UV-absorbing Species Research Articles

Biochemical and physical–chemical characterisation of leaf proteins extracted from Cichorium endivia leaves

This study provides a detailed characterisation of a leaf protein concentrate (LPC) extracted from Cichorium endivia leaves using a pilot scale process. This concentrate contains 74.1% protein and is mainly composed of Ribulose-1,5-BISphosphate Carboxylase/Oxygenase (RuBisCO). We show that the experimentally determined extinction coefficient (around 5.0 cm−1 g−1 L depending on the pH) and refractive index increment (between 0.27 and 0.39 mL g−1) are higher than the predicted ones (about 1.6 cm−1 g−1 L and 0.19 mL g−1, respectively). In addition, the UV–visible absorption spectra show a maximum at 258 nm. These data suggest the presence of non-protein UV-absorbing species. Chromatographic separation of the concentrate components in denaturing conditions suggests that RuBisCO SC may be covalently bounded to few phenolic compounds. Besides, the solubility of LPC proteins is higher than 90% above pH 6. Such high solubility could make LPC a good candidate as a functional food ingredient.

Floral UV Features of Plant Species From a Neotropical Savanna.

Despite the wide interest in flower colours, only after the end of the nineteenth-century studies started to comprise floral UV reflection, which is invisible to humans but visible to the major groups of pollinators. Many flowers and inflorescences display colour patterns, an important signal for pollinators, promoted by the presence of at least two different colours within flowers or inflorescences, including colours in the UV waveband. For Neotropical savanna plant species, we characterised floral UV features using UV-photography and reflectance measurements. We tested (i) whether floral UV features were constrained by their shared ancestry, (ii) whether floral UV features were associated with pollinators, and (iii) whether floral UV features were associated with floral traits mediating these interactions, including floral resource, type of attraction unit and presence/absence of non-UV colour patterns. Of 80 plant species, ca. 70% were UV-patternless, most of them UV-absorbing. Approximately 30% presented one of three types of UV-patterns: bullseye, contrasting corolla markings oriented toward floral resources or contrasting reproductive structures, which were all considered as floral guides. Floral UV features were phylogenetically constrained and were associated with pollinators, floral resources and attraction unit, but not with non-UV colour patterns. UV-patternless flowers were associated with most of the pollination systems, while UV-patterned flowers were mainly associated with bee-pollination. UV-absorbing flowers comprised the only category with hawkmoth- and butterfly-pollinated flowers, and a high percentage of hummingbird-pollinated species. Nocturnal pollinated species were also commonly UV-absorbing, except for one UV-reflecting bat-pollinated species and one beetle-pollinated species with UV-reflecting stigmas. All types of floral UV features were associated with nectar; however, flowers with contrasting reproductive structures were mainly associated with pollen. There was an association between UV-absorbing species and the presence of inflorescences and intermediate attraction units. Our results evince that phylogenetic relatedness can constraint floral UV features’ diversification, but combinations of evolutionary and ecological processes may be expected in this scenario.

PHOTOCATALYTIC SOLAR OXIDATION OF ACESULFAME-K. EFFECT OF INITIAL pH, CATALYST DOSE AND OXIDANT CONCENTRATION

Aqueous acesulfame-K was oxidized in a pilot solar photocatalytic reactor, equipped with a compound parabolic collector (CPC). The reactor has an area of 0.40 m2 of CPC collectors with geometrical concentration ratio of 1 and a simple mechanical configuration. The experiments of photocatalytic oxidation of acesulfame-K were performed by treating 2 L of water with an initial concentration of 15 mg/L. The photocatalyst used was P25 (Aeroxide TiO2) from Evonik. One of the goals of the research work was to find a set of conditions to efficiently remove this emerging concern pollutant. Results allowed proposing a set of reaction conditions that lead to a high removal of acesulfame-K. In addition, the experimental design allowed determining the effect of initial pH as well as the impact of initial concentrations of photocatalyst and chemical oxidant. The removal efficiency of acesulfame-K and related UV-absorbing species reached values up to 96-99 % and there was not a quantifiable amount of intermediate products (analyzed as UV absorbing species). Despite reaction time was fixed in 3 h for all the experiments, oxidation efficiencies higher than 95 % were reached at 2 h of reaction or even before.

