Evaluation of activity of selected antioxidants on proteins in solution and in emulsions

Protection against protein oxidation by lipophilic and hydrophilic antioxidants in model systems using bovine serum albumin (BSA) in solution alone, or in an emulsion with linolenic acid methyl ester (LnMe) was found to be strongly dependent on the oxidation initiator. Tocopherol, Trolox, or the carotenoids astaxanthin and canthaxanthin were incubated with BSA or BSA/LnMe and oxidation was initiated either with the water-soluble azo-initiator 2,2′ azo-bis-(2-amidinopropane) hydrochloride (AAPH), or FeCl₃ and ascorbate, or the Fenton system using FeCl₂/EDTA/H₂O₂, or with the singlet oxygen generating species anthracene-9,10-dipropionic acid disodium 1,4 endoperoxide (NDPO₂).

The results show that all the antioxidants tested were inefficient in the system with FeCl₃/ascorbate. However, with the other initiating agents, the hydrophilic antioxidant, Trolox, was the most effective in preventing both protein and lipid oxidation. In contrast the lipophilic antioxidants were ineffective in preventing oxidation of BSA in aqueous solution, but did show some moderate antioxidative activity on protein and lipid in the BSA/LnMe system. Using the singlet oxygen generating system it was also demonstrated that Trolox always provided better protection of the protein than tocopherol and the carotenoids in both the BSA and the BSA/LnMe systems. In conclusion, prevention of protein oxidation using a water-soluble antioxidant has a protective effect on the lipid fraction and this approach deserves further attention in complex biological systems.     

Hyaluronic Acid Degradation by Ascorbic Acid and Influence of Iron

The effects of ascorbic acid, iron and ADP on hyaluronic acid, a compound present in inflamed joints, were investigated in an in vitro system. Ascorbic acid induces degradation of hyaluronic acid which increased in the presence of FeCl, and which is additionally stimulated by ADP chelated ferric ions. The hyaluronic acid degrading reactions induced by the Fe-III/ADP/ascorbic acid system were inhibited by catalase and formate to various extents whereas the presence of superoxide dismutase did not exert any inhibitory effect. Desferrioxamine, a specific iron chelator, completely inhibited hyaluronic acid depolymerisation by ascorbic acid as well as in combination with FeCl₃ or FeCl₃/ADP, respectively. We suggest that the ultimate hyaluronic acid degrading species is OH', generated via the Fe-III/ADP catalysed Haber Weiss reaction. There is also an indication for the involvement of perferryl or/and ferryl species in the degradation process.     

Comparison of the reaction products of oleic acid ozonized in BCl3-, HCl- and BF3-MeOH media

In BF₃-MeOH medium, the principal ozonolysis reaction products of oleic acid were methyl nonanoate (MMC₉) and dimethyl azelate (DMC₉) in yields of 98% with formation of minor secondary reaction products (methyl octanoate, nonaldehyde with nonaldehyde dimethyl acetal and dimethyl suberate, plus the C₉ half-ester-aldehyde, with its corresponding acetal). The gas-liquid chromatographic analysis of the ozonolysis reaction products in BCl₃- and HCl-MeOH revealed the existence of 4 major components with low yields of methyl nonanoate and dimethyl azelate (45–50%). The two other major reaction products, isolated by a combination of thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC), were identified as the chlorinated acetals, 1,1-dimethoxy-2-chloro-nonane and the 8-chloro-9,9-dimethoxy methyl nonanoate.     

Reactions of 6,6-dimethyl-2,2-bipyridyl with iron(II) in aqueous and non-aqueous media

Reaction of anhydrous FeCl₂ with 6,6^′-dimethyl-2,2^′-bipyridyl (dmby) in non-aqueous media gives the yellow, high spin, tetrahedral complex FeCl₂(dmby), which is characterized crystallographically, magnetically and by ¹H NMR spectroscopy. In contrast, reaction of FeCl₂ · 4H₂O with dmby in 0.1 M hydrochloric acid, the method of choice for preparing 3:1 and 2:1 iron(II) complexes of 2,2^′-bipyridyl, gives [H₂dmby][FeCl₄] and [Hdmby][FeCl₄], in which the dmby has been protonated. These complexes are also characterized crystallographically.     

