Oxidation of catechol in plants : III. Purification and properties of the diphenylene dioxide 2,3-quinone-forming enzyme system from Tecoma leaves

In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricalcium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120-fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of the enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35 °C. The Km for catechol was determined as 4 × 10−4 Image . The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activity. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show any peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.

Diketopyrrolopyrrole-Based Donor-Acceptor Copolymers as Organic Sensitizers for Dye Sensitized Solar Cells

In this work diketopyrrolopyrrole based copolymers (PDPP-BBT and TDPP-BBT) containing a donor-acceptor structural unit have been explored as organic Sensitizers for quasi-solid state dye Sensitized solar cells. Polymer-sensitized solar cells (PSSC) fabricated utilizing PDPP-BBT and TDPP-BBT as the active layer resulted in a typical power conversion efficiency of 1.43% and 2.41%, respectively. The power conversion efficiency of PSSCs based on TDPP-BBT With use of TiCl4-modified TiO2 photoanode was about 3.06%, attributed to the reduced back recombination reaction and more charge carriers in the external Circuit.

1-Anilino-8-naphthalene sulfonate (ANS) binding to proteins revisited: Investigation of dye binding to molten globule states by electrospray ionization mass spectrometry

The binding of 1-anilino-8-naphthalene-sulfonic acid to globular proteins at acidic pH has been investigated by electrospray ionization mass spectrometry (ESIMS). Mass spectra of apomyoglobin recorded in the pH range 2−7 establish that maximal ANS binding is observed at pH 4.0. As many as seven distinct species may be observed in the gas phase which correspond to protein molecules containing one to six molecules of bound ANS. At neutral pH only a single molecule of ANS is bound. In the case of cytochrome c, maximal binding is observed at pH 4.0, with five molecules being bound. Binding is suppressed at neutral pH. In both cases ESIMS demonstrates maximal ANS binding at pH values where the proteins have been reported to exist in molten globule states. ANS binding is not observed for lysozyme, which has a tightly folded structure over the entire pH range. Reduction of disulfide bonds in lysozyme leads to the detection of ANS-bound species at neutral pH. Binding is suppressed at low pH due to complete unfolding of the reduced protein. The results suggest that ESIMS may provide a convenient method of probing the stoichiometry and distribution of dye complexes with molten protein globules

Studies on nucleotidases in plants: Fluorescence and kinetic properties of nucleotide pyrophosphatase from mung bean (Phaseolus aureus) seedlings

Nucleotide pyrophosphatase of mung bean seedlings has earlier been isolated in our laboratory in a dimeric form (Mr 65,000) and has been shown to be converted to a tetramer by AMP and to a monomer by p-hydroxymercuribenzoate. All the molecular forms were enzymatically active with different kinetic properties. By a modified procedure using blue-Sepharose affinity chromatography, we have now obtained a dimeric form of the enzyme which is desensitized to AMP interaction. The molecular weight of the desensitized form of the enzyme was found to be the same as that of the native dimeric enzyme. However, the desensitized enzyme functioned with a linear time course, contrary to the biphasic time course exhibited by the native enzyme. In addition, it was not converted to a tetramer on the addition of AMP, had only one binding site for adenine nucleotides, and p-hydroxy-mercuribenzoate had no effect on the time course of the reaction or on the molecular weight of the enzyme. The temperature optimum of the desensitized enzyme was found to be 67 °C in contrast to the optimum of 49 °C for the native dimer. Fifty percent of the tryptophan residues of the desensitized enzyme were not accessible for quenching by iodide. Fluorescence studies gave Kd values of 0.34, 2.2, and 0.8 mImage for AMP, ADP, and ATP, which were close to the Ki values of 0.12, 2.2, and 0.9 mImage , respectively, for these nucleotides. The binding and inhibition studies with AMP and its analogs showed that the 6-amino group and the 5′-phosphate group were essential for the inhibition of the enzyme activity.

Overwintering ecology of northern field layer plants : snow and photosynthesis in Vaccinium vitis-idaea L.

