Ternary copper(II) complexes involving 2-(aminomethyl)-benzimidazole and some bio-relevant ligands. Equilibrium studies and kinetics of hydrolysis for glycine methyl ester under complex formation

Formation equilibria of copper(II) complexes of 2-(aminomethyl)-benzimidazole (AMBI) and the ternary complexes Cu(AMBI)L (L = amino acid, amide, dicarboxylic acid or DNA constituents) have been investigated. Ternary complexes of amino acids or amides are formed by a simultaneous mechanism. Amino acids form the complex Cu(AMBI)L, whereas amides form two complex species Cu(AMBI)L and Cu(AMBI)(LH₋₁). The ternary complexes of copper(II) with AMBI and dicarboxylic acids or DNA units are formed by a stepwise mechanism, whereby binding of copper(II) to AMBI is followed by ligation of the dicarboxylic acids or DNA components. The values of Δ log K indicate that the ternary complexes containing aromatic amino acids are significantly more stable than the complexes containing alkyl- and hydroxyalkyl-substituted amino acids. This may be taken as an evidence for a stacking interaction between the aromatic moiety of AMBI and the aromatic side chains of the bio-active ligands. The solid complexes Cu(AMBI)L where L = 1,1-cyclobutanedicarboxylic acid (CBDCA) and malonic acid were separated and identified by elemental analysis and infrared spectroscopy and magnetic moment. The decomposition course and steps for the isolated complexes were analyzed and the kinetic parameters of the non-isothermal decomposition were calculated. The hydrolysis of glycine methyl ester (MeGly) is catalyzed by the Cu(AMBI)²⁺ complex. The kinetic data is fitted assuming that the hydrolysis reaction proceeds in two steps. The first step, involving coordination of the amino acid ester by the amino and carbonyl groups, is followed by rate-determining attack by OH⁻ ion. The second step involves the equilibrium formation of the hydroxo-complex Cu(AMBI)(MeGly)(OH) followed by intramolecular OH⁻ attack.     

Salivary amino acids in Lygus species (Heteroptera:Miridae)

Free and protein-bound amino acids were investigated in the phytophagous bug Lygus rugulipennis and its salivary gland. Over 38 substances were separated. The total content of amino compounds in the insects was about 1400 μmol/g fr. wt (16% by weight), of which 97% was amino acid residues in proteins.The salivary glands, which comprise about 1.5% of the live weight of the insects, contain 3.5% of the total free amino acids and 1% of the whote insect. Free and protein-bound amino acids comprise, respectively, about 1.4 and 11.6% of the fresh weight of the gland. The total concentration of free amino acids in the saliva was estimated to range from 0.5 to 2.2% by weight (ca. 0.1 M).The composition of free amino acids in the salivary gland of Lugus varies markedly. In four studied species (L. rugulipennis, L. gemellatus, L. pratensis, L. punctatus), the most abundant compounds were proline, arginine, lysine, leucine, glutamic acid, methionine sulphoxide and glycerophosphoethanolamine. In whole specimens of L. rugulipennis the predominant free amino acids were proline, alanine, taurine, glutamic acid, glutamine and methionine sulphoxide. The most abundant amino acids in proteins were glutamic and aspartic acid, glycine, alanine and leucine. The results indicate that the amino acid composition in the salivary glands of Lygus species does not differ markedly from that of the whole insect. The functions of salivary amino acids are discussed.     