Optimization of Biomass Conversion to Levulinic Acid in Acidic Ionic Liquid and Upgrading of Levulinic Acid to Ethyl Levulinate

Levulinic acid (LA) is a versatile platform chemical that can be derived from biomass as an alternative to fossil fuel resources. Herein, the optimization of LA production from glucose and oil palm fronds (OPF) catalyzed by an acidic ionic liquid; 1-sulfonic acid-3-methyl imidazolium tetrachloroferrate ([SMIM][FeCl₄]) have been investigated. Response surface methodology based on Box-Behnken design was employed to optimize the LA yield and to examine the effect and interaction of reaction parameters on the LA production. The reaction parameters include reaction temperature, reaction time, feedstock loading, and catalyst loading. From the optimization study, the predicted mathematical models for LA production from glucose and OPF covered more than 90 % of the variability in the experimental data. At optimum conditions, 69.2 % of LA yield was obtained from glucose, while 24.8 % of LA yield was attained from OPF and registered 77.3 % of process efficiency. The recycled [SMIM][FeCl₄] gave sufficient performance for five successive cycles. Furthermore, the optimum LA produced from glucose and OPF can be directly converted to ethyl levulinate through esterification over the [SMIM][FeCl₄] catalyst. This study highlights the potential of [SMIM][FeCl₄] for biorefinery processing of renewable feedstocks at mild process conditions.     

Corn stover pretreatment by inorganic salts and its effects on hemicellulose and cellulose degradation

Inorganic salts, NaCl, KCl, CaCl₂, MgCl₂, FeCl₂, FeSO₄, FeCl₃, and Fe₂(SO₄)₃, were studied as catalysts for the degradation of hemicellulose in corn stover. FeCl₃ significantly increased the hemicellulose degradation in aqueous solutions heated between 140 and 200 °C with high xylose recovery and low cellulose removal, amounting to 90% and <10%, respectively. Hemicellulose removal increased 11-fold when the corn stover was pretreated with 0.1 M FeCl₃ compared to pretreatment with hot water under otherwise the same conditions, which was also 6-fold greater than pretreatment with dilute sulfuric acid at the same pH. Optimum pretreatment conditions were found where the corn stover was pretreated with 0.1 M FeCl₃ at 140 °C for 20 min. Under such conditions, 91% of hemicellulose was removed, and the recovery of monomeric and oligomeric xylose in liquid fraction achieved 89%, meanwhile, only 9% of cellulose was removed.     

Lipid Peroxidation. Definition of Experimental Conditions for Selective Study of the Propagation and Termination Phases

To find experimental conditions to selectively study the propagation phase of lipoperoxidation we studied the lipoperoxidation, catalyzed by FeCl₂, of liposomes in a buffering condition where Fe²⁺ autoxidation and oxygen active species generation does not occur. Liposomes from egg yolk phosphatidylcholine. prepared by vortex mixing, do not oxidize Fe²⁺: on the contrary they oxidize Fe²⁺ when prepared by ultrasonic irradiation. Dimyristoyl phosphatidylcholine liposomes prepared by ultrasonic irradiation do not oxidize Fe²⁺. During sonication polyunsaturated fatty acid residues autoxidize and lipid hydroperoxides (LOOH) are generated. Only when LOOH are present in the liposimes Fe²⁺ oxidizes and its rate of oxidation depends on the amount of LOOH in the assay. The reaction results in the generation of both LOOH and thiobarbituric acid reactive material (TBAR): it is inhibited by butylated hydroxytoluene and has a acidic pH optimum; it is not inhibited by catalase and OH' scavengers. The reaction studied. thus, appears to be the chain branching and propagation phase of lipoperoxidation. When we studied the dependence of Fe²⁺ oxidation, LOOH and TBAR generation on FeCl₂ concentration, we observed that at high FeCl₂ concentrations the termination phase of lipoperoxidation was prevalent. Thus. by selecting the appropriate FeCl₂ concentration the proposed experimental system allows study of either the propagation or the termination phase of lipoperoxidation.     

Preparation and separation of methyl ethers of D-arabinose

All possible methyl ethers of methyl β-d-arabinofuranoside and methyl β-d-arabinopyranoside were prepared by partial methylation using Kuhn's method (Ag₂O and MeI in N,N-dimethylformamide) and Haworth's method (Me₂SO₄ and 40% NaOH in water). The ratios of the methyl ethers obtained from the furanoside were found to be considerably more dependent on the method of methylation than those from the pyranoside. All of the methyl ethers of d-arabinofuranose and d-arabinopyranose are separable by thin-layer chromatography on silica gel and paper electrophoresis in borate buffer.     

