistent with the experimental observation of both conformations. To analyze the proton relay from Tyr21 to the flavin via Gln63, the free-energy profiles for Gln63 rotation were calculated on the ground state, the locally excited state of the flavin, and the charge-transfer state associated with electron transfer from Tyr21 to the flavin. For the Trp in /Met out conformation, the hydrogen-bonding pattern conducive to the proton relay is not thermodynamically favorable on the ground state but becomes more favorable, corresponding to approximately half of the configurations sampled, on the locally excited state. The calculated energy gaps between the locally excited and charge-transfer states suggest that electron transfer from Tyr21 to the flavin is more facile for configurations conducive to proton transfer. When the active site conformation is not conducive to PCET from Tyr21, Trp104 can directly compete with Tyr21 for electron transfer to the flavin through a nonproductive pathway, impeding the signaling efficiency.

Human serum albumin (HSA) is the most abundant protein in the circulatory system. Oxidized albumin was identified in the skin of patients suffering from vitiligo, a depigmentation disorder in which the protection against ultraviolet (UV) radiation fails because of the lack of melanin. Oxidized pterins, efficient photosensitizers under UV-A irradiation, accumulate in the skin affected by vitiligo. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to induce structural and chemical changes in HSA under UV-A irradiation. Our results showed that Ptr is able to photoinduce oxidation of the protein in at least two amino acid residues: tryptophan (Trp) and tyrosine (Tyr). HSA undergoes oligomerization, yielding protein structures whose molecular weight increases with irradiation time. The protein cross-linking, due to the formation of dimers of Tyr, does not significantly affect the secondary and tertiary structures of HSA. Trp is consumed in the photosensitized process, and N-formylkynurenine was identified as one of its oxidation products. The photosensitization of HSA takes place via a purely dynamic process, which involves the triplet excited state of Ptr. The results presented in this work suggest that protein photodamage mediated by endogenous photosensitizers can significantly contribute to the harmful effects of UV-A radiation on the human skin.

This paper reports a novel nanosensor for tyrosine based on photoinduced electron-transfer (PET) between NaYF4:Yb, Tm upconversion nanoparticles (UCNPs) and melanin-like polymers. Melanin-like films were obtained from catalytic oxidation of tyrosine by tyrosinase, and deposited on the surface of UCNPs, and then quenched the fluorescence of UCNPs. Under the optimized conditions, the fluorescence quenching of UCNPs showed a good linear response to tyrosine concentration in the range of 0.8-100 μΜ with a detection limit of 1.1 μΜ. Meanwhile, it showed good sensitivity, stability and has been successfully applied to the detection of tyrosine in human serum., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Ultraviolet radiation (UVR) exposure is a major risk factor for age-related cataract, a protein-aggregation disease of the human lens often involving the major proteins of the lens, the crystallins. γD-Crystallin (HγD-Crys) is abundant in the nucleus of the human lens, and its folding and aggregation have been extensively studied. Previous work showed that HγD-Crys photoaggregates in vitro upon exposure to UVA/UVB light and that its conserved tryptophans are not required for aggregation. Surprisingly, the tryptophan residues play a photoprotective role because of a distinctive energy-transfer mechanism. HγD-Crys also contains 14 tyrosine residues, 12 of which are organized as six pairs. We investigated the role of the tyrosines of HγD-Crys by replacing pairs with alanines and monitoring photoaggregation using light scattering and SDS-PAGE. Mutating both tyrosines in the Y16/Y28 pair to alanine slowed the formation of light-scattering aggregates. Further mutant studies implicated Y16 as important for photoaggregation. Mass spectrometry revealed that C18, in contact with Y16, is heavily oxidized during UVR exposure. Analysis of multiple mutant proteins by mass spectrometry suggested that Y16 and C18 likely participate in the same photochemical process. The data suggest an initial photoaggregation pathway for HγD-Crys in which excited-state Y16 interacts with C18, initiating radical polymerization.

Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 Å, which occurs by a multistep proton-coupled electron-transfer mechanism. Using a photooxidant-labeled β2 subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α2:β2 complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface, Y356F(β) and Y731F(α), this oxidation is identified to be localized on Y356. The rate of Y356 oxidation depends on the presence of Y731 across the interface. This observation supports the proposal that unidirectional PCET across the Y356(β)-Y731(α)-Y730(α) triad is crucial to radical transport in RNR.

Free radicals and other oxidation products were characterized on α-lactalbumin with electron spin resonance (ESR), immuno-spin trapping, and mass spectrometry (MS) after riboflavin-mediated oxidation. Radicals were detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in immuno-spin trapping with both enzyme-linked immunosorbent assay (ELISA) and Western blotting and further characterized with mass spectrometry. A DMPO-trapped radical was identified at His68 and another at one of the tyrosine residues, Tyr50 or Tyr36, respectively, generated by a type II or I mechanism. Not all tyrosyl radicals were trapped, as the secondary oxidation product, 3,4-dihydroxyphenylalanine (DOPA), was detected by mass spectrometry at Tyr18 and Tyr50. A further oxidation of DOPA resulted in the DOPA o-semiquinone radical, which was characterized by ESR. Both surface exposure and the neighboring residues in the local environment of the tertiary structure of α-lactalbumin seem to play a role in the generation of DMPO trapped radicals and secondary oxidation products.

Age-onset cataracts are believed to be expedited by the accumulation of UV-damaged human γD-crystallins in the eye lens. Here we show with molecular dynamics simulations that the stability of γD-crystallin is greatly reduced by the conversion of tryptophan to kynurenine due to UV-radiation, consistent with previous experimental evidences. Furthermore, our atomic-detailed results reveal that kynurenine attracts more waters and other polar sidechains due to its additional amino and carbonyl groups on the damaged tryptophan sidechain, thus breaching the integrity of nearby dry center regions formed by the two Greek key motifs in each domain. The damaged tryptophan residues cause large fluctuations in the Tyr-Trp-Tyr sandwich-like hydrophobic clusters, which in turn break crucial hydrogen-bonds bridging two β-strands in the Greek key motifs at the "tyrosine corner". Our findings may provide new insights for understanding of the molecular mechanism of the initial stages of UV-induced cataractogenesis.

