Deactivation kinetics and response surface analysis of the stability of alpha-L-rhamnosidase from Penicillium decumbens.

The stability of the mixed enzyme preparation Naringinase from Penicillium decumbens was studied in dependence of the temperature, the pH value, and the enzyme concentration by means of response surface methodology. Deactivation kinetics by formation of an intermediate state was proposed for fitting deactivation data. Empirical models could then be constructed for prediction of deactivation rate constants, specific activity of intermediate state, and half-life values under different incubation conditions. From this study, it can be concluded that (1) Naringinase is most stable in the pH range of 4.5-5.0, being quite sensitive to lower pHs (<3.5) and (2) the glyco-enzyme is a rather thermo-stable enzyme preserving its initial activity for long times when incubated at its optimal pH up to temperatures of 65 degrees C. Enriched alpha-L-rhamnosidase after column treatment and ultrafiltration presented similar deactivation kinetics pattern and half-life values as the unpurified enzyme. Thus, any influence of low molecular weight substances on its deactivation is most probably negligible. The intermediate state of the enzyme may correspond to unfolding and self-digestion of its carbohydrate portion, lowering its activity relative to the initial state. The digestion- and unfolding-grade of this intermediate state may also be controlled by the pH and temperature of incubation.

New insights into the mechanisms of the thermal Fenton reactions occurring using different iron(II)-complexes.

Although the Fenton reagent (a mixture of hydrogen peroxide and an iron(II) salt) has been known for more than a century, the manifold mechanisms occurring during the thermal Fenton reaction are still under discussion. Indeed, this discussion served as a powerful driving force for the steadily increasing insight into the field of inorganic radical and electron transfer chemistry. In this work, an experimental approach towards the elucidation of the first steps taking place in the reaction between several iron(II)-complexes and hydrogen peroxide (H2O2) in water at pH = 3.0 is presented. 2,4-xylidine (2,4-dimethylaniline) reacts differently with reactive intermediates via the addition or hydrogen abstraction by the hydroxyl radical (HO*) or electron transfer reactions to higher valent iron-species, such as a hydrated ferryl-complex (Fe(IV)). The chemical reactivity of the employed iron(II)-complexes with H2O2 differed strongly depending on their ground-state one-electron oxidation potentials. The results are interpreted in accordance with the paradigm originally developed by Goldstein et al. which is based on the evidence obtained from the Marcus theory that outer-sphere electron transfer reactions between metal complexes are not likely to occur because they are too slow. Therefore, most of the "Fenton-reagents" form transient metal complexes, which can be described as [LnFe-H2O2]m+. They form, depending on the reaction conditions, either the hydroxyl radical or higher-valent iron complex species.

Degradation of polyvinyl alcohol (PVA) by homogeneous and heterogeneous photocatalysis applied to the photochemically enhanced Fenton reaction.

The reaction mechanism of the oxidative degradation of polyvinyl alcohol (PVA) by the photochemically enhanced Fenton reaction was studied using a homogeneous (Fe2+(aq) + H2O2) and a heterogeneous reaction system (iron(III)-exchanged zeolite Y+ H2O2). In the homogeneous Fenton system, efficient degradation was observed in a batch reactor, equipped with a medium pressure mercury arc in a Pyrex envelope and employing 80% of the stoichiometric amount of H2O2 required for the total oxidation of PVA and a concentration ratio as low as I mole of iron(II) sulfate per 20 moles of PVA sub-units (C2H40). Model PVA polymers of three different molecular weights (15,000, 49,000 and 100,000 g mol(-1)) were found to follow identical degradation patterns. Strong experimental evidence supports the formation of supermacromolecules (MW: 1-5 x 10(6) g/mol) consisting of oxidized PVA and trapped iron(III) at an early reaction stage. Low molecular weight intermediates, such as oxalic acid, formic acid or formaldehyde were not found during PVA degradation in the homogeneous Fenton system, and we may deduce that the manifold of degradation reactions is mainly taking place within the super-macromolecules from which CO2 is directly released. However, in the heterogeneous Fenton system, the reaction behavior was found to be distinctly different: a decrease of the molecular weights of all three tested monodisperse PVA samples was observed by the broadening of the GPC-traces during irradiation, and oxalic acid was formed. The results lead to the mechanistic hypothesis that during the heterogeneous Fenton process, the cleavage of the PVA-chains may occur at random positions, the reactive centres being located inside the iron(III)-doped zeolite Y photocatalysts.

Optimal experimental design and artificial neural networks applied to the photochemically enhanced Fenton reaction.

Among advanced oxidation processes (AOPs), the photochemically enhanced Fenton reaction may be considered as one of the most efficient for the degradation of contaminants in industrial wastewater. This process involves a series of complex reactions. Therefore, an empirical model based on artificial neural networks has been developed for fitting the experimental data obtained in a laboratory batch reactor for the degradation of 2,4-dimethyl aniline (2,4-xylidine), chosen as a model pollutant. The model describes the evolution of the pollutant concentration during irradiation time as a function of the process conditions. It has been used for simulating the behavior of the reaction system in sensitivity studies aimed at optimizing the amounts of reactants employed in the process, an iron(III) salt and hydrogen peroxide, as well as the temperature. The results show that the process is most sensitive to the concentration of iron(III) salt and temperature, whereas the concentration of hydrogen peroxide has a minor effect.

Role for nucleolin/Nsr1 in the cellular localization of topoisomerase I.