Characterization of amino Acid efflux from isolated soybean cells

Cells from reproductive soybean (Glycine max [L.] Merr.) plants were isolated using a mechanical-enzymic technique that produced a high yield of uniform, physiologically active cells. Cells were incubated in a pH 6.0 buffered solution and subjected to various treatments in order to determine the nature of net amino acid efflux. Total net amino acid (ninhydrinreactive substances) efflux was not affected by the following conditions: (a) darkness, (b) aeration, (c) K⁺ concentrations of 0.1, 1.0, 10, or 100 millimolar and (d) pH 4, 5, 6, 7, or 8. The Q₁₀ for net amino acid efflux between 10°C and 30°C was 1.6. Thus, it seems that net amino acid efflux requires neither current photosynthetic energy nor a pH/ion concentration gradient. Amino acid analyses of the intra-and extracellular fractions over time showed that each amino acid was exported linearly for at least 210 minutes, but that export rate was not necessarily related to internal amino acid pools. Amino acids that were exported fastest were alanine, lysine, leucine, and glycine. Addition of the inhibitor p-chloromercuriphenyl sulfonic acid, 3(3,4-dichlorophenyl)-1,1-dimethylurea, or carbonylcyanide p-trifluoromethoxyphenylhydrazone increased the rate of total amino acid efflux but had specific effects on the efflux of certain amino acids. For example, p-chloromercuriphenyl sulfonic acid greatly enhanced efflux of γ-aminobutyric acid, which is not normally exported rapidly even though a high concentration normally exists within cells. The data suggest that net amino acid efflux is a selective diffusional process. Because net efflux is the result of simultaneous efflux and influx, we propose that efflux is a facilitated diffusion process whereas influx involves energy-dependent carrier proteins.     

There are more small amino acids and fewer aromatic rings in proteins of ionizing radiation-resistant bacteria

The identification of specific amino acids (AAs) or groups of functionally important AA residues in ionizing radiation-resistant bacteria (IRRB) is an important challenge in understanding the biological basis of resistance to ionizing radiation (IR; X-rays and gamma-rays). To address this problem, we compared homologous sites in multiple alignments of proteins of IRRB and IR-sensitive bacteria (IRSB) using the DeltaProt Toolbox. Substitution patterns were used as evidence for selection of certain AAs over others. Our results show that, in contrast to aromatic AAs, small/tiny AAs tend to be preferred in IRRB compared to IRSB. In agreement with previous experimental data showing that oxidation of AA residues is causative in the killing of irradiated cells and that IR resistance is correlated with the accumulation of divalent manganese ions (Mn²⁺)–peptide–orthophosphate (Pi) complexes, we proposed a chemical interpretation based on the Hard and Soft (Lewis) Acids and Bases (HSAB) concept. These findings should assist future efforts in selecting mutations for rational design of proteins with enhanced IR tolerance properties.     

Action of Mercury on the Photosynthetic Apparatus of Spinach Chloroplasts

In chloroplasts of Spinacea oleracea L., Hg2+ ions interact with some sites in the photosynthetic electron transport chain: (l) with the intermediates Z+/D+ situated in the D1 and D2 proteins and with the manganese cluster in the oxygen evolving complex which are located on the donor side of photosystem (PS) 2, (2) with the chlorophyll a dimer in the core of PS1 (P700). P700 is oxidized in the dark by HgCl2. The Hg2+ ions form organometallic complexes with amino acids contained in chloroplast proteins.     

Role of the Interchangeable Cations on the Sorption of Fumaric and Succinic Acids on Montmorillonite and its Relevance in Prebiotic Chemistry

It has been proposed that clays could have served as key factors in promoting the increase in complexity of organic matter in primitive terrestrial and extraterrestrial environments. The aim of this work is to study the adsorption–desorption of two dicarboxylic acids, fumaric and succinic acids, onto clay minerals (sodium and iron montmorillonite). These two acids may have played a role in prebiotic chemistry, and in extant biochemistry, they constitute an important redox couple (e.g. in Krebs cycle) in extant biochemistry. Smectite clays might have played a key role in the origins of life. The effect of pH on sorption has been tested; the analysis was performed by UV–vis and FTIR-ATR spectroscopy, X-ray diffraction and X-ray fluorescence. The results show that chemisorption is the main responsible of the adsorption processes among the dicarboxylic acids and clays. The role of the ion, present in the clay, is fundamental in the adsorption processes of dicarboxylic acids. These ions (sodium and iron) were selected due to their relevance on the geochemical environments that possibly existed into the primitive Earth. Different mechanisms are proposed to explain the sorption of dicarboxylic acids in the clay. In this work, we propose the formation of complexes among metal cations in the clays and dicarboxylic acids. The organic complexes were probably formed in the prebiotic environments enabling chemical processes, prior to the appearance of life. Thus, the data presented here are relevant to the origin of life studies.