Background: Photoageing of skin is thought to be caused by protein denaturation, which can be induced by ultraviolet radiation. Previous studies have also reported that inflammation is related to protein denaturation; however, the influence of inflammation on skin ageing has not been explored in detail., Aim: To investigate the possible connection between inflammation and protein denaturation, which might lead to skin ageing, we focused on halogenated tyrosine as a denatured substance produced during the inflammation process., Methods: We measured halogenated tyrosine in aged human skin. Inflammatory cells and halogenated tyrosine were detected by immunohistochemistry using antibodies to mast-cell tryptase, neutrophilic myeloperoxidase and halogenated tyrosine. Finally, using elastic van Gieson (EVG) staining, we investigated whether the sites of halogenated tyrosine coincided with the sites at which proteins were denatured., Results: Immunohistochemical analysis indicated that both inflammatory cells and halogenated tyrosines increased with ageing in both photoexposed and photoprotected skin. EVG staining confirmed that the localization of halogenated tyrosine was close to the sites at which protein was denatured., Conclusions: Our investigations indicate a possible connection between skin ageing and inflammation, suggesting that halogenated tyrosine could be a useful marker of ageing skin., (© The Author(s). CED © 2012 British Association of Dermatologists.)

Background: Melanocytes are producing melanin after UV irradiation as a defense mechanism. However, UV-induced damage is involved in melanoma initiation, depending on skin phototype. Melanocytes seem to be extremely susceptible to free radicals. Their main enzymatic antioxidants are superoxide dismutase and catalase., Aim: To study how melanin synthesis modulates the activity of the oxidative stress defense enzymes and cell proliferation after UV induced cell damage., Methods: Normal human melanocyte cultures from fair skin individuals were exposed to high levels of L-tyrosine and irradiated, with 20, 30, 40 mJ/cm2 UVA, and respective UVB. Proliferation was measured using a MTS assay; viability was assessed by trypan blue exclusion dye method. Spectrophotometrical methods were used to determine total melanin content, the enzymatic activity of tyrosinase, superoxide dismutase and catalase., Results: Tyrosine had a negative effect on proliferation, enhanced with time elapsed. Overall, UV irradiation decreased proliferation. UVA increased proliferation relative to UVB in the cultures exposed for a longer time to high (2 mM) tyrosine concentration. There were no proliferation differences between UVA and UVB irradiation in lower tyrosine concentration exposed melanocytes. Both, UV irradiation and tyrosine increased melanogenesis. Exposure of the melanocytes to increased levels of tyrosine in medium (0.5 mM and 1 mM) and UV irradiation enhanced the activity of superoxide dismutase and catalase. The enzymes showed a high activity rate in melanocytes while exposed for a short time to 2 mM tyrosine, but their activity was dramatically decreased with longer tyrosine exposure and UV irradiation., Conclusion: Our data indicate that in low phototype melanocytes, melanogenesis, either following UV irradiation, or tyrosine exposure, especially in high concentrations, was detrimental for the cells by reducing the activity of catalase and superoxidedismutase, the natural antioxidants. UVA was more efficient in stimulating the activity of superoxide dismutase and catalase but also in depleting the reserves of the enzymatic defense against oxidative stress, especially catalase, than UVB. This physiologic response to UV light can be considered as an adjunctive risk factor for people with low phototype for developing a melanoma, when exposed to UV irradiation.

We employed X-irradiation to activate a caged amino acid with a 2-oxoalkyl group. We designed and synthesized tyrosine derivative caged by a 2-oxoalkyl group (Tyr(Oxo)) to evaluate its radiolytic one-electron reduction characteristics in aqueous solution. Upon hypoxic X-irradiation, Tyr(Oxo) released a 2-oxopropyl group to form the corresponding uncaged tyrosine. In addition, radiolysis of dipeptides containing Tyr(Oxo) revealed that the efficiency of radiolytic removal of 2-oxopropyl group increased significantly by the presence of neighboring aromatic amino acids.

The effects of neighboring residues and formulation variables on tyrosine oxidation were investigated in model dipeptides (glysyl tyrosine, N-acetyl tyrosine, glutamyl tyrosine, and tyrosyl arginine) and tripeptide (lysyl tyrosyl lysine). The tyrosyl peptides were oxidized by light under alkaline conditions by a zero-order reaction. The rate of the photoreaction was dependent on tyrosyl pK(a), which was perturbed by the presence of neighboring charged amino acid residues. The strength of light exposure, oxygen headspace, and the presence of cationic surfactant, cetyltrimethylammonia chloride had a significant effect on the kinetics of tyrosyl photo-oxidation. Tyrosine and model tyrosyl peptides were also oxidized by hydrogen peroxide/metal ions at neutral pH. Metal-catalyzed oxidation followed first-order kinetics. Adjacent negatively charged amino acids accelerated tyrosine oxidation owing to affinity of the negative charges to metal-ions, whereas positively charged amino acid residues disfavored the reaction. The oxidation of tyrosine in peptides was greatly affected by the presence of adjacent charged residues, and the extent of the effect depended on the solution environment.

By the action of UVA light, fluphenazine reacted with nucleophiles through a mechanism involving defluorination of its trifluoromethyl group, giving rise to carboxylic acid derivatives that were easily detected by electrospray mass spectrometry. This photoreaction took place with alcohols, sulphydryls, and amines. When irradiation of fluphenazine was carried out in the presence of an amino acid at pH 7.4, the alpha-amino group was covalently bound to the drug. With amino acids possessing a further nucleophilic residue on the side chain, such as lysine, tyrosine, and cysteine--but not serine--both groups reacted, resulting in a fluphenazine-amino acid-fluphenazine diadduct. The same occurred with the physiological peptide glutathione (gamma-glutamylcysteinylglycine). By means of MALDI mass spectrometry, it was shown that fluphenazine also covalently bound to peptides and proteins such as calmodulin. This binding may result in the formation of antibodies, ultimately leading to the destruction of the granulocytes and thus suggesting that photoactivation of this drug may play a role in its clinical side effects, such as agranulocytosis.

