A mass spectrometric investigation on the possible role of tryptophan and 7-hydroxytryptophan in melanogenesis

The activity of tyrosinase and peroxidase + H2O2 in promoting melanogenesis from tryptophan (Trp) and 7-hydroxytryptophan (7-HTP) has been investigated. The reaction samples have been drawn at different reaction times and analysed by MALDI mass spectrometry. The data obtained showed that tryptophan undergoes, under tyrosinase and peroxidase action, an oligomerization process mainly due to the reaction of anthranilic acid (AA) and Trp. However, analysing the UV and fluorescence data, it is seen that the oligomers cannot belong to the melanin pattern, but their possible role in melanogenesis is not to be excluded. Once it reacts with the two enzymes, 7-hydroxytryptophan leads to dark brown products, indicating its possible role in melanin production. In contrast to what was observed in the case of 5-hydroxytryptophan, for which oligomers were constituted by 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5-HT) units, the MALDI data indicate a sharply different behaviour for 7-HTP. In fact, in the case of 5-hydroxytryptophan, oligomerization takes place through the formation of 5-hydroxytryptamine and the oligomerization products are due to mixed 5-HTP-5-HT oligomers. In the case of 7-hydroxytryptophan, the formation of 7-hydroxytryptamine (7-HT) is also observed, but it does not seem to play any role; the only oligomerization products formed are due to the reaction of 7-hydroxytryptophan and AA. The data so obtained indicate that 7-hydroxytryptophan acts like an effective melanin precursor in the presence of both tyrosinase and peroxidase + H2O2.     

An investigation on the possible role of melatonin in melanogenesis

The possible role of melatonin in melanogenesis was investigated by performing reactions of melatonin with peroxidase + H2O2 or H2O2 only, in the presence or absence of UV irradiation. Samples of the reaction mixtures were drawn at different times (from 15 to 480 min), the enzyme (when present) was removed by ultrafiltration and the samples so obtained were analyzed by MALDI/MS.The results show that melatonin undergoes oligomerization reaction with peroxidase + H2O2, leading to heptameric species. For high reaction times the MALDI/MS data do not show the formation of larger oligomers, but UV-vis spectroscopy indicates that the oligomerization processes proceed. The failure of MALDI-TOF in the identification of larger oligomers was related to the chemical-physical and morphological behavior of melanins.In the case of UV irradiation, the formation of species originating from the O- and O2 addition to melatonin, which activate new oligomerization channels, has been observed.     

Identification and reactivity of the triplet excited state of 5-hydroxytryptophan

Both the neurotransmitter serotonin and the unnatural amino acid 5-hydroxytryptophan (5HT), contain the 5-hydroxyindole chromophore. The photochemistry of 5HT is being investigated in relation to the multiphoton excitation of this chromophore to produce a characteristic photoproduct with green fluorescence ('hyperluminescence'). Laser flash photolysis (308 nm) of 5HT in aqueous solution at neutral pH produces both the neutral 5-indoloxyl radical (lambda(max) 400-420 nm) and another transient absorption with lambda(max) 480 nm and lifetime of 2 micros in deaerated solutions. Based on quenching by oxygen and beta-carotene, the species at 480 nm is identified as the triplet excited state of 5HT. In acidic solution a new oxygen-insensitive intermediate with lambda(max) 460 is assigned to the radical cation of 5HT. Time-resolved measurements of luminescence at 1270 nm have shown that the triplet state of 5HT is able to react with oxygen to form singlet excited oxygen (1O2*) with a quantum yield of approximately 0.1. However, 5HT has also been found to be an effective quencher of singlet oxygen with a second order rate constant of 1.3 x 10(8) dm3 mol(-1) s(-1). The results are discussed in the light of recent observations on the multiphoton-excited photochemistry of serotonin.     

The influence of melanins on the photoperoxidation of lipids

The influence of synthetic Dopa-melanin, natural-A, AB-melanin and pheomelanin has been studied for the following processes: (1) microsomal and liposomal lipid peroxidation induced by illumination with UV- and visible light; (2) interaction of melanin with the superoxide anion; and (3) chemiluminescence intensity of phospholipids in the presence and absence of melanins. It has been shown that (a) melanins can function as a scavenger of the superoxide anion radical O2-. (pseudo-superoxide dismutase) and (b) melanins protect microsomes and liposomes against lipid peroxidation which is due to the absorption of light energy (filter effect). Our data show that in the presence of melanins lipid peroxidation is significantly lower in comparison with the phospholipids incubated in the absence of melanins (except pheomelanin). The level of photoperoxidation of lipids was studied by means of the chemiluminescence method and by measurement of the malondialdehyde product. The results obtained by both methods indicate that melanin can effectively influence the process of lipid peroxidation. Mechanisms involved in the physical (optical screening) and photochemical reactions are proposed.     