Nucleolin functions in ribosome biogenesis and contains an acidic N terminus that binds nuclear localization sequences. In previous work we showed that human nucleolin associates with the N-terminal region of human topoisomerase I (Top1). We have now mapped the topoisomerase I interaction domain of nucleolin to the N-terminal 225 amino acids. We also show that the Saccharomyces cerevisiae nucleolin ortholog, Nsr1p, physically interacts with yeast topoisomerase I, yTop1p. Studies of isogenic NSR1(+) and Deltansr1 strains indicate that NSR1 is important in determining the cellular localization of yTop1p. Moreover, deletion of NSR1 reduces sensitivity to camptothecin, an antineoplastic topoisomerase I inhibitor. By contrast, Deltansr1 cells are hypersensitive to the topoisomerase II-targeting drug amsacrine. These findings indicate that nucleolin/Nsr1 is involved in the cellular localization of Top1 and that this localization may be important in determining sensitivity to drugs that target topoisomerases.

Nonradiative and radiative deactivation of singlet molecular oxygen (O2(a1deltag)) in micellar media and microemulsions.

The effects of microheterogeneous media (micelles and microemulsions) on the lifetime and, to our knowledge for the first time, on the emission of singlet molecular oxygen (O2 (a1Ag), denoted as 1O2) were investigated. Micellar media and various types of microemulsions based on anionic (sodiumdodecyl sulfate), cationic (cetyltrimethylammonium chloride) or nonionic (Triton X-100) surfactants were formulated for this purpose. The nonradiative and radiative deactivation rate constants (k(d) and k(e), respectively) were determined in selected microheterogeneous media and in the pure solvents used for their formulation, by combining steady-state and time-resolved 1O2, luminescence detection techniques. We have shown that a simple additive model, as used in homogeneous mixtures of solvents, was inadequate for predicting values of k(d) and k(e) in organized media. In contrast, both 1O2 lifetimes (taudelta = 1/k(d)) and k(e) in the microheterogeneous systems investigated could be predicted with good precision from the composition of the media and the taudelta and k(e) values in the pure solvents, using a two-pseudophase kinetic model for the 1O2 distribution. Such a model takes into account the average times spent by 1O2 in the aqueous and lipophilic pseudo-phases of the organized media, the corresponding equilibrium constant (Keq) depending on the nature of the system.

Effect of the microenvironment on the efficiency of singlet oxygen (O2(1 delta g)) production by photosensitizing anti-inflammatory drugs.

The influence of the medium on the quantum yields of singlet oxygen (O2(1 delta g)) production (phi delta) by a series of photosensitizing non-steroidal anti-inflammatory drugs (NSAID) derived from 2-arylpropionic acid (APA) has been investigated. Four-component oil-in-water and water-in-oil microemulsions, based on anionic and cationic surfactants, have been employed as the simplest models to mimic more complex biological environments. phi delta values have been determined by monitoring the singlet oxygen (1O2) luminescence at 1270 nm upon continuous excitation of the drugs under air-equilibrated conditions. Results indicate that phi delta values are highly affected by the medium, being higher in microheterogeneous systems than in (homogeneous) solution. Some of the anti-inflammatory derivatives are very efficient 1O2 sensitizers: e.g., values of apparent phi delta as high as 0.86 (+/- 0.04) and 0.70 (+/- 0.03) have been found for tiaprofenic acid and suprofen, respectively. The location of the drugs in the interfacial region of the microemulsions combined with their high phi delta values suggest that type II reactions may play a significant role in the overall photodynamic process in more complex organized media, such as biological membranes.

Antioxidant activity of flavonoids: efficiency of singlet oxygen (1 delta g) quenching.

Flavonoids, polyphenolic pigments widely present in plants, have been reported to act as scavengers of various oxidizing species. However, most often an overall antioxidant effect was measured. In this paper we report the results of a systematic study of the reactivity of 13 selected flavonoids (from the flavonol, flavone, flavanone and flavane families) with singlet oxygen (1O2(1 delta g)) in order to establish a structure-activity relationship. The rate constants of the chemical reaction of these flavonoids with 1O2(k r) and their rate constants of 1O2 physical quenching (kq) have been determined by kinetic measurements and near-IR singlet oxygen luminescence. The efficiency of the physical quenching is mainly controlled by the presence of a catechol moiety on ring B, whereas the structure of ring C (particularly the presence of a hydroxyl group activating the double bond) is the main factor determining the efficiency of the chemical reactivity of these compounds with 1O2. The total reactivity factor determining the efficiency of the chemical reactivity of these compounds with 1O2. The total reactivity scale is dominated by kq, which is in general higher than kr. (+)-Catechin is the most efficient quencher of the series.

Primary effects of singlet oxygen sensitizers on eggs and embryos of sea urchins.

Photodynamic effects of rose bengal, a well-known singlet oxygen sensitizer, and of haematoporphyrin derivative, the most widely used sensitizer in photodynamic therapy of tumours, could be visualized using sea urchin eggs and embryos. This biological material is a valuable model for the analysis of mechanisms and/or sites of the photodynamic action occurring in any living tissue. Depending on the sensitizer used, singlet oxygen may be identified as the main mediator of the cytotoxic effects observed. Besides observations made on the living, in particular within the context of fertilization ability of the egg cell, gross damages of the cells are morphologically analysed by scanning electron microscopy. The results support the working hypothesis explaining the different susceptibility of healthy and tumour cells for photosensitization as a cell cycle phenomenon.