The BLUF (sensor of blue light using FAD) domain is a blue light receptor possessing a flavin molecule as an active cofactor. A conserved Tyr residue located adjacent to flavin has been proposed to be a key amino acid in the mechanism of the photoreaction of the BLUF domain. We have studied the structure of this key Tyr residue and the relevance to the photoreaction in the BLUF protein of the cyanobacterium Thermosynechococcus elongatus, TePixD, by means of Fourier transform infrared (FTIR) difference spectroscopy and density functional theory (DFT) calculations. Light-induced FTIR difference spectra of unlabeled and [4-13C]Tyr-labeled TePixD in H2O and D2O revealed that the nuCO/deltaCOH vibrations of a photosensitive Tyr side chain are located at 1265/1242 cm-1 in the dark-adapted state and at 1273/1235 cm-1 in the light-induced signaling state. These signals were assigned to the vibrations of Tyr8 near flavin from the absence of the effect of [4-13C]Tyr labeling in the Tyr8Phe mutant. DFT calculations of H-bonded complexes of p-cresol with amides as models of the Tyr8-Gln50 interactions showed that Tyr8 acts as a H-bond donor to the Gln50 in both of the dark and light states. Further DFT analysis suggested that this H-bond is strengthened upon photoconversion to the light state accompanied with a change in the H-bond angle. The change in the H-bond structure of Tyr8 is coupled to the flavin photoreaction probably through the Tyr8-Gln50-flavin H-bond network, suggesting a significant role of Tyr8 in the photoreaction mechanism of TePixD.

Purpose: Ceruloplasmin (CP), an important serum antioxidant, was previously found to reduce the incidence of ventricular fibrillation (VF) induced by ischemia and reperfusion in isolated rat hearts. The present study investigated whether CP sterilized by gamma-irradiation maintains its antiarrhythmic capacity and in vitro antioxidant properties., Materials and Methods: Isolated rat hearts submitted to regional ischemia (15 min), were reperfused (10 min) with native CP or with CP irradiated at various doses (1-3 kGy) in the absence or presence of tyrosine (Tyr)., Results: All untreated hearts showed VF at reperfusion, which were all irreversible ventricular fibrillation (IVF). No IVF were found in hearts treated with native CP or gamma-irradiated CP. Cardioprotection afforded by irradiated CP (with or without Tyr) was slightly higher than that obtained with native CP. No VF at all (100% prevention) was found in hearts treated with CP irradiated alone or in the presence of tyrosine at 3 kGy. Tyrosine and irradiated tyrosine had no cardiotoxic or protective effects on reperfusion-induced arrhythmias. The Oxygen Radical Absorbing Capacity (ORAC), measured in vitro with beta-phycoerythrin (beta-PE) fluorescent indicator, was slightly higher for gamma-irradiated CP in the presence of Tyr., Conclusions: Ceruloplasmin sterilized by gamma-irradiation maintains antioxidant and antiarrhythmic effects in the post-ischemia reperfused isolated rat heart.

Reactive nitrogen species, produced during the process of inflammation induced by various factors including UV radiation, modify amino acids in crucial proteins. It is assumed that skin tissue is more likely to be modified, as it is located at the outer layer of a body that is exposed to UV radiation on a daily basis. To investigate the influence of the modified tyrosine on UV-exposed skin, we detected the nitrotyrosine or halogenated tyrosine and dityrosine in photo-aged model mice. The back skin of mice was exposed to a dose of 10 J cm(-2) day(-1) every day for 15 weeks. Samples exhibiting typical symptoms of photo aging were provided to the immunofluorescence study. The quantification of modified proteins was accomplished through a chemical analytical method known as HPLC-tandem mass spectrometry. Analysis of the irradiated skin samples showed that all modified tyrosine except nitrotyrosine demonstrated statistically significant increases. The molecular weights of major modified proteins, confirmed as 25-50 kDa, were measured using Western blot analysis with an anti-nitrotyrosine antibody. Furthermore, the immunofluorescence study verified that the localization of myeloperoxidase conformed to that of nitrotyrosine. This result suggests that the modified tyrosine was produced during the process of inflammation by UV irradiation. In this study, we used a low dose of UV irradiation to which we are exposed in daily life. Our results suggest that UV exposure in daily life may induce the production of modified tyrosines and skin aging.

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH* radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH. radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.

Three long-wavelength absorbing dipeptide-modified hypocrellin B (HB) derivatives, Gly-HB, Tyr-HB, and Trp-HB, were prepared for application in photodynamic therapy (PDT). Their abilities to produce free radicals and singlet oxygen were compared in detail with EPR technique, and their binding interactions with calf thymus DNA (CT DNA) were studied by absorption spectra and DNA melting temperature measurements. Tyr-HB and Trp-HB distinguish themselves from Gly-HB and HB remarkably by their significantly improved efficiencies to generate semiquinone anion radicals, superoxide anion radicals, and hydroxyl radicals, as well as their affinity to CT DNA, as the result of the electron-donating properties and intercalating abilities of tyrosine and tryptophan groups. Tyr-HB and Trp-HB show remarkably enhanced photodamage capabilities on CT DNA than their parent HB in aerobic conditions. Moreover, they possess moderate photodamage abilities on CT DNA even in anaerobic conditions, indicating the role of Type I mechanism in their photodynamic behaviors.