PHOTOCATALYTIC SOLAR OXIDATION OF ACESULFAME-K. EFFECT OF INITIAL pH, CATALYST DOSE AND OXIDANT CONCENTRATION

Aqueous acesulfame-K was oxidized in a pilot solar photocatalytic reactor, equipped with a compound parabolic collector (CPC). The reactor has an area of 0.40 m2 of CPC collectors with geometrical concentration ratio of 1 and a simple mechanical configuration. The experiments of photocatalytic oxidation of acesulfame-K were performed by treating 2 L of water with an initial concentration of 15 mg/L. The photocatalyst used was P25 (Aeroxide TiO2) from Evonik. One of the goals of the research work was to find a set of conditions to efficiently remove this emerging concern pollutant. Results allowed proposing a set of reaction conditions that lead to a high removal of acesulfame-K. In addition, the experimental design allowed determining the effect of initial pH as well as the impact of initial concentrations of photocatalyst and chemical oxidant. The removal efficiency of acesulfame-K and related UV-absorbing species reached values up to 96-99 % and there was not a quantifiable amount of intermediate products (analyzed as UV absorbing species). Despite reaction time was fixed in 3 h for all the experiments, oxidation efficiencies higher than 95 % were reached at 2 h of reaction or even before.

Picosecond Pulse Radiolysis Study on the Radiation-Induced Reactions in Neat Tributyl Phosphate.

The ultrafast radiolytic behavior of tributyl phosphate, TBP, has been investigated using 7 ps electron pulses with 7 MeV kinetic energy, from which two key species have been observed and characterized: the TBP solvated electron (eTBP-) and the TBP triplet excited state TBP* (3a) or its fragmentation products. The eTBP- exhibits a broad absorption band in the visible and near-infrared (NIR) spectrum, with a maximum beyond our 1500 nm detection limit. Nitromethane was used to scavenge eTBP- to confirm its absorption spectrum and to determine its associated rate coefficient, 1.0 × 1010 M-1 s-1. The electron's molar extinction coefficients were found by an isosbestic method using biphenyl as a solvated electron scavenger. The time-dependent radiolytic yield of eTBP- was also determined directly from 7 ps to 7 ns and compared with those in water, tetrahydrofuran, and diethyl carbonate. In less than 10 ns, the decay is not due to the reaction with other solvent molecules and is instead predominantly due to the reactions with cations issued from the proton transfer by the TBP radical cation (TBP•+). In addition to eTBP-, another absorption band, stable up to 7 ns, was identified in the visible range. This has been attributed mainly to the TBP triplet excited state, TBP*(3a), by a combination of molecular modeling methodologies. Interestingly, we did not observe any absorption band in the visible nor in the NIR range arising from TBP•+. Calculations suggest that TBP•+ undergoes rapid proton transfer to yield a UV-absorbing species, TBP(-H+). Experimental results and supporting molecular simulations provide detailed identification of the earliest species yielded from the radiolysis of neat TBP.

Use of a heated graphite scrubber as a means of reducing interferences in UV-absorbance measurements of atmospheric ozone

Abstract. A new solid-phase scrubber for use in conventional ozone (O3) photometers was investigated as a means of reducing interferences from other UV-absorbing species and water vapor. It was found that when heated to 100–130 °C, a tubular graphite scrubber efficiently removed up to 500 ppb ozone and ozone monitors using the heated graphite scrubber were found to be less susceptible to interferences from water vapor, mercury vapor, and aromatic volatile organic compounds (VOCs) compared to conventional metal oxide scrubbers. Ambient measurements from a graphite scrubber-equipped photometer and a co-located Federal equivalent method (FEM) ozone analyzer showed excellent agreement over 38 days of measurements and indicated no loss in the scrubber's ability to remove ozone when operated at 130 °C. The use of a heated graphite scrubber was found to reduce the interference from mercury vapor to ≤ 3 % of that obtained using a packed-bed Hopcalite scrubber. For a series of substituted aromatic compounds (ranging in volatility and absorption cross section at 253.7 nm), the graphite scrubber was observed to consistently exhibit reduced levels of interference, typically by factors of 2.5 to 20 less than with Hopcalite. Conventional solid-phase scrubbers also exhibited complex VOC adsorption and desorption characteristics that were dependent upon the relative humidity (RH), volatility of the VOC, and the available surface area of the scrubber. This complex behavior involving humidity is avoided by use of a heated graphite scrubber. These results suggest that heated graphite scrubbers could be substituted in most ozone photometers as a means of reducing interferences from other UV-absorbing species found in the atmosphere. This could be particularly important in ozone monitoring for compliance with the United States (U.S.) Clean Air Act or for use in VOC-rich environments such as in smog chambers and monitoring indoor air quality.