Coordination chemistry of acrylamide 6: Formation and structural characterization of [Fe(O-OC(NH2)CHCH2)6][Fe2OCl6]

The new acrylamide iron(II)/iron(III) complex [Fe(O-OC(NH₂)CHCH₂)₆][Fe₂OCl₆] (1) was obtained by the reaction of a mixture of anhydrous FeCl₂ and anhydrous FeCl₃ with acrylamide (molar ratio 1:2:6) in 98% pure commercial nitromethane under nitrogen atmosphere. According to an X-ray structural analysis, the acrylamide ligands in the cation are coordinated via the amide-oxygen atoms. The formation of the (μ-oxo)bis[trichloroferrate(III)]²⁻ anion presumably resulted from partial hydrolysis of FeCl₃ or [FeCl₄]⁻ by small amounts of water in the nitromethane and/or by the nitromethane itself.     

Microbial flocculant from Arcuadendron sp. TS-49

Summary A flocculant was purified from the culture broth of Archuadendron sp. TS-49 by a series of precipitations with acetone, 60% ammonium sulfate-butanol, salting-out by dialysis, and cetylpyridinium chloride. The flocculating activity was observed most highly at pH 3.0 and markedly enhanced by the addition of salts, especially in the case of FeCl₃ or FeSO₄. This bioflocculant efficiently flocculated all tested solids, including various microorganisms and organic/ inorganic materials. Qualitative analyses of the bioflocculant showed that it might contain hexosamine, uronic acid, neutral sugar, and protein.     

Improved degradation of lignocellulosic biomass pretreated by Fenton-like reaction using Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> magnetic nanoparticles

The ability of Fe₃O₄ Fenton-like reaction to produce glucose from lignocellulosic biomass was investigated. Fe₃O₄ magnetite nanoparticles were chemically synthesized from iron salts (a mixture of 1 M FeCl₂ and 2M FeCl₃) using an ammonia solution (30% NH₄OH). The synthesized Fe₃O₄ nanoparticles were further characterized by X-ray photoelectron spectroscopy, energy dispersive X-ray spectroscopy, scanning electron microscopy, and transmission electron microscopy. Reed stems and rice straw biomasses pretreated with optimized Fenton-like reagents (Fe₃O₄ and H₂O₂) increased glucose production by 177 and 87%, respectively, compared to the control without the catalysts.     

Study of the role of iron in the anticryptococcal activity of human serum and fluconazole

Anticryptococcal activity of human serum and apotransferrin in RPMI 1640 was studied in vitro. The effects of varying concentrations of FeCl₃ on this activity was investigated. Possible synergy of serum and apotransferrin with fluconazole was also measured. The fungistatic activity of human serum, whether lyophilized, stored at 4 °C, fresh frozen or purchased from commercial sources vs. Cryptococcus neoformans was comparable. There was no significant loss of fungistatic activity after freezing and thawing the serum up to 10 times. The fungistatic activity of human serum was similar when tested in different tissue culture media with the exception of Medium 199. The addition of apotransferrin (2.0 or 0.2 mg/ml) to RPMI 1640 had an inhibitory effect on cryptococcal growth. This effect was reversed by 20 M of FeCl₃ at both apotransferrin concentrations. By contrast, addition of FeCl₃ to human serum and RPMI 1640 did not reverse inhibition of growth. Fluconazole synergized with the human serum preparations described, but not with pooled commercial serum, for fungicidal activity. Synergistic activity of fluconazole and human serum was not affected by the addition of FeCl₃. Apotransferrin did not show any synergistic fungicidal activity with fluconazole.     

Active Transport of Iron in Bacillus megaterium: Role of Secondary Hydroxamic Acids

Kinetics of radioactive iron transport were examined in three strains of Bacillus megaterium. In strain ATCC 19213, which secretes the ferric-chelating secondary hydroxamic acid schizokinen, ⁵⁹Fe³⁺ uptake from ⁵⁹FeCl₃ or the ferric hydroxamate Desferal-⁵⁹Fe³⁺ was rapid and reached saturation within 3 min. In strain SK11, which does not secrete schizokinen, transport from ⁵⁹FeCl₃ was markedly reduced; the two ferric hydroxamates Desferal-⁵⁹Fe³⁺ or schizokinen-⁵⁹Fe³⁺ increased both total ⁵⁹Fe³⁺ uptake and the ⁵⁹Fe³⁺ appearing in a cellular trichloroacetic acid-insoluble fraction, although 10 min was required to reach saturation. Certain characteristics of transport from both ferric hydroxamates and FeCl₃ suggest that iron uptake was an active process. The growth-inhibitory effect of aluminum on strain SK11 was probably due to the formation of nonutilizable iron-aluminum complexes which blocked uptake from ⁵⁹FeCl₃. Desferal or schizokinen prevented this blockage. A strain (ARD-1) resistant to the ferric hydroxamate antibiotic A22765 was isolated from strain SK11. Strain ARD-1 failed to grow with Desferal-Fe³⁺ as an iron source, and it was unable to incorporate ⁵⁹Fe³⁺ from this source. Growth and iron uptake in strain ARD-1 were similar to strain SK11 with schizokinen-Fe³⁺ or the iron salt as sources. It is suggested that the ferric hydroxamates, or the iron they chelate, may be transported by a special system which might be selective for certain ferric hydroxamates. Strain ARD-1 may be unable to recognize both the antibiotic A22765 and the structurally similar chelate Desferal-Fe³⁺, while retaining its capacity to utilize schizokinen-Fe³⁺.     