Radiation-induced hydroxylation of 3-nitrotyrosine (3-NY) and its derivatives in aqueous solution were investigated as a function of gamma-radiation dose. Irradiated 3-NY, 3-nitrotyrosine ethyl ester (3-NYE) and 3-NY containing peptide Gly-nitroTyr-Gly were separated and analyzed with reverse-phase HPLC and UV-Vis absorption spectroscopy. The structures of the hydroxylated products were confirmed by electrospray ionization mass spectrometry and (1)H-NMR spectrometry. The amounts of the hydroxylated products in irradiated 3-NY and Gly-nitroTyr-Gly solutions increased with increasing radiation dose. Tandem electrospray ionization mass spectrometry (ESI-Mass(2)) was performed to investigate the hydroxylation of peptide Gly-nitroTyr-Gly. These studies showed that the hydroxylation occurred at 3-NY residue. We also found that the identification of 3-NY hydroxylation in peptide could be identified by ion scanning for the specific immonium ion at m/z 197.0.

Understanding the molecular determinants of ultraviolet (UV) response may lead to the development of novel targets; and therefore, better approaches for the management of cancers, which mainly arise due to the exposure of skin to UV (particularly its UVB spectrum). Signal transducer and activator of transcription (Stat) proteins have been shown to activate multiple signaling pathways to contribute to oncogenesis. Here, we studied the regulation of Stat3 during UVB exposure-mediated responses in the skin of SKH-1 hairless mouse, a model regarded to possess relevance to human situations. Our data demonstrated that a single UVB (180 mJ/cm(2)) exposure to the skin of SKH-1 hairless mice resulted in significant upregulation in (i) protein levels of Stat3 and (ii) phosphorylation of Stat3 at tyrosine(705). Further, the activation of Stat3 was found to be associated with a decrease in apoptotic response of UVB and a gradual time-dependent increase in leukocyte infiltration and hyperplasia. In conclusion, we have demonstrated, for the first time, that UVB exposure to skin resulted in an activation of pro-survival protein Stat3. Based on our observation, we suggest that Stat3 could serve as a target for the management of UVB exposure-mediated damages including skin cancer.

Rhenium(I) polypyridyl complexes have been designed for the intramolecular photogeneration of tyrosyl radical. Tyrosine (Y) and phenylalanine (F) have each been separately appended to a conventional Re(I)(bpy)(CO)(3)CN framework via an amide linkage to the bipyridine (bpy) ligand. Comparative time-resolved emission quenching and transient absorption spectra of Re(bpy-Y)(CO)(3)CN and Re(bpy-F)(CO)(3)CN show that Y is oxidized only upon its deprotonation at pH 12. In an effort to redirect electron transport so that it is more compatible with intramolecular Y oxidation, polypyridyl Re(I) complexes have been prepared with the amide bond functionality located on a pendant phosphine ligand. A [Re(phen)(PP-Bn)(CO)(2)](PF(6)) (PP = bis(diphenylphosphino)ethylene) complex has been synthesized and crystallographically characterized. Electrochemistry and phosphorescence measurements of this complex indicate a modest excited-state potential for tyrosine oxidation, similar to that for the (bpy)Re(I)(CO)(3)CN framework. The excited-state oxidation potential can be increased by introducing a monodentate phosphine to the Re(I)(NN)(CO)(3)(+) framework (NN = polypyridyl). In this case, Y is oxidized at all pHs when appended to the triphenylphosphine (P) of [Re(phen)(P-Y)(CO(3))](PF(6)). Analysis of the pH dependence of the rate constant for tyrosyl radical generation is consistent with a proton-coupled electron transfer (PCET) quenching mechanism.

A compact, inexpensive detector for proteins has been constructed based on two-photon excitation of fluorescence from phenylalanine, tyrosine, and tryptophan. The fluorescence was excited by a solid-state microchip laser operating at 532 nm. Detection limits for phenylalanine, tyrosine, and tryptophan were 62 microM, 2.0 microM, and 470 nM, respectively, in a volume of 3 fL. The detection limit for a test protein, bovine serum albumin, was 130 nM.

Thyronine derivatives are essential indicators of thyroid gland diseases in clinical diagnosis and are currently used as standards for developing ordinary biochemical assays. Photooxidation of gland hormones of the thyronine (TN) family and structurally related compounds (TN, 3,5-diiodothyronine,3,3',5-triiodothyronine and 3,3',5,5'-tetraiodothyronine or thyroxine) was studied using rose bengal, eosin and perinaphthenone (PN) as dye sensitizers. Tyrosine (Tyr) and two iodinated derivatives (3-iodotyrosine and 3,5-diiodotyrosine) were also included in the study for comparative purposes. Irradiation of aqueous solutions of substrates containing xanthene dyes with visible light triggers a complex series of competitive interactions, which include the triplet excited state of the dye (3Xdye*) and singlet molecular oxygen [O2(1Deltag)]-mediated and superoxide ion-mediated reactions. Rate constants for interaction with the 3Xdye*, attributed to an electron transfer process, are in the order of 10(8)-10(9) M-1 s-1 depending on the dye and the particular substrate. The photosensitization using PN follows a pure Type-II (O2(1Deltag) mediated) mechanism. The presence of the phenolic group in Tyr, TN and iodinated derivatives dominates the kinetics of photooxidation of these compounds. The reactive rate constants, k(r), and the quotient between reactive and overall rate constants (k(r)/k(t) values, in the range of 0.7-0.06) behave in an opposite fashion compared with the overall rate constants and oxidation potentials. This apparent inconsistency was interpreted on the basis of an internal heavy atom effect, favoring the intersystem-crossing deactivation route within the encounter complex with the concomitant reduction of effective photooxidation.