Time-resolved sub-ppm CH3 detection in a shock tube using cavity-enhanced absorption spectroscopy with a ps-pulsed UV laser

In this study we report a novel UV laser absorption diagnostic for sensitive and time-resolved measurement of methyl radicals (CH3) in shock tube kinetics studies, utilizing a frequency-quadrupled, high-repetition-rate (78 MHz) ps-pulsed Ti-sapphire laser targeted at the strong rovibronic transition of CH3 at 216.62 nm. This diagnostic employs a cavity-enhanced absorption spectroscopy (CEAS) strategy that amplifies the absorbance signal by a gain factor of about 50, which enables sub-ppm detection limit of CH3 to be achieved under typical shock conditions of 1200 K < T < 2500 K and P ∼ 1 atm, in a shock tube of ∼15 cm inner diameter. The use of a pulsed laser also enables an on-axis CEAS configuration, which yields a time resolution of 2–4 µs. For example, CH3 measurements were performed in shock tube decomposition experiments of dilute ethane and methane mixtures, demonstrating the substantial improvement in detection sensitivity of the current diagnostic compared to conventional single-pass absorption measurements. This new diagnostic promises to be a powerful tool for future shock tube chemical kinetics studies, and can be potentially extended to the detection of other UV-absorbing species.

Optical soundings of cars' exhaust and urban atmosphere with laser-plasma light and open-path spectrophotometry

Optical soundings of i.c. engines' exhaust and urban atmosphere have been carried out, using an Open-Path UV Spectrophotometer, based on a Laser-Produced Plasma as the light source. The measured optical depths are numerically inverted in terms of known species, by means of the Non-negative Least-Squares (NLS) algorithm. Concentrations of major UV-absorbing species are evaluated at the tailpipe of in-use vehicles (NO, NO2, EC) and in urban atmosphere over a 200m-long optical path (O2, O3. NO2, EC, nitrates). Also, we report on the detection in both environments of an additional compound, the nanometric organic carbon (NOC), known to form in fuel-rich flames, which is not considered yet in ordinary emissions inventories. Also, the problem of dilution/dispersion of primary species is addressed, by tracking the combustion-formed CO2 at the tailpipe and in urban atmosphere.

Determination of different classes of amines with capillary zone electrophoresis and contactless conductivity detection

The use of contactless conductivity detection for the determination of different organic amines in CE was successfully demonstrated. Aliphatic non UV-absorbing species could be determined along absorbing compounds by measuring the conductivity of their protonated forms. The species tested included short-chained aliphatic primary, secondary and tertiary amines, branched aliphatic amines, diamines, hydroxyl-substituted amines as well as species incorporating aromatic and non-aromatic cyclic moieties. Highest sensitivity was obtained with BGE solutions containing solely acetic acid. A concentration of 0.5 M at a pH value of 2.5 was used. Detection limits were in the order of 1 microM. Complete separation of cis- and trans-1,2-diaminocyclohexane could be achieved by adding 18-crown-6 as modifier to the electrolyte solution.