     

Cytotoxicity and DNA binding property of the dimers of triphenylethylene–coumarin hybrid with one amino side chain

Novel dimers of triphenylethylene–coumarin hybrid containing one amino side chain were designed and synthesized by the condensation of four dicarboxylic acids with the amino monomeric hybrids catalyzed by HATU and DIPEA at room temperature. The adding order of the reactants had a significant effect on the condensation reaction when the malonic acid was used. The dimeric compounds 7a and 7b linked by the malonic acid, showed a broad-spectrum and good anti-proliferative activity against four tumor cells and low cytotoxicity in osteoblast. UV–vis, fluorescence, and circular dichroism (CD) spectroscopies and thermal denaturation exhibited that compounds 7b, 8b, 9b, and 10b had significant interactions with Ct-DNA by the intercalative mode of binding. Both the DNA binding properties and the anti-proliferative activities would be enhanced by dimerization of the monomeric hybrid with one amino side chain, and were significantly affected by the length of the linker (dicarboxylic acids).     

Reversal of Copper Inhibition in Chloroplast Reactions by Manganese

In the Mehler reaction, a Hill reaction utilizing molecular oxygen as the electron acceptor, rates of net oxygen uptake are stimulated by added manganous ions. Both whole cell photosynthesis and the Mehler reaction are inhibited by copper. Copper inhibition of the Mehler reaction can be reversed by manganese salts. Glutathione. which alone has no effect on Mehler reaction rates, enhances the effect of manganese in reversing copper inhibition. The effects of added Cu²⁺, Cu²⁺ and Mn²⁺, or Cu²⁺, Mn²⁺, and glutathione exhibit no induction phenomena when measured manometrically. Furthermore, the order of addition of these factors is unimportant: final rates are dependent only on the composition of reaction mixtures. Compared to the Mehler reaction, conventional Hill reactions are less sensitive to copper poisoning, while certain chloroplast mediated photoxidations (e.g. the photoxidation of diketogulonic acid) are far more sensitive. In all of the chloroplast mediated photoreactions tested, manganese is effective in reducing the sensitivity to copper poisoning.     

Purification and characteristics of a low-molecular-weight peptide possessing oxidative capacity for phenol from Phanerochaete chrysosporium

A new low-molecular-weight peptide with phenol oxidase activity, named Pc factor, was isolated and purified from liquid culture of a white-rot basidiomycete Phanerochaete chrysosporium. Its molecular weight was about 600 Da estimated by gel-filtration. Three amino acids Glu, Gly and Val were detected in hydrolysate. Absorption peaks corresponding to amino acids and peptide were observed by UV and IR spectra analysis. And the signal of Cα of amino acid was also detected by ¹³C-NMR method. Pc factor had high thermostability and remained active in weakly alkalescent pH range. It could chelate Fe³⁺ and reduce it to Fe²⁺, but no hydroxyl radical HO▪ could be detected during the reaction process. It could oxidize phenolic lignin-model compounds such as 2,6-dimethoxyphenol (2,6-DMP), 2,2¢-azinobis (3-ethylbenzathiazoline-6-sulfinic acid) (ABTS) and syringaldazine in the absence of Mn²⁺ and H₂O₂. These characteristics differed greatly from those of manganese peroxi-dases. The oxidative catalysis of Pc factor can be enhanced by certain metal ions such as Cu²⁺ and Mn²⁺ etc., and O2 molecule was necessary for this reaction. In summary, Pc factor may function as an electron carrier in this novel oxidation-reduction system.     