The photophysics of the single tyrosine in bovine ubiquitin (UBQ) was studied by picosecond time-resolved fluorescence spectroscopy, as a function of pH and along thermal and chemical unfolding, with the following results: First, at room temperature (25 degrees C) and below pH 1.5, native UBQ shows single-exponential decays. From pH 2 to 7, triple-exponential decays were observed and the three decay times were attributed to the presence of tyrosine, a tyrosine-carboxylate hydrogen-bonded complex, and excited-state tyrosinate. Second, at pH 1.5, the water-exposed tyrosine of either thermally or chemically unfolded UBQ decays as a sum of two exponentials. The double-exponential decays were interpreted and analyzed in terms of excited-state intramolecular electron transfer from the phenol to the amide moiety, occurring in one of the three rotamers of tyrosine in UBQ. The values of the rate constants indicate the presence of different unfolded states and an increase in the mobility of the tyrosine residue during unfolding. Finally, from the pre-exponential coefficients of the fluorescence decays, the unfolding equilibrium constants (KU) were calculated, as a function of temperature or denaturant concentration. Despite the presence of different unfolded states, both thermal and chemical unfolding data of UBQ could be fitted to a two-state model. The thermodynamic parameters Tm = 54.6 degrees C, DeltaHTm = 56.5 kcal/mol, and DeltaCp = 890 cal/mol//K, were determined from the unfolding equilibrium constants calculated accordingly, and compared to values obtained by differential scanning calorimetry also under the assumption of a two-state transition, Tm = 57.0 degrees C, DeltaHm= 51.4 kcal/mol, and DeltaCp = 730 cal/mol//K., (Copyright 2004 Biophysical Society)

Laser flash photolysis at 266 nm of 3-nitrotyrosine and N-acetyl-3-nitrotyrosine ethyl ester generates an oxidizing species, which shows all of the characteristics of a hydroxyl radical. This species reacts with Br(-) to yield Br(2).-, via an intermediate, that is kinetically identified as HOBr.-. Moreover, the formation of Br(2).- can be suppressed by methanol; competition kinetics yield relative rate constants for the reaction of the reactive species with Br(-) and methanol that are similar to those for the hydroxyl radical. Parallel time-resolved UV/vis spectroscopy suggests the formation of phenoxyl radicals, consistent with the formation of hydroxyl radicals. Laser flash photolysis at 355 nm also generates reactive intermediates that oxidize Br(-) to Br(2).- but appear not to be hydroxyl radicals.

Melanosomes scavenged tyrosyl radical that was generated by ultraviolet irradiation of tyrosine. Purified mushroom tyrosinase also removed tyrosyl radical in a dose-dependent manner. To elucidate the underlying mechanism, we analyzed the reaction of mushroom tyrosinase with tyrosyl radical generated by horseradish peroxidase and hydrogen peroxide. Resting tyrosinase, which contained a small amount of oxytyrosinase, did not oxidize tyrosine to DOPAchrome until horseradish peroxidase exhausted H(2)O(2) and thereafter the enzyme recovered its full activity. During the inhibition period most tyrosine was converted to dityrosine, suggesting that only a small amount of tyrosyl radical was enough to interact with a fraction of tyrosinase which was in the active oxy-form. When horseradish peroxidase and H(2)O(2) were added to oxytyrosinase, which was prepared by allowing it to turn over beforehand, DOPAchrome production was abolished with an accelerated consumption of H(2)O(2). Dityrosine formation was totally suppressed and tyrosine concentration stayed constant during the inhibition period with a concomitant production of O(2). The results are accounted for by a mechanism in which tyrosyl radical is reduced to tyrosine by oxytyrosinase and the resulting met-form reacts with H(2)O(2) to return to the oxy-form.

A power-like decay function, characterized by the mean excited-state lifetime and relative variance of lifetime fluctuation around the mean value, was applied in analysis of fluorescence decays measured with the aid of time-correlated single photon counting. We have examined the fluorescence decay, in neutral aqueous medium, of tyrosine (L-tyrosine and N-acetyl-L-tyrosinamide), and of the tyrosine residues in a tryptophan-free protein, the enzyme purine nucleoside phosphorylase from Escherichia coli in a complex with formycin A (an inhibitor), and orthophosphate (a co-substrate). Tryptophan fluorescence decay was examined in neutral aqueous medium for L-tryptophan, N-acetyl-L-tryptophanamide, and for two tryptophan residues in horse liver alcohol dehydrogenase. To detect solvent effect, fluorescence decay of Nz-acetyl-L-tryptophanamide in aqueous medium was compared with that in dioxan. Hitherto, complex fluorescence decays have usually been analyzed with the aid of a multiexponential model, but interpretation of the individual exponential terms (i.e., pre-exponential amplitudes and fluorescence lifetimes), has not been adequately characterized. In such cases the intensity decays were also analyzed in terms of the lifetime distribution as a consequence of an interaction of fluorophore with environment. We show that the power-like decay function, which can be directly obtained from the gamma distribution of fluorescence lifetimes, is simpler and provides good fits to highly complex fluorescence decays as well as to a purely single-exponential decay. Possible interpretation of the power-like model is discussed.

Objective: To evaluate the absorbance of ultraviolet radiation (UVR) in the aqueous humor of various animal species in relation to the ambient radiation of their respective habitats, and to identify substances responsible for this absorbance. Representatives of all five classes (fish, amphibian, reptile, bird, and mammal) have been tested., Methods: Absorbance was recorded using a spectrophotometer. The ascorbic and uric acid concentrations were determined by HPLC, and the amino acid profiles with an automatic analyzer. Screening for potential UV-absorbing substances was performed by HPLC and a total of 12 species were examined, 7 of them birds., Results: UV-absorbing substances in the aqueous humor were proteins, tryptophan, tyrosine and ascorbic and uric acid. In addition, an unknown UV-absorbing component present in bird aqueous humor caused a high, red-shifted UV-absorbance spectrum, particularly in tentatively heavily exposed species such as goose when migrating at 10,000 m altitude. By comparison, the UV absorbance above the 288-nm wavelength was low in the aqueous humor of fish, frogs, aquatic mammals and two ground-living birds. The crocodile, whose aqueous humor contained significant amounts of both ascorbic and uric acid, revealed a concentration mechanism for ascorbic acid., Conclusions: The UV absorbance of aqueous humor varies considerably from one species to the next, and independent of class. It is noteworthy that the species being at highest risk for high-dose UV exposure, the migrating goose, showed the most red-shifted spectrum., (Copyright 2003 S. Karger AG, Basel)