Reversed-phase HPLC with UV detection for the determination of N-acetylaspartate and creatine

Reversed-phase HPLC with UV detection for the determination of N-acetylaspartate and creatine

Isolation and characterization of a new ribosome inactivating protein, momorgrosvin, from seeds of the monk's fruit Momordica grosvenorii

Momorgrosvin, a single-chained glycoprotein with a molecular weight of 27.7 kilodaltons and an isoelectric point of about 9 was isolated from the seeds of Momordica grosvenorii (Family Cucurbitaceae). The isolation procedure entailed acetone precipitation, affinity chromatography on Hi Trap Blue, cation exchange chromatography on Resource S and size exclusion chromatography on Superose 12. The sequence of the first eighteen N-terminal amino acid residues of momorgrosvin exhibited homology to those of RIPs from other Momordica species. Momorgrosvin inhibited protein synthesis in the rabbit reticulocyte lysate system with an IC 50 of 0.3 nM and displayed RNA N-glycosidase activity giving rise to the diagnostic Endo's band at a concentration as low as 9 nM. The protein acted on tRNA to produce acid-soluble uv-absorbing species.

Speciation of Trace Elements, Binding and Non-binding with Proteins in Human Blood Serum, by Surfactant-Mediated HPLC with Element-Selective Detection by ICP-MS

The total concentrations of some trace elements in blood serum are generally used as convenient parameters in medical diagnosis because of their extreme importance for diagnosis of health and disease,1 as well as their easy accessibility. However, most of the trace elements in blood serum are combined with biological substances to have their own specific functions. Therefore, more information about chemical species of the elements, in addition to the total concentrations,2,3 is required for the investigations on the biological and physiological functions of trace elements as well as for biomedical diagnosis of the physical conditions. Chemical species of trace elements in blood serum are generally divided into two chemical forms. 2 One is protein-associated species, which play important roles in enzymatic activities, and in transportation and storage of trace elements. The other is protein-non-associated species, which are related to membrane transport or excretion of the elements. In a previous work, 4 the present authors developed a surfactant-mediated chromatographic separation system which allowed simultaneous multi-component separation, based on the mixed-mode separation properties such as size-exclusion, electrostatic and hydrophobic interactions. In this system, large-molecule species (MW 6500‐2000000) were eluted within 3 min of elution time, while small-molecule species (MW< 1000) were eluted after 3.5 min. Thus, the surfactant-mediated separation system could be used for direct injection analysis of the serum samples to separate small-molecule drugs rapidly from proteins in human blood serum without any pretreatment, 4 where the UV absorption detector was employed. Since the surfactant-mediated separation column also allows us to couple with inductively coupled plasma mass spectrometry (ICP-MS) for simultaneous multielement detection, the HPLC/ICP-MS combined system using the surfactant-mediated separation column can be applied to speciation of trace elements in blood serum with direct sample injection. In the present paper, we report a preliminary study on speciation of trace elements binding and non-binding with proteins in human blood serum which was carried out by using the surfactant-mediated HPLC/ICP-MS system. The HPLC system was composed of a pump (Model LC10AD; Shimadzu, Kyoto), a sample injector (Model V7, Pharmacia-LKB, Uppsala, Sweden) with a 20 µl sample loop, and a UV absorption detector (Model 870-UV; Jasco, Tokyo). The UV detector was used for detection of UV-absorbing species. In the present experiment, an ODS column (L-column; 4.6 mm i. d. × 250 mm long; Chemical Inspection and Testing Institute, Tokyo) dynamically coated with 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS), zwitterionic bile acid derivative, was employed as the separation column. The CHAPS-coated ODS column was prepared in a manner similar to that used in the previous paper. 4 The mobile phase of 0.2 mM Tris‐HCl buffer solution (pH 7.4) was used at a flow rate of 0.7 ml min ‐1 , into which 0.2 mM CHAPS was added to prevent the degradation of the separation column. For the on-line element-selective detection, an ICP-MS instrument (Model SPQ8000A, Seiko Instruments, Chiba) was used with direct introduction of the eluent from the column into the nebulizer of ICP-MS via a Teflon tubing. The ICP-MS instrument was operated using a time-sequential program, which allowed simultaneous multielement chromatogram measurements. The human blood serum samples were obtained from healthy volunteers. The sampling and pretreatment procedures of the serum samples were performed in a manner similar to those in the previous paper. 5 The CHAPS-coated ODS column with diverse physicochemical properties can separate a variety of solutes, such as inorganic anions, amino acids, aromatic compounds, and