Purification and characteristics of a low-molecular-weight peptide possessing oxidative capacity for phenol from Phanerochaete chrysosporium

A new low-molecular-weight peptide with phenol oxidase activity, named Pc factor, was isolated and purified from liquid culture of a white-rot basidiomycete Phanerochaete chrysosporium. Its molecular weight was about 600 Da estimated by gel-filtration. Three amino acids Glu, Gly and Val were detected in hydrolysate. Absorption peaks corresponding to amino acids and peptide were observed by UV and IR spectra analysis. And the signal of Cα of amino acid was also detected by ¹³C-NMR method. Pc factor had high thermostability and remained active in weakly alkalescent pH range. It could chelate Fe³⁺ and reduce it to Fe²⁺, but no hydroxyl radical HO˙ could be detected during the reaction process. It could oxidize phenolic lignin-model compounds such as 2,6-dimethoxyphenol (2,6-DMP), 2,2′-azinobis (3-ethylbenzathiazoline-6-sulfinic acid) (ABTS) and syringaldazine in the absence of Mn²⁺ and H₂O₂. These characteristics differed greatly from those of manganese peroxidases. The oxidative catalysis of Pc factor can be enhanced by certain metal ions such as Cu²⁺ and Mn²⁺ etc., and O₂ molecule was necessary for this reaction. In summary, Pc factor may function as an electron carrier in this novel oxidation-reduction system.     

Contribution of amino compounds to dissolved organic nitrogen in forest soils

Dissolved organic nitrogen (DON) may play an important role in plantnutrition and nitrogen fluxes in forest ecosystems. In spite of the apparentimportance of DON, there is a paucity of information concerning its chemicalcomposition. However, it is exactly this chemical characterization that isrequired to understand the importance of DON in ecosystem processes. Theprimaryobjective of this study was to characterize the distribution of free aminoacidsand hydrolyzable peptides/proteins in the DON fraction of Oa horizon leachatesalong an extreme edaphic gradient in northern California. Insitu soil solutions were extracted by centrifugation from Oahorizonscollected beneath Pinus muricata (Bishop pine) andCupressus pygmaea (pygmy cypress) on slightlyacidic/fertile and highly acidic/infertile sites. DON accounted for 77 to99% of the total dissolved nitrogen in Oa horizon leachates. Nitrogen infree amino acids and alkyl amines ranged from 0.04–0.07 mgN/L on the low fertility site to 0.45–0.49 mg N/L onthe high fertility site, and accounted for 1.5 to 10.6% of the DON fraction.Serine, glutamic acid, leucine, ornithine, alanine, aspartic acid andmethylamine were generally the most abundant free amino compounds. Combinedamino acids released by acid hydrolysis accounted for 48 to 74% of theDON, suggesting that proteins and peptides were the main contributor to DON inOa horizon leachates. Together, nitrogen from free andcombined amino compounds accounted for 59 to 78% of the DON. Most of theDON was found in the hydrophobic fraction, which suggests the presence ofprotein/peptide-polyphenol complexes or amino compounds associated withhumic substances. Because free and combined amino acids can be an importantnitrogen source for some plants, soil DON may play an important role in plantnutrition and ecosystem function.     

A comparison of manganese sources using tomato plants grown on marl,peat and sand soils

Summary The availability of manganous oxide, manganese dioxide and MnEDTA was compared to that of manganous sulfate at several equivalent rates on a Mn-deficient peaty muck, marl and on Mn-sufficient Scranton sand. Tomato plants grown in pot studies were analyzed for manganese and iron. Mn-deficiency leaf symptoms were in general agreement with the manganese level in the plant and the Fe/Mn ratio. Iron intake and manganese intake showed some evidence of inverse relationships. Manganous sulfate was slightly more available than manganous oxide which, in turn, was more available than MnO₂ or MnEDTA.     