Near-IR (NIR) excitation at liquid He temperatures of photosystem II (PSII) membranes from the cyanobacterium Synechococcus vulcanus or from spinach poised in the S2 state results in the production of a g = 2.035 EPR resonance, reminiscent of metalloradical signals. The signal is smaller in the spinach preparations, but it is significantly enhanced by the addition of exogenous quinones. Ethanol (2-3%, v/v) eliminates the ability to trap the signal. The g = 2.035 signal is identical to the one recently obtained by Nugent et al. by visible-light illumination of the S1 state, and preferably assigned to S1Y(Z*) [Nugent, J. H. A., Muhiuddin, I. P., and Evans, M. C. W. (2002) Biochemistry 41, 4117-4126]. The production of the g = 2.035 signal by liquid He temperature NIR excitation of the S2 state is paralleled by a significant reduction (typically 40-45% in S. vulcanus) of the S2 state multiline signal. This is in part due to the conversion of the Mn cluster to higher spin states, an effect documented by Boussac et al. [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007], and in part due to the conversion to the g = 2.035 configuration. Following the decay of the g = 2.035 signal at liquid helium temperatures (decay halftimes in the time range of a few to tens of minutes depending on the preparation), annealing at elevated temperatures (-80 degrees C) results in only partial restoration of the S2 state multiline signal. The full size of the signal can be restored by visible-light illumination at -80 degrees C, implying that during the near-IR excitation and subsequent storage at liquid helium temperatures recombination with Q(A-) (and therefore decay of the S2 state to the S1 state) occurred in a fraction of centers. In support of this conclusion, the g = 2.035 signal remains stable for several hours (at 11 K) in centers poised in the S2...Q(A) configuration before the NIR excitation. The extended stability of the signal under these conditions has allowed the measurement of the microwave power saturation and the temperature dependence in the temperature range of 3.8-11 K. The signal intensity follows Curie law temperature dependence, which suggests that it arises from a ground spin state, or a very low-lying excited spin state. The P1/2 (microwave power at half-saturation) value is 1.7 mW at 3.8 K and increases to 96 mW at 11 K. The large width of the g = 2.035 signal and its relatively fast relaxation support the assignment to a radical species in the proximity of the Mn cluster. The whole phenomenology of the g = 2.035 signal production is analogous to the effects of NIR excitation on the S3 state [Ioannidis, N., Nugent, J. H. A., and Petrouleas, V. (2002) Biochemistry 41, 9589-9600] producing an S2'Y(Z*) intermediate. In the present case, the intermediate is assigned to S1Y(Z*). The NIR-induced increase in the oxidative capability of the Mn cluster is discussed in relation to the photochemical properties of a Mn(III) ion that exists in both S2 and S3 states. The EPR properties of the S1Y(Z*) intermediate cannot be reconciled easily with our current understanding of the magnetic properties of the S1 state. It is suggested that oxidation of tyr Z alters the magnetic properties of the Mn cluster via exchange of a proton.

The UV-A (320-400 nm) component of sunlight is a significant damaging factor of plant photosynthesis, which targets the photosystem II complex. Here we performed a detailed characterization of UV-A-induced damage in photosystem II membrane particles using EPR spectroscopy and chlorophyll fluorescence measurements. UV-A irradiation results in the rapid inhibition of oxygen evolution accompanied by the loss of the multiline EPR signal from the S(2) state of the water-oxidizing complex. Gradual decrease of EPR signals arising from the Q(A)(-)Fe(2+) acceptor complex, Tyr-D degrees, and the ferricyanide-induced oxidation of the non-heme Fe(2+) to Fe(3+) is also observed, but at a significantly slower rate than the inhibition of oxygen evolution and of the multiline signal. The amplitude of Signal II(fast), arising from Tyr-Z degrees in the absence of fast electron donation from the Mn cluster, was gradually increased during the course of UV-A treatment. However, the amount of functional Tyr-Z decreased to a similar extent as Tyr-D as shown by the loss of amplitude of Signal II(fast) that could be measured in the UV-A-treated particles after Tris washing. UV-A irradiation also affects the relaxation of flash-induced variable chlorophyll fluorescence. The amplitudes of the fast (600 micros) and slow (2 s) decaying components, assigned to reoxidation of Q(A)(-) by Q(B) and by recombination of (Q(A)Q(B))(-) with donor side components, respectively, decrease in favor of the 15-20 ms component, which reflects PQ binding to the Q(B) site. In the presence of DCMU, the fluorescence relaxation is dominated by a 1 s component due to recombination of Q(A)(-) with the S(2) state. After UV-A radiation, this is partially replaced by a much faster component (30-70 ms) arising from recombination of Q(A)(-) with a stabilized intermediate PSII donor, most likely Tyr-Z degrees. It is concluded that the primary damage site of UV-A irradiation is the catalytic manganese cluster of the water-oxidizing complex, where electron transfer to Tyr-Z degrees and P(680)(+) becomes inhibited. Modification and/or inactivation of the redox-active tyrosines and the Q(A)Fe(2+) acceptor complex are subsequent events. This damaging mechanism is very similar to that induced by the shorter wavelength UV-B (280-320) radiation, but different from that induced by the longer wavelength photosynthetically active light (400-700 nm).

The effects of fast neutrons (10-100 Gy) on chromatin extracted from normal (liver of Wistar rats) and tumor (Walker carcinosarcoma maintained on Wistar rats) tissues were compared. The spectroscopic assays used were (i) chromatin intrinsic fluorescence, (ii) time-resolved fluorescence of chromatin - proflavine complexes, and (iii) fluorescence resonance energy transfer (FRET) between dansyl chloride and acridine orange coupled to chromatin. For both normal and tumor chromatin, the intensity of intrinsic fluorescence specific for acidic and basic proteins decreased with increasing dose. The relative contributions of the excited-state lifetime of proflavine bound to chromatin were reduced upon fast-neutron irradiation, indicating a decrease in the proportion of chromatin DNA available for ligand binding. The Forster energy transfer efficiencies were also modified by irradiation. These effects were larger for chromatin from tumor tissue. In the range 0-100 Gy, fast neutrons induced alterations in DNA and acidic and basic proteins, as well as in global chromatin structure. The radiosensitivity of chromatin extracted from tumor tissue seems to be higher than that of chromatin extracted from normal tissue, probably because of its higher euchromatin (loose)--heterochromatin (compact) ratio.