Intrinsic ribonuclease activities in ribonuclease and ribosome-inactivating proteins from the seeds of bitter gourd

Alpha- and beta-momorcharins are ribosome-inactivating proteins present in the seeds of the bitter gourd ( Momordica charantia). Both of them possess ribonuclease activity which may account for some of their biological properties. However, the activity is weak and hence it is important to confirm that the ribonuclease activity observed is not due to any contamination. To this end, the ribonuclease from the seeds of M. charantia (RNase-MC) was purified and compared with the ribonuclease activity of the momorcharins. Purification was achieved by ion-exchange chromatographies on DEAE–cellulose, SP-Sepharose and Mono-S. RNase-MC had a molecular mass of 22 kDa. It acted on tRNA to release acid-soluble UV-absorbing species with a pH optimum around 6.0–6.5. When polyhomoribonucleotides were used as substrates, it was found that RNase-MC acted preferentially on polyU but exerted much weaker activity on polyC, polyG and polyA. Chromatographic analysis of the reaction product indicated that mono- and oligo-ribonucleotides, but not free base, were generated from polyU, suggesting that the enzymatic action involved ribonucleolytic cleavage. RNase-MC exhibited a much more potent (at least 1000-fold higher) ribonuclease activity than α- and β-momorcharins. RNase-MC, α-momorcharin and β-momorcharin were separable on Mono-S, indicating that the ribonuclease activities present in the three proteins were distinct entities.

A Glow Discharge−Conductivity Detector for Carbohydrates in Aqueous Chromatography

A new glow discharge-conductivity detector is shown to respond to low nanomolar and femtomole quantities of carbohydrates in aqueous high-performance liquid chromatography. Its sensitivity is comparable to those of the most sensitive detection methods available for carbohydrate analysis. The atmospheric pressure argon glow discharge is sustained between two metallic electrodes and is operated at the output of a liquid chromatography system. The glow discharge induces oxidation in the eluting carbohydrates, forming acid(s) in amounts proportional to carbohydrate concentration. The relative acid concentration is monitored with a conductivity detector placed downstream from the discharge region. A detailed description of the detector and the parameters that control sensitivity is presented. The formation of hydrogen peroxide, hydronium ion, and UV-absorbing species in aqueous flowing glucose solutions is also studied using the glow discharge-conductivity detector. A comparison between these products and the products formed under similar conditions using high-energy radiation is discussed.

Application of capillary electrophoresis in atmospheric aerosol analysis: determination of cations

The feasibility of using capillary electrophoresis (CE) with a new electrolyte system for the analysis of alkali metal, alkaline-earth metal cations and ammonium in atmospheric aerosols has been demonstrated. 1,1′-Di- n-heptyl-4,4′-bipyridinium (DHPB) hydroxide was used as the UV-absorbing species, which allowed for the indirect UV detection of cations at 280 nm, while glycine, 18-crown-6 ether and methanol were employed to improve the separation of the studied cations. The precision of migration time and peak area was better than 0.2% and 5%, respectively. Separation efficiencies were between 90 000 and 600 000 theoretical plates per meter, and detection limits between 9 and 60 ng/ml. With the described electrolyte composition it is also possible to determine manganese and cadmium, and short-chain aliphatic amines. The comparison of CE results with ion chromatography obtained for cations in atmospheric aerosols is presented.

Ser-143 Is an Essential Active Site Residue in Histidine Ammonia Lyase of Pseudomonas putida

Site directed mutagenesis was used to investigate the role of Ser-143 in enzyme activity and as a point for attack by cyanide or L-cysteine, two irreversible inhibitors of histidine ammonia-lyase (histidase). Two mutant proteins, a S143A substitution and an A142S-S143A transposition,were made. Both mutant histidases completely lost all enzymatic activity. Western blots with anti-histidase antibodies revealed that the mutant proteins were being expressed at a level equal to that of the wild-type protein. The purified mutant proteins could not incorporate [ 14C]cyanide nor could they generate the UV-absorbing species normally observed when L-cysteine modifies wild-type histidase. These results support the hypothesis that Ser-143 is the binding site for an as yet unidentified histidase cofactor.