Effect of Zinc, Copper, and Iron on Ochratoxin A Production

The effect of zinc, copper, and iron levels on production of ochratoxin A by Aspergillus ochraceus Wilhelm in a synthetic medium in a shake culture was investigated. Optimal concentrations of ZnSO₄, CuSO₄, and FeCl₃ for ochratoxin A production were 0.055 to 2.2 mg/liter, 0.004 to 0.04 mg/liter, and 1.2 to 24 mg/liter, respectively. Zinc and copper levels greater than optimum reduced the rate of ochratoxin accumulation without altering either glutamate or sucrose utilization. Ochratoxin A production was correlated with rapid utilization of sucrose by the fungus and decreasing pH of the medium. Most of the glutamic acid was removed from the medium prior to ochratoxin production. There was no correlation between mycelial dry weight and ochratoxin A production.     

Bioemulsifier production by Aspergillus niger MYA 135: presumptive role of iron and phosphate on emulsifying ability

Microbial emulsifiers are compounds employed in primary mechanisms for bioremediation of petroleum and other hydrocarbon pollutants from the environment. Although emulsifiers of biological origin are produced by microorganisms generally in response to growth on hydrocarbons, Aspergillus niger MYA 135 produced a bioemulsifier during fermentation in a sucrose-based culture medium at an initial pH of 5.0 and at 30°C. The production of bioemulsifiers can be strongly influenced by environmental factors. In this connection, a study of the effect of initial pH, the incubation temperature and presence of CaCl₂ or FeCl₃ in the culture medium was conducted. Emulsification index was increased by 112 and 206% at an initial pH 2.0 or in medium supplemented with FeCl₃, respectively. On the other hand, emulsifying ability of Aspergillus niger supernatants was detected during the exponential phase, suggesting that bioemulsifiers accumulation and microbial growth would be related. Interestingly, this study suggests that iron and/or phosphate ions would play a key role in maintaining the emulsifying ability. Finally, factorial design was also employed to study the effects of the initial pH, the presence of FeCl₃ and the concentration of KH₂PO₄ on the emulsification index.     

Effects of Environmental and Nutritional Factors on Production of the Polyketide Phytotoxin Coronatine by Pseudomonas syringae pv. Glycinea

Pseudomonas syringae pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The effects of environmental, nutritional, and host factors on growth and coronatine production by PG4180 were examined by varying the components of a defined basal medium which contained the following nutrients per liter: glucose (10 g), NH₄Cl (1 g), MgSO₄ · 7H₂O (0.2 g), KH₂PO₄ (4.1 g), K₂HPO₄ · 3H₂O (3.6 g), and FeCl₃ (2 μM). Bacterial growth was recorded as dry weight, and coronatine production was measured by high-performance liquid chromatography. Both growth and the quantity of coronatine synthesized were significantly affected by carbon source, nutrient levels (glucose, NH₄Cl, phosphate, Mg, and SO₄), amino acid supplements, and the presence of complex carbon and nitrogen sources. The yield of coronatine generally declined when conditions were varied from those in the basal medium. Coronatine production and growth were not affected when the pH was adjusted from 6.5 to 7.8. Increases in the osmolarity of the basal medium significantly decreased coronatine production without affecting growth. The addition of plant extracts, plant-derived secondary metabolites, or zinc did not affect growth or coronatine production, while the addition of millimolar levels of KNO₃ or micromolar levels of FeCl₃ significantly enhanced coronatine production. The yield of coronatine was maximized after a 7-day incubation at 18°C and 280 rpm. The results of the present study were used to formulate a medium which allowed for enhanced coronatine production in nearly all strains of P. syringae tested. A rapid method for extracting coronatine from small volumes of culture supernatant was also developed.     