Winter is a significant period for the seasonality of northern plants, but is often overlooked when studying the interactions of plants and their environment. This study focuses on the effects of overwintering conditions, including warm winter periods, snow, and snowmelt on boreal and sub-Arctic field layer plants. Wintertime photosynthesis and related physiological factors of evergreen dwarf shrubs, particularly of Vaccinium vitis-idaea, are emphasised. The work combines experiments both in the field and in growth chambers with measurements in natural field conditions. Evergreen dwarf shrubs are predominantly covered by snow in the winter. The protective snow cover provides favourable conditions for photosynthesis, especially during the spring before snowmelt. The results of this study indicate that photosynthesis occurs under the snow in V. vitis-idaea. The light response of photosynthesis determined in field conditions during the period of snow cover shows that positive net CO2 exchange is possible under the snow in the prevailing light and temperature. Photosynthetic capacity increases readily during warm periods in winter and the plants are thus able to replenish carbohydrate reserves lost through respiration. Exposure to low temperatures in combination with high light following early snowmelt can set back photosynthesis as sustained photoprotective measures are activated and photodamage begins to build up. Freezing may further decrease the photosynthetic capacity. The small-scale distribution of many field layer plants, including V. vitis-idaea and other dwarf shrubs, correlates with the snow distribution in a forest. The results of this study indicate that there are species-specific differences in the snow depth affinity of the field and ground layer species. Events and processes taking place in winter can have a profound effect on the overall performance of plants and on the interactions between plants and their environment. Understanding the processes involved in the overwintering of plants is increasingly important as the wintertime climate in the north is predicted to change in the future.

Studies in biogas technology. Part III. Thermal analysis of biogas plants

A thermal model for a conventional biogas plant has been developed in order to understand the heat transfer from the slurry and the gas holder to the surrounding earth and air respectively. The computations have been performed for two conditions : (i) when the slurry is at an ambient temperature of 20°C, and (ii) when it is at 35°C, the optimum temperature for anaerobic fermentation. Under both these conditions, the gas holder is the major “culprit” with regard to heat losses from the biogas plant. The calculations provide an estimate for the heat which has to be supplied by external means to compensate for the net heat losses which occur if the slurry is to be maintained at 35°C. Even if this external supply of heat is realised through (the calorific value of) biogas, there is a net increase in the biogas output, and therefore a net benefit, by operating the plant at 35°C. At this elevated temperature, the cooling effect of adding the influent at ambient temperature is not insignificant. In conclusion, the results of the thermal analysis are used to define a strategy for operating biogas plants at optimum temperatures, or at higher temperatures than the ambient.

Dye sensitized visible light degradation of phenolic compounds

The present research work reports the eosin Y (EY) and fluorescein (FL) sensitized visible light degradation of phenol, 4-chlorophenol (CP), 2,4-dichlorophenol (DCP) and 2,4,6-trichlorophenol (TCP) using combustion synthesized nano-TiO2 (CS TiO2). The rate of degradation of the phenolic compounds was higher in the presence of EY/CS TiO2 compared to FL/CS TiO2 system. A detailed mechanism of sensitized degradation was proposed and a mechanistic model for the rate of degradation of the phenolic compound was derived using the pyramidal network reduction technique. It was found that at low initial dye concentrations, the rate of degradation of the phenolic compound is first order in the concentration of the dye, while at high initial dye concentrations, the rate is first order in the concentration of the phenolic compound. The order of degradation of the different phenolic compounds follows: CP > TCP > DCP > phenol. The different phenolic and dye intermediates that were formed during the degradation were identified by liquid chromatography-mass spectrometry (LC-MS) and the most probable pathway of degradation is proposed. (C) 2010 Elsevier B.V. All rights reserved.

Studies on molecular evolution and structural features of double-headed inhibitors of ?-amylase and trypsin in plants

Plant seeds usually have high concentrations of proteinase and amylase inhibitors. These inhibitors exhibit a wide range of specificity, stability and oligomeric structure. In this communication, we report analysis of sequences that show statistically significant similarity to the double-headed alpha-amylase/trypsin inhibitor of ragi (Eleusine coracana). Our aim is to understand their evolutionary and structural features. The 14 sequences of this family that are available in the SWISSPROT database form three evolutionarily distinct branches. The branches relate to enzyme specificities and also probably to the oligomeric state of the proteins and not to the botanical class of the plant from which the enzymes are derived. This suggests that the enzyme specificities of the inhibitors evolved before the divergence of commercially cultivated cereals. The inhibitor sequences have three regions that display periodicity in hydrophobicity. It is likely that this feature reflects extended secondary structure in these segments. One of the most variable regions of the polypeptide corresponds to a loop, which is most probably exposed in the native structure of the inhibitors and is responsible for the inhibitory property.