Cell culture media (RPMI 1640, Dulbecco's Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5'-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS). The production of ROS was light dependent. The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin. These observations point to exposure of cells to ROS under in vitro culture conditions.

Redox state-dependent changes in the relative orientation of the phenol side chain and the peptide group in model tyrosine have been characterized using specific 2H isotopic labelling and X-band electron paramagnetic resonance (EPR) spectroscopy. Tyrosyl radicals were generated by UV photolysis of tyrosine trapped in rigid polycrystalline basic-aqueous medium at T < or = 170 K. Ring-2H(4) and beta-2H(2) substitutions on tyrosine were used to enhance the lineshape contributions from beta-hydrogen or ring-hydrogen hyperfine interactions, respectively. The EPR lineshape at 120 K of the trapped ring-2H(4)-tyrosyl radical is altered dramatically after annealing at 235 K. In contrast, the lineshape of the beta-2H(2)-tyrosyl radical is impervious to annealing. The effect of annealing on the lineshape therefore arises from a change in the isotropic hyperfine coupling between unpaired pi-electron spin density at the ring carbon atom C(1) and the beta-hydrogen nuclei, which is caused by rotational relaxation of the ring and peptide group about the C(1)-C(beta) bond. EPR simulations indicate angular distributions of the peptide group (R-) of 0 degrees < or = theta(R) < or = 30 degrees and 0 degrees < or = theta(R)< or = 18 degrees in the rigid and relaxed radical states, respectively. Redox-induced changes in the C(1)-C(beta) rotamer distribution must be accounted for in assessments of stable amino acid side chain equilibrium structures, and may influence catalytic tyrosyl radical/tyrosine function in enzymes.

Irradiation of N-(tyrosyl)-N'-(5-azido-2-nitrobenzoyl)-1,2-diaminoethane (I) initiates chemical reactions that lead to different products depending on the experimental conditions. All of these products are attributed to the reactions of triplet 4-nitrobenzoyl nitrene (4NBN). The reactions of triplet 4NBN with the tyrosyl residue result in the formation of two distinct products: compound II, which is unstable in aqueous solution, and the stable compound cyclo-[1-(4'-nitro-3'-benzoyl)-2-(aminotyrosyl)-N,N'-ethylenediami ne] (III). The formation of II is detected only in aerobic conditions. The unstable photoproduct II converts almost completely into compound III when its solution is concentrated. The photoproducts II and III have absorption spectra that are close to those of the photolabelled peptides. This finding is important for speculating about the chemical nature of the photomodification products of protein tyrosyl residues by the arylazide group.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry experiments were applied to study the influence of ultraviolet (UV) irradiation in melanogenesis. Samples were prepared starting from three different precursors, tyrosine, Dopa and dopamine, in the presence or absence of tyrosinase, the enzyme responsible for the synthesis of melanin. Enzymatic reactions were carried out for 10, 30, 60 and 120 min under UV irradiation at 365 nm, and aliquots were then immediately ultrafiltered and lyophilized. Samples obtained by irradiation of tyrosine solution revealed the formation of 5,6-dihydroxyindole (DHI) oligomers up to pentamers at 120 min; the reaction kinetics were markedly enhanced in the presence of tyrosinase. In the case of Dopa, UV irradiation favored melanogenesis only in the presence of the enzyme; in this case, many reaction pathways were activated, originating various oligomeric species of Dopa, DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Conversely, when dopamine was used as tyrosinase substrate under UV light, mechanisms of melanogenesis different from those generated by simple enzymatic reaction without irradiation were not activated, as the same oligomeric species were present., (Copyright 2000 John Wiley & Sons, Ltd.)

We observed emission from the tyrosine derivative N-acetyl-L-tyrosinamide (NATyrA) when excited with the fundamental output of a femtosecond Ti:Sapphire laser from 780 to 855 nm. The dependence on incident laser power indicates a three-photon process. The emission spectra and intensity decay in glycerol-water (30:70) at 5 degrees C were found to be identical for one- and three-photon excitation. Also the excitation spectrum of three-photon-induced fluorescence of NATyrA corresponds to the one-photon excitation spectrum. The time-zero or fundamental anisotropy spectrum was reconstructed from the frequency-domain anisotropy decays. The three-photon anisotropies are similar or larger than the one-photon anisotropies. These three-photon anisotropies are surprising given the near zero values known for tyrosine with two-photon excitation. The observations indicate that one- and three-photon excitation directly populates the same singlet excited states(s). However, the origin of the anisotropies with multi-photon excitation of tyrosine remain unclear and unpredictable.

This paper studies the dye-sensitized photooxidation of tyrosine (tyr) and tyr di- and tripeptides (tyr-tyr and tyr-tyr-tyr) mediated by singlet molecular oxygen (O2[1 delta g]) in alkaline media. Photooxidation quantum efficiencies (phi r) were obtained by determining the overall and reactive rate constants of interaction with the oxidative species, employing the time-resolved O2(1 delta g) phosphorescence detection method and static-photolysis actinometric method, respectively. The interaction of O2(1 delta g)-tyr derivatives occurs through an intermediate encounter complex with polar character. Ionization of the phenolic OH group of tyr derivatives and the polarity of the solvent favors the overall interaction. Nevertheless, phi r values decrease when changing from water to MeCN-water medium. This indicates that the reactive deactivation of the encounter complex, probably an entropy-controlled step, may be affected by solvent polarity in the same way as those processes in which charges are neutralized along the reaction pathway. Photooxidation quantum efficiencies indicate that the contribution to O2(1 delta g) physical quenching (a second alternative deactivation route for the encountered complex [O2(1 delta g)-tyr derivatives]) increases with the complexity of the peptide. As a result, the selfprotection of the peptidic entity against physical quenching also increases. The information obtained from the fractional consumption mol O2/mol tyr derivative (in tyr, the di- and tripeptides and the respective methyl ester of tyr and the tripeptide), together with the evolution (either consumption and/or generation) of primary amino groups upon photosensitized irradiation of the same compounds clearly indicates that the photooxidation of di- and tri-tyr peptides proceeds with the breakage of peptidic bonds. As a consequence, in the final balance each tyr unity behaves as an independent photooxidizable target.

