Activite des complexes acides humiques-invertase: Influence des cations floculants

-Humic acid (AH) and invertase (I) may be complexed with one of the following cations Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Al³⁺ or Fe³⁺ in order to study their catalytic activity and longevity. Provided that enough cations are supplied there is an immediate, almost total flocculation with Cu²⁺, Al³⁺ and Fe³⁺, or a slow flocculation with Ca²⁺, Co²⁺ and Mn²⁺ or no flocculation at all with Mg²⁺, depending on the reactivity between organic matter and cation. The six kinds of complexes have very different enzymatic activities in magnitude and longevity.No correlation was found between enzymatic activity and the atomic weight and valency of the cation used to prepare the complex. There is a pH at which flocs occur that is closely related to the pH of the metal chloride and to the affinity of each cation for precipitate; this pH value largely determines the structure of the humic material and consequently the catalytic activity of the enzymatic complex produced. For Cu²⁺, Al³⁺ and Fe³⁺ complexes, the pH values are low, between 1.7 and 3.2 and correspond to high conversion rates if the AH-I-cation reaction time is short; in cases where the reaction time is more protracted, enzyme molecules bound to the outer surface of the micellae are denatured by these very low pH values and the complexes irreversibly lose much of their activity. For Ca²⁺, Co²⁺ and Mn²⁺, the pH values lie between 4.30 and 5.65 and correspond to nearly ten times lower enzymatic activities; here, however, a prolonged reaction enhances activity. For Mg²⁺, the pH is 6.25–6.45; for this value, the humic material remains highly dispersed in buffer and the complex AH_Mg-I cannot be separated.The possibility of a relation between the number of enzyme molecules retained in complexes and the cationic radii on the one hand, and between the longevity of the complexes and the bound-state of humic acid-flocculating cation, on the other hand, is suggested.     

Humic acid association with peroxidase and catalase

Laboratory studies showed that humic acids can form stable associations with catalase and peroxidase. The humic—enzyme complex was isolated by gel chromatography and was shown to retain a considerable portion of the activity of the pure enzyme. The complex was much more stable towards heat and enzymatic degradation than the free enzyme. Formation of stable humic—enzyme association may be one of the mechanisms through which cell-free enzymes may be preserved in soils and sediments. It is suggested that the enzyme is physically trapped in the humic macromolecular structure rather than being ionically bound.     

Formation of [14C]cellulase-humic complexes and their stability in soil

[¹⁴C]cellulase was extracted from the culture medium of Trichoderma viride and an attempt made to complex it with humic acid by adsorption. The results showed that the humic acid extracted from soil does not form a stable complex with [¹⁴C]cellulase. In contrast, the flocculation of humic acid by 24 mM Ca²⁺ in the presence of the cellulase resulted in the formation of stable humic-cellulase complexes. These complexes showed great resistance to proteolysis and storage at high temperatures. DEAE cellulose chromatography of cellulase-humic complexes revealed that cellulase could not be separated from the humic acid. Enzyme activity was only eluted along with humic acid upon increasing gradient concentration from 1.0 to 1.5 m NaCl. Furthermore, in order to test their stability, the enzyme-humic complexes were incorporated into fresh soil for 90 days. The cellulase-humic complexes were then extracted from soil. Fractionation of the extract on DEAE cellulose and G 100 Sephadex revealed that cellulase activiiy could not be separated from humic acid and was again eluted in the form of enzyme-humic complexes. This confirmed the stability of cellulase-humic complexes in soil.     

Infrared spectra of Cu2+ Pb2+ and Ca2+ complexes of soil humic substances

An infrared spectroscopic investigation of the complexes of Cu²⁺, Pb²⁺, and Ca²⁺ with humic and fulvic acids demonstrated the participation of OH and CO groups in addition to COOH in the binding of heavy-metal cations. The degree to which metal-carboxylate linkages are ionic or covalent cannot be accurately determined from the positions of antisymmetric and symmetric carboxylate stretching vibrations due to interference from covalent bonding with other groups. The apparent order of the reaction of three divalent cations with humic and fulvic acids was Cu²⁺ > Pb²⁺ > Ca²⁺.     

铁铝氧化物和高岭石对转化酶的吸附及活性的影响

Experiments were conducted to study the influences of synthetic bayerite, non-crystalline aluminum oxide (N-AlOH), goethite, non-crystalline iron oxide (N-FeOH) and kaolinite on the adsorption, activity, kinetics and thermal stability of invertase. Adsorption of invertase on iron, aluminum oxides fitted Langmuir equation. The amount of invertase held on the minerals followed the sequence kaolinite > goethite > N-AlOH > bayerite > N-FeOH. No correlation was found between enzyme adsorption and the specific surface area of minerals examined. The differences in the surface structure of minerals and the arrangement of enzymatic molecules on mineral surfaces led to the different capacities of minerals for enzyme adsorption. The adsorption of invertase on bayerite, N-AlOH, goethite, N-FeOH and kaolinite was differently affected by pH. The order for the activity of invertase adsorbed on minerals was N-FeOH > N-AlOH > bayerite > reak goethite > kaolinite. The inhibition effect of minerals on enzyme activity was kaolinite > crystalline oxides > non-crystalline oxides. The pH optimum of iron oxide- and aluminum oxide-invertase complexes was similar to that of free enzyme (pH 4.0), whereas the pH optimum of kaolinite-invertase complex was one pH unit higher than that of free enzyme. The affinity to substrate and the maximum reaction velocity as well as the thermal stability of combined invertase were lower than those of the free enzyme.     