Fluorescence of wool grease

Wool yolk is highly fluorescent. The amount of wool yolk and intensity of wool yolk fluorescence varies along a Merino wool fibre. The fluorescence intensity of the yolk increases on travelling from tip to the root. Two components-‘grease’ and chlorophyll-contribute to the fluorescence of wool grease. The ‘grease’ fluorescence reveals the presence of a number of chromophores/components. An attempt to separate the chemical constituents and identify them was only partly successful as each of nine fractions obtained by preparatory thin-layer chromatography exhibited similar fluorescence properties. There are two types of chlorophyll presentchlorophyll a and chlorophyll b. Chlorophyll could be detected in all the wool greases examined. Artificial UV irradiation gave rise to a number of complex photochemical reactions including the destruction of chlorophyll. After an initial decrease in fluorescence intensity (for unknown reasons), there is a change of non-fluorescing visible-absorbing species to fluorescing UV-absorbing species before a final change in the latter to the former. The relative size of the changes was different for two different wool greases examined. In-situ studies using blue light as the irradiation source suggest that it also causes a (slower) decrease in fluorescence intensity, but proportionally faster for the ‘grease’ component than for the chlorophyll component.

125I-labelled aprotinin as reagent for simultaneous determination of serine proteinases by hydrophobic interaction chromatography

A previously described method for determining more than one serine proteinase simultaneously by hydrophobic interaction chromatography of their complexes with aprotinin is inapplicable when other UV-absorbing species are co-eluted. The suitability of125I-labelled aprotinin has therefore been tested as a reagent in the analysis of mixtures containing trypsin, α-chymotrypsin and kallikrein taken as models, in the presence of ribonuclease and lysozyme. A new procedure is described which, without introducing changes in the chromatographic separation, allows direct determination of serine proteinases in terms of molarity. Results obtained in experiments with solutions containing from 0.20 to 30.00 nmol of each serine proteinase are reported.

Identification of Ser 143 as the Site of Modification in the Active Site of Histidine Ammonia-Lyase

Histidine ammonia-lyase (histidase) from Pseudomonas putida was irreversibly inactivated by L-cysteine at pH 10.5 in the presence of oxygen. Inactivation was accompanied by the formation of a new uv-absorbing species centered around 340 nm. L-[ 35S]cysteine labeling experiments revealed that 4 mol of L-cysteine was bound per mole of enzyme tetramer upon complete modification. However, the radiolabel was dissociated from the protein under denaturing conditions without loss of the 340-nm absorbance. Prior inactivation of histidase by cyanide, borohydride, or bisulfite precluded the formation of the 340-nm species in subsequent L-cysteine modification experiments. This suggests a common target site for modification of histidase by all of these reagents. Based on its strong absorbance at 340 nm an octapeptide was isolated from L-cysteine-inactivated histidase following trypsin and staphylococcal V8 protease digestion. Electrospray MS/MS revealed that this peptide (Gly 138SerValGlyAlaSerGlyAsp 145) contained an unidentified modification of mass 184 Da located on Ser 143. This peptide and the serine residue are conserved in all histidases and phenylalanine ammonia-lyases for which the amino acid sequence is available. Ser 143 represents the binding site for an electrophilic cofactor required for histidase activity.

Separation and identification of desferrioxamine and its iron chelating metabolites by high-performance liquid chromatography and fast atom bombardment mass spectrometry: Choice of complexing agent and application to biological fluids

An HPLC-based method capable of separating desfer-rioxamine (DFO) and its iron chelating metabolites from uv-absorbing species present in biological fluids has been developed. This method relies on the use of nitrilotriacetic acid (NTA) as the complexing agent in the mobile phase, instead of EDTA, previously used in HPLC methods. The use of NTA ensures that iron contamination present in buffers and bound to the column does not interfere with analysis. The disadvantages of using EDTA are discussed. The identity of the iron chelating metabolites of DFO present in the urine of patients with β-thalassemia major has been established using FAB mass spectrometry. The metabolism of DFO, reported in this study, takes place almost exclusively at the N-terminal region of the molecule and is in many respects similar to the degradation of the amino acid lysine. In addition, a metabolite which corresponds to N-hydroxylation of the terminal amino group has been identified.