Gibberellin-induced inhibition and promotion of sprouting in aerial tubers of Begonia evansiana Andr. in relation to photoperiodic treatment and tuber stage

Gibberellic-acid (GA₃) treatment, when applied within a period ranging from the start of short-day (SD) treatment until about 10 SD, GA₃ strongly inhibited formation of aerial tubers in response to SD and brought about sprouting of developing aerial tubers. In contrast, when applied after about 10 SD or more, GA₃ hastened the completion of the dormant state in the tubers and prolonged their dormancy. The dormancy-promoting effect of GA₃ on detached tubers increased with their degree of maturation. Application of growth retardants N-dimethylaminosuccinamic acid (B-9), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride (AMO-1618) and 2-chloroethyltrimethylammonium chloride (CCC) to the cuttings delayed the onset of dormancy in the aerial tuber. When the retardants were applied to detached aerial tubers, however, such a delay of dormancy was not observed, and GA₃ application did not inhibit sprouting in aerial tubers detached from CCC-treated cuttings.Abbreviations GA gibberellin - GA₃ gibberellic acid - SD short day(s) - LD long day(s) - SDP short-day plant - LDP long-day plant - CCC 2-chloroethyltrimethylammonium chloride - B-9 N-dimethylaminosuccinamic acid - AMO-1618 2-isopropyl-4-dimethyl-amino-5-methylphenyl-1-piperidine carboxylate methyl chloride     

Protein oxidation by the cytochrome P450 mixed-function oxidation system

This mini-review summarizes results of studies on the oxidation of proteins and low-density lipoprotein (LDL) by various mixed-function oxidation (MFO) systems. Oxidation of LDL by the O₂/FeCl₃/H₂O₂/ascorbate MFO system is dependent on all four components and is much greater when reactions are carried out in the presence of a physiological bicarbonate/CO₂ buffer system as compared to phosphate buffer. However, FeCl₃ in this system could be replaced by hemin or the heme-containing protein, hemoglobin, or cytochrome c. Oxidation of LDL by the O₂/cytochrome P450 cytochrome c reductase/NADPH/FeCl₃ MFO system is only slightly higher (25%) in the bicarbonate/CO₂ buffer as compared to phosphate buffer, but is dependent on all components except FeCl₃. Omission of FeCl₃ led to a 60% loss of activity. These results suggest that peroxymonobicarbonate and/or free radical derivatives of bicarbonate ion and/or CO₂ might contribute to LDL oxidation by these MFO systems.     

Iron(III) complexes using NNS reduced Schiff bases and NNOS coordinating tetradentate ligands: Synthesis, structure and catecholase activity

The orange-red colored complexes of the type [Fe(L_SB)Cl₃], 1, have been synthesized in excellent yields by reacting FeCl₃·6H₂O with L_SB in methanol. Here, L_SB is (2-(ethylthio)-N-(pyridin-2-ylmethyl)ethanamine), (L_SB¹) and (2-(benzylthio)-N-(pyridin-2-ylmethyl)ethanamine) (L_SB²). Similarly, FeCl₃·6H₂O reacted with 2-(((2-(ethylthio) ethyl) (pyridin-2-ylmethyl)amino)methyl)phenol (HL¹), 2-(((2-(ethylthio)ethyl)(pyridin-2 ylmethyl)amino)methyl)-4-nitrophenol (HL²), 4-chloro-2-(((2-(ethylthio)ethyl)(pyridin-2-ylmethyl)amino)methyl)phenol (HL³), 2-(((2-(benzylthio)ethyl)(pyridin-2-ylmethyl) amino)methyl)phenol (HL⁴), 2-(((2-(benzylthio)ethyl)(pyridin-2-ylmethyl)amino)methyl) -4-nitrophenol (HL⁵), and 4-chloro-2-(((2-(benzylthio)ethyl)(pyridin-2-ylmethyl)amino) methyl)phenol (HL⁶) to give dichloro complexes of the type [Fe(L)Cl₂], 2. The solid and solution structure of the complexes, as well as their properties, were probed using X-ray diffraction, spectroscopic and electrochemical methods. The Mössbauer spectral study at 80 K for complexes reveals the existence of (III) oxidation state and high-spin state of the metal center in the complex. Dioxygenase activity of the complexes has been studied and both 1 and 2 have been found to display the intradiol-cleaving pathway. However, no extradiol cleavage products have been isolated.     

Natural Chlorinated Auxins Labelled with Radioactive Chloride in Immature Seeds

Immature seeds were harvested from 15 species grown in perlite/vermiculite containing ³⁶Cl^-, but with very low levels of cold Cl^-. Autoradiograms of one- and two-dimensional thin layer chromatograms of butanol extracts of lyophilized seeds indicated several radioactive compounds besides the ³⁶Cl^- in many species. In pea the radioactivity cochromatographed with 4-(or 6-)chloroindolyl-3-acetic acid and its methyl ester; in other species radioactivity was found near these chlorinated indolyl-acetic acid markers.