In vivo investigation of the role of SfmO2 in saframycin A biosynthesis by structural characterization of the analogue saframycin O

Saframycin A(SFM-A),a tetrahydroisoquinoline antibiotic isolated from Streptomyces lavendulae,shows potent anti-proliferation activities against a variety of tumor cell lines,and shares the core structure with ecteinascidin 743(ET-743),the anticancer drug for soft-tissue sarcoma.Characterization of the SFM-A biosynthetic gene cluster revealed three nonribosomal peptide synthetase genes and a series of genes encoding oxygenases.To investigate the function of sfmO2 gene,encoding a FAD-dependent monooxygenase/hydroxylase,we constructed the gene replacement mutant(△sfmO2) strain S.lavendulae TL2007 and the corresponding gene complementation mutant strain S.lavendulae TL2008.A novel compound,SFM-O,was isolated from the △sfmO2 replacement mutant strain and its structure was characterized by comparison to the HRMS and NMR spectra of SFM-A.These findings indicated that SfmO2 is responsible for the oxidation of ring A in the biosynthetic pathway of SFM-A,and the new compound SFM-O could be considered as an advanced intermediate in the semisynthesis of ET-743.     

Combinatorial solid-phase synthesis of 6-hydroxy-1,2,3,4-tetrahydro-beta-carbolines from l-5-hydroxytryptophan

A library of biologically relevant 6-hydroxy-tetrahydro-beta-carbolines (6-OH-THBCs) based on the L-5-OH-tryptophan scaffold was prepared. A solid-phase synthesis was developed, utilizing aminomethyl polystyrene resin and solid-phase-optimized reactions, such as Pictet-Spengler condensation. The library was designed such that three points of diversity would be readily introduced, making the strategy potentially suitable for generation of a large number of compounds.     

An investigation on the production of maize-wheat-bread

Zusammenfassung Das Problem, die Proteine von Mais so zu verändern, da\ sie sich in ihrem Theologischen Verhalten dem Weizengluten annähern und die Produktion eines geeigneten Mais- oder Mais-Weizenbrotes ermöglichen, wurde experimentell in Angriff genommen. Die rheologische Veränderung des Maisteiges, erkennbar durch Weichwerden des Teiges, wurde durch Einwirkung von thermophilen cocci bei einer optimalen Temperatur von 45 C nach 2–3 Stunden erzielt. Nach der bakteriellen Einwirkung erfolgte in jeder Versuchsreihe die Zumischung von Weizenteig unter Zusatz von Backhefe und darauf folgende Brotgärung. Der Zusatz von Sojamehl ist notwendig. Soja- und Weizenteig besitzen eine im Mikroskop sichtbare Netzstruktur. Maisteig zeigt diese Struktur nicht, wohl aber der gemischte Maisteig. Um die Gärungstoleranz der bakteriellen Einwirkung zu bestimmen, mu\ eine Theologische Methode gefunden werden, welche die Messung des Weichheitsgrades des Maisteiges in einfacher Weise ermöglicht.

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Hans Zocher to his 70th birthday.     

329 - Investigation on the electrochemical properties of catecholmines and melanins

The melanization process was investigated using electrochemical methods.On the a.c. and multisweep curves the particular stages occur as separated, consecutively rising and decreasing peaks, corresponding to gradual association of precursor molecules.An interesting effect is the rise of d.c. waves at potentials characteristic for H₂O₂ (appearing usually if O₂ is present in the solution) in deoxygenated solutions of melanins. The differences in the curves of catechol-melanin and indole-melanin permit to ditinguish these compounds using polarographic methods.The result of practical importance is the possibility of a fast determination of small amounts of adrenaline and noradrenaline by use of multisweep methods.     

An investigation into the role of chloro-substituents in hydrogen-bonded crystals: The crystal structures of the dichlorophenols

A graphical analysis has been made of hydrogen-bonding patterns and non-bonded interactions in the crystal structures of the six dichlorophenols. This shows that hydrogen bonding is the primary interaction in stabilising all the isomers, and that C?C and Cl?Cl non-bonded interactions are also important in stabilising structures characterised by a short (≈ 4 Ã) axis.     

Laboratory investigation on the role of organics in atmospheric nanoparticle growth

The uptake of organic vapors by 4-20 nm H(2)SO(4) particles has been investigated to assess the role of organics in atmospheric nanoparticle growth. Sulfuric acid nanoparticles are generated from homogeneous binary nucleation of H(2)SO(4) and H(2)O vapors in a laminar flow chamber. The growth factor of H(2)SO(4) nanoparticles exposed to methyglyoxal, ethanol, 1-butanol, 1-heptanol, and 1-decanol is measured using a nanotandem differential mobility analyzer (nano-TDMA). The measured growth factor is close to unity when nanoparticles are exposed to methylglyoxal, ethanol, 1-butanol, 1-heptanol, and 1-decanol, indicating no apparent growth within the experimental uncertainty. In addition, spectroscopic evolution of functional groups in H(2)SO(4) particles of ～40 nm diameter size, deposited on ZnSe crystal and subsequently exposed to glyoxal and 2,4-hexadienal, is studied using attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FT-IR). The ATR-FT-IR measurements present the first spectroscopic signatures of high molecular weight aldol and oligomer products and show that polymerization and oligomerization reactions are partially reversible. The implications of the present results to nanoparticle growth in the atmosphere are discussed.     