Effect of Organic Acids on Reactions Catalyzed by Manganese Peroxidase from Phanerochaete chrysosporium

The effect of several organic acids on the oxidation of Mn(II) catalyzed by manganese peroxidase was studied. Reactivities of manganese peroxidase and chemically prepared Mn(III) organic acid complexes towards phenolic compounds were compared. If lactate appears to be the best complexant for manganese peroxidase activity, chemically prepared Mn(III)—lactate complex is a less effective oxidant towards phenolic compounds than other Mn(III)—complexes. Our results agree with the hypothesis that certain organic acids are involved in the catalytic cycle of manganese peroxidase. Malonate and lactate seem to be the most attractive complexants for practical applications of manganese peroxidase and were used in enzymatic treatment of hardwood kraft pulp. Bleaching of kraft pulp was studied and after alkaline extraction, a significant decrease of kappa number was measured. The bleaching was enhanced in lactate buffer.     

Determination of structure and composition of suberin from the roots of carrot, parsnip, rutabaga, turnip, red beet, and sweet potato by combined gas-liquid chromatography and mass spectrometry

Suberin from the roots of carrots (Daucus carota), parsnip (Pastinaca sativa), rutabaga (Brassica napobrassica), turnip (Brassica rapa), red beet (Beta vulgaris), and sweet potato (Ipomoea batatas) was isolated by a combination of chemical and enzymatic techniques. Finely powdered suberin was depolymerized with 14% BF₃ in methanol, and soluble monomers (20-50% of suberin) were fractionated into phenolic (<10%) and aliphatic (13-35%) fractions. The aliphatic fractions consisted mainly of ω-hydroxyacids (29-43%), dicarboxylic acids (16-27%), fatty acids (4-18%), and fatty alcohols (3-6%). Each fraction was subjected to combined gas-liquid chromatography and mass spectrometry. Among the fatty acids very long chain acids (>C₂₀) were the dominant components in all six plants. In the alcohol fraction C₁₈, C₂₀, C₂₂, and C₂₄ saturated primary alcohols were the major components. C₁₆ and C₁₈ dicarboxylic acids were the major dicarboxylic acids of the suberin of all six plants and in all cases octadec-9-ene-1, 18-dioic acid was the major component except in rutabaga where hexadecane-1, 16-dioic acid was the major dicarboxylic acid. The composition of the ω-hydroxyacid fraction was quite similar to that of the dicarboxylic acids; 18-hydroxy-octadec-9-enoic acid was the major component in all plants except rutabaga, where equal quantities of 16-hydroxyhexadecanoic acid and 18-hydroxyoctadec-9-enoic acid (42% each) were found. Compounds which would be derived from 18-hydroxyoctadec-9-enoic acid and octadec-9-ene-1, 18-dioic acid by epoxidation, and epoxidation followed by hydration of the epoxide, were also detected in most of the suberin samples. The monomer composition of the six plants showed general similarities but quite clear taxonomic differences.     

Factors affecting the metal ion-hydroxamate interactions II: effect of the length of the connecting chain on the Fe(III), Mo(VI) and V(V) complexation of some new desferrioxamine B (DFB) model dihydroxamic acids