A new post-column photolysis technology has been developed based on the use of a low pressure, low temperature UV lamp and TiO2 coated knitted reaction coil. As a test case the developed technique was used for the determination of 3-nitro-L-tyrosine by liquid chromatography with electrochemical detection. Different photolysis lamps and reactor tubing lengths were evaluated in terms of their effect on the separation efficiency and/or photolysis efficiency. A detection limit of 0.5 nM (10 fmol) for 3-nitro-L-tyrosine was achieved under optimized conditions, with a linear correlation coefficient of R2 = 0.9898 over a concentration range of 2-100 microM. Pre-injection photolysis of 3-nitro-L-tyrosine indicated that dihydroxyphenylalanine is the main photolysis product. In general, use of the photoreactor prior to liquid chromatography is an excellent method for exploring photodegradation products of an analyte in conjunction with the full range of available liquid chromatography detectors.

A liquid chromatographic method has been developed for the determination of 3-nitro-L-tyrosine. Different detection methods, including UV, oxidative and redox electrochemistry, and postcolumn photolysis followed by electrochemical detection, have been optimized and compared in terms of analysis time, detection limit and dynamic range. It was demonstrated that liquid chromatography with postcolumn photolysis followed by electrochemical detection is the most effective method, with an analysis time of 5 min, detection limit of 0.01 pmol, and a linear dynamic range from 2 nM to 100 microM.

We have demonstrated the feasibility of preparing caged peptides by derivatizing a single amino acid side chain in peptides up to 20 amino acids long. Two peptides are illustrated whose activities are reduced by nearly 2 orders of magnitude using this caging approach. The specific strategy described here of derivatizing tyrosine side chains with a charged caging moiety should be generally applicable in the preparation of caged peptides that have a critical tyrosine residue (e.g., LSM1) or that have critical hydrophobic patches (e.g., RS-20). Other amino acid side chains are also accessible via this caging strategy. Derivatives of threonine, serine, lysine, cysteine, glutamate, aspartate, glutamine, and asparagine can be prepared and site specifically inserted into peptides in an analogous manner. The caged peptides synthesized and purified by the methods described here are compatible with biological samples, including living cells, and have been used to demonstrate the central importance of calmodulin, MLCK, and, by inference, myosin II in ameboid locomotion in polarized eosinophil cells. Photoactivation of peptides within cells should provide a wealth of new information in future investigations by allowing specific protein activities to be knocked out in an acute and spatially defined way.

Radiation-induced formation of 3,4-dihydroxyphenylalanine (DOPA) in Tyr and Tyr-containing peptides and proteins was investigated as a function of X-irradiation dose. Irradiated Tyr (0-30 Gy) and the acid hydrolysates of irradiated peptide and protein (0-240 Gy) were conjugated with dansyl chloride. The dansylated amino acids were analyzed by reversed-phase HPLC using fluorescence detection. Formation of DOPA, determined by integrated peak area, increased with dose. Analysis of the major product from irradiated tripeptide Tyr-Gly-Gly detected Gly and DOPA (2:1). Extension of the model study to irradiated BSA and RNase A showed correlation of DOPA formation with Tyr modification up to 120 Gy. Higher dose induced further transformation of DOPA. The fluorescence signal of dansylated DOPA was linear from 1.5 nmol to 0.5 pmol (correlation coefficient of 0.999, n = 3). The detection limit allows the detection of 1 molecule of DOPA/300 molecules of BSA in 5 micrograms of dansylated hydrolysate. Most standard amino acid analysis techniques are limited to detect normal residues of protein. Protein-bound DOPA has been suggested to have a role in the replenishment of reduced transition metal ion involved free-radical-generating system in vivo. Sensitive analysis of protein-bound DOPA will be useful to study amplification of the radical-damaging event.

The present experiment indicates that oligopeptides are easily produced in solid state from mixtures of simple amino acids by irradiating with high energy charged particles. We investigated such amino acids and their mixtures as tryptophan, tyrosine and glycine. The thin films was irradiated with protons (6.6 MeV). Such dipeptides as Trp-Trp, Gly-Tyr, Tyr-Gly, and Tyr-Tyr have been detected as products of irradiation. Cosmic rays might be an effective energy source for abiotic formation of bioorganic compounds on the surface of small bodies in the solar system on early stage of formation of planets as well as at present day.

Optic characteristics of solutions of aromatic amino acids containing fluorine products were examined by adding iodine solution. Iodine exerted variously directed effects on the intensity of photochemiluminescence of tryptophan, thyrosine, and beta-indolacetic acid solutions. Extinction of the luminescence of beta-indolacetic acid accumulating in an organism during tumors may be used as an additional test for the differential diagnosis of cancer.

Exposure to ionizing radiation leads to formation of covalent crosslinks between DNA and proteins. The nature, extent and site of the modifications are not well understood due to the difficulty in assessing free radical-induced damage in biopolymers. Electrospray ionization mass spectrometry (ESI-MS) permits direct analyses of intact oligopeptides, permitting characterization of the radiation-induced DNA-protein covalently crosslinked constituents. Our first application of this methodology to free radical-induced damage was in a model system where angiotensin, a small 10-amino acid peptide, is irradiated at various doses in the presence of excess thymine. The relative yield of crosslinks, which ranged from 0.1 to 15%, was linearly related to radiation dose for doses from 0.1 to 100 Gy. Detection of thymine-tyrosine moieties in this model system was possible at doses as low as 0.1 Gy with a signal-to-noise ratio of 4 to 1. ESI-MS revealed that the site of crosslink was located exclusively on the tyrosine residue as expected.