An investigation on the role of 3-hydroxykynurenine in pigment formation by matrix-assisted laser desorption/ionization mass spectrometry

In order to investigate the role of tryptophan and its metabolites in biogenesis of melanins, a study on the enzymatic reaction of 3-hydroxykynurenine with tyrosinase and peroxidase was performed. The reaction at different pH values was monitored by sampling at different times, with ultrafiltration used before analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The data obtained in this way showed that oligomerization processes take place with both enzymes, but with different behaviour, also depending on pH. 3-Hydroxykynurenine in the presence of tyrosinase at pH 6.0 leads to formation of xanthommatin, and at pH 8.0 hydroxanthommatin is formed in the first step of the reaction followed by formation of black-brown pigments. In contrast, the formation of oligomerization products by peroxidase action is observed in high yields under both acidic and basic conditions; however, at pH 6.0, a more extensive oligomerization process is observed. Thus peroxidase is able to activate oligomerization analogous to that observed in the case of tyrosinase without depending on the variation of pH. Due to the early formation of decarboxylated hydroxykynurenine, hydroxanthommatin and decarboxylated hydroxanthommatin, the enzymatic reaction leads to mixed oligomers, which can be considered as precursors of new pathways in pigment production.     

An investigation of the relationship between microstructure and permeation properties of ZSM-5 membranes

The influence of membrane microstructure on the transport properties of ZSM-5 membranes was investigated. Two zeolite membranes with (1 0 1)- and (0 0 2)-orientations were grown layer-by-layer onto seeded alumina support. The membrane morphology was kept constant as well as the shape of the individual crystal grains that made up the polycrystalline zeolite membrane layer. The membrane microstructure were characterized and quantified using six microstructural parameters that include membrane thickness (τ), grain size (d), grain morphology (M), zeolite population (N), crystal intergrowth (I_c) and film orientation. Eight different gases including He, H₂, N₂, Ar, CH₄, n-C₄H₁₀, i-C₄H₁₀ and SF₆ were used as molecular probes to investigate the transport processes through the membrane of different thicknesses. By maintaining a comparable non-zeolite flow, it was demonstrated that the (1 0 1)- and (0 0 2)-oriented ZSM-5 membranes have comparable transport resistance. Also, the results of the multi-thickness comparison using the different sized molecular probes indicate a strong similarity in the transport mechanism and diffusion pathway through these two membranes. The experiment suggests that the grain boundary is the main non-zeolite diffusion pathway in the membrane and their elimination through grain growth can result in better membrane performance.     

Fluorescence properties of recombinant tropomyosin containing tryptophan, 5-hydroxytryptophan and 7-azatryptophan

Tropomyosin mutants containing either tryptophan (122W), 5-hydroxytryptophan (5OH122W) or 7-azatryptophan (7N122W) have been expressed in Escherichia coli and their fluorescence properties studied. The fluorescent amino acids were located at position 122 of the tropomyosin primary sequence, corresponding to a solvent-exposed position c of the coiled-coil heptapeptide repeat. The emission spectrum of the probe in each mutant is blue-shifted slightly with respect to that of the probe in water. The fluorescence anisotropy decays are single exponential, with a time constant of 2-3 ns while the fluorescence lifetimes of the probes incorporated into the proteins, in water, are nonexponential. Because tryptophan in water has an intrinsic nonexponential fluorescence decay, it is not surprising that the fluorescence decay of 122W is well described by a triple exponential. The fluorescence decays in water of the nonnatural amino acids 5-hydroxytryptophan and 7-azatryptophan (when emission is collected from the entire band) are single exponential. Incorporation into tropomyosin induces triple-exponential fluorescence decay in 5-hydroxytryptophan and double-exponential fluorescence decay in 7-azatryptophan. The range of lifetimes observed for 5-hydroxyindole and 5-hydroxytryptophan at high pH and in the nonaqueous solvents were used as a base with which to interpret the lifetimes observed for the 5OH122W and indicate that the chromophore exists in several solvent environments in both its protonated and unprotonated forms in 5OH122W.     

An investigation into the role of metastable argon atoms in the afterglow plasma of a low pressure discharge

An investigation into the behaviour of metastable argon atoms in a low pressure (250 Pa) pulsed electrical discharge was undertaken in an effort to find the cause of the persisting emission from sputtered metal atoms in the afterglow of an atomic fluorimeter. Results obtained by time-resolved emission and absorption measurements of several argon and copper spectral lines indicate that low energy electrons in the afterglow are converted to high energy electrons via the recombination of electrons with argon ions and the subsequent collisions of pairs of metastable argon atoms. The high energy electrons excite the sputtered metal atoms to give rise to a slow decaying emission tail in the afterglow. A probable change in the electron energy distribution in the afterglow may also have an effect on the observed emission. This phenomenon may be reduced by the use of a suitable quenching gas.