Three new dihydroxamic acids (HO(CH₃)NCO-(CH₂)₂-CO-NH-(CH₂)_x-CON(CH₃)OH where the x values are 4; 3 and 2, and the compounds are abbreviated as 2,4-DIHA, 2,3-DIHA and 2,2-DIHA), containing the peptide group in a certain position to one of the two functional groups and in different distances to the other one, were synthesized and their complexation with Fe(III), Mo(VI) and V(V) was studied by pH-potentiometric, spectrophotometric and in some cases by CV methods to evaluate the redox behaviour of the Fe(III) complexes and assess their potential biological activity as siderophore models. All these compounds are structural models for the natural siderophore, desferrioxamine B (DFB). The results were compared to those of the complexes of 2,5-DIHA having the same connecting chain structure and length as DFB has, and the effects of the length of the connecting chain on the co-ordination mode and on the stability of the complexes formed were evaluated.Very similar stability of the mono-chelated complexes formed with all these dihydroxamic acids was found. All the results obtained suggest that one dihydroxamic acid (even the 2,2-DIHA) is able to complete the four coordination sites of a MoO₂ ²⁺ core forming simple mononuclear complexes. Favoured monomeric structures of the bis-chelated complexes of these dihydroxamic acids are also suggested with V(V) having the smallest ionic radius among the three metal ions studied. In the case of iron(III), however, clear indication was obtained for the slightly different complexation behaviour of 2,2-DIHA. Namely, the formation of the mononuclear bis-chelated complex with this shortest ligand seems to have sufficient strain to induce the formation of bimetallic species such as [Fe(2,2-DIHA)₂Fe)]²⁺.     

Physiological amino acids in brain tissue of the lepismatids,Lepisma saccharina and Thermobia domestica (Insecta,Thysanura)

1. The free (physiological) amino acid profile in the brain tissue of Lepisma saccharina and Thermobia domestica (Thysanura, Lepismatidae) was investigated.2. Nitrogenous waste products (ammonia and urea) together constituted 28% of the total ninhydrinpositive compounds for both species.3. GABA accounted for 3% of the total ninhydrin-positive compounds in the brain of L. saccharina, and T. domestica.4. Glutamate comprised 7% of the total ninhydrin-positive compounds in the brain of L. saccharina, and 8% in T. domestica.5. The most abundant amino acids were alanine, proline, arginine and taurine.6. The possible role of amino acids as neurotransmitters and/or modulators of neurotransmitter activity is discussed as well as the comparative free amino acid composition of the brain tissues of various arthropod groups.     

Production of New Unsaturated Lipids during Wood Decay by Ligninolytic Basidiomycetes

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.     

Evidence of in situ uptake and incorporation of bicarbonate and amino acids by a hydrothermal vent mussel

The hydrothermal vent mussel Bathymodiolus sp. is demonstrated to incorporate inorganic CO₂ from sea water. After ≈24 h incubation with H¹⁴CO⁻₂ the major part of the radioactivity is incorporated into macromolecules mostly in proteins but also in a notable lipidic fraction. 77 to 98% of this radioactivity is found in the gill and autoradiographs show that CO₂ fixation is only observed in cells containing high concentrations of bacteria. The results endorse the hypothesis that the associated bacteria might provide a nutritional source for the mussel.The mussel is also able to absorb and incorporate dissolved amino acids. Heterotrophic processes involving dissolved organic matter may interfere with the autotrophic pathways. Beside its capability of feeding on particulate material, the mussel may be thus able to live on reduced carbon and nitrogen compounds synthesized by its associated bacteria as well as on dissolved organic compounds present in sea water. The effective participation of the different processes is probably related to the ecological conditions experienced by the mussel in vent areas.     

An Electron Spin Resonance Study of Manganese in Wild-Type and Mutant Strains of Chlamydomonas reinhardi

Changes in the intensity of the electron spin resonance signal of divalent manganese were found to occur in suspensions of wild-type Chlamydomonas reinhardi. The observed manganese signal decreased in the light and increased in the dark. Through the use of a continuous-flow system it was possible to determine that the manganous ions responsible for the observed signal were localized solely in the medium. Changes in the signal intensity associated with wild-type cells were independent of the ability of fragments prepared from these cells to perform the Hill reaction with 2,6-dichlorophenol-indophenol (DPIP) as the oxidant.

The manganese signal changes were still evident, though smaller, in cell suspensions of wild-type cells treated with 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, and in mutant strains unable to carry out the Hill reaction, ac-115 and ac-141.

From these data it is concluded that the changes in intensity of the manganese resonance are not related to the function of manganese in photosynthesis but may reflect the capacity of cells for ion uptake in the light.