Measurement of electrostatic and site-specific associations of alkali metal cations with humic acid

A discontinuous acidimetric titration method incorporating ultrafiltration was developed to measure the association of a soil humic acid with Li⁺ t, Na⁺ and K⁺ (pH 3 to 8). In addition, possible site-specific binding of these alkali metal cations was investigated using desorption experiments at pH 1. Li, Na and K cations behaved equivalently in the titrations and the amounts of these cations associated with the humic acid was measurable at all pH values between 3 and 8. Up to 90% of the total alkali metal cation was humate-associated at pH 8. The absolute amount of humic-associated cation did not depend on the alkali metal cation concentration, but rather on the solution alkalinity. In addition, the net charge of the humate polyanion made a negligible contribution to the electroneutrality of the bulk solution under all conditions. These results are consistent with a diffuse layer model of hydrated humic acid in which the alkali metal cations neutralize the humic charge. The association of Na+ andK+ with humic acid at pH 1 was successfully described by a Langmuir adsorption model. The number of sites per g of humic acid was very small, and greater for K+ than for Nat. Lithium cations exhibited no detectable humic association at pH 1. These differences suggest that humic acids may have a small number of specific binding sites for which the size of the hydrated cation is important.     

Interaction of the 2,4-dichlorophenoxyacetic acid herbicide with soil organic matter moieties: a theoretical study

The determination of the structure of humic substances from soils and natural waters is an intriguing problem in soil science. Humic substances consist of molecules covering a broad distribution of molecular size and involving different functional groups. Taking this into account, we have chosen smaller model systems with functional groups typically present in humic substances. We investigated theoretically, by quantum chemical calculations, the environmental effects on the complexes formed from the interaction of 2,4‐dichlorophenoxyacetic acid and its anion with acetaldehyde, methanol, methylamine, protonated methylamine, acetic acid and water. The important case of a cation bridge mechanism, with Ca²⁺ as the bridging cation, is also included into the set of model reactions. It is found that this cation bridge belongs to one of the most stable mechanisms of fixation of organic compounds in soils. According to our calculations the hydroxyl group forms the most stable complexes with 2,4‐D in a polar solvent environment.     

Formation of metal-humic acid complexes by titration and their characterization by differential thermal analysis and infrared spectroscopy

Humic acid (HA) extracted from a Eustis loamy sand (Psammentic Paleudult, Red Yellow Podzolic soil) was flocculated by titration with Al³⁺-, Fe³⁺-, Cu²⁺-, Zn²⁺-, Mn²⁺-, Ba²⁺-, Ca²⁺-, and Mn²⁺-chloride solutions, respectively, to determine possible development of metal-HA complexes, as reported by Flaig et al. (1975), and Tiurin and Kononova (1962). Titration was conducted with HA solutions with an initial pH 11.5 or 7.0. The results indicated that the cations used, except Mg²⁺, yielded insoluble complexes with HA, irrespective of initial pH. After titration, the pH of the metal-HA flocs was 6.0–7.0, which was expected in view of the presence of cation exchange and buffering capacity of HA compounds. More complex formation through electrovalent and covalent bonding by COO^? and phenolic OH groups of the HA molecule was only attained by the use of HA solutions with pH 11.5. On the other hand, less complex formation occurred by the use of HA solutions with an initial pH 7.0, through electrovalent bonding by COO^? groups. Differential thermal analysis (d.t.a.) curves of HA showed shifts in temperatures of the main decomposition peak as a result of flocculation with the different metals. Based on the type of the cations involved, the metal-humic acid flocs could be listed in the following decreasing order of thermal stability: Al³⁺ = Zn²⁺ = Mg²⁺ ≥ HA > Ca²⁺ > Ba²⁺ > Fe³⁺ > Cu²⁺ > Mn²⁺. A systematic relationship could not be found indicating that trivalent ions resulted in the formation of thermally less stable metal-humic acid flocs than divalent ions, as has been reported for HA-metal complexes. Physical mixtures of HA and metal hydroxides exhibited d.t.a. features resembling those of original (nontreated) HA, but not those of the HA-metal flocs.Infrared spectroscopy revealed increased absorption for COO^? vibrations at 1620 and 1400cm^?1 in spectrograms of metal-HA flocs compared to that of original humic acid, a phenomenon explained by many authors to be caused by bonding of the metal ions in hydrated form to the carboxyl or phenolic hydroxyl groups or both of the humic acid molecule. HA-flocs formed from solutions with an initial pH 11.5 had identical i.r. spectra compared with those formed from solutions with an initial pH 7.0.     

Solubilization of iron by water‐extractable humic substances

The capability of water‐extractable humic substances (WEHS) to solubilize Fe from sparingly soluble Fe‐hydroxide was studied. Addition of WEHS (1.7 mmol organic C l^—1) to a dialysis tube containing labeled insoluble Fe‐hydroxide caused an increase in the amount of ⁵⁹Fe measured in the external solution. The humic fraction was also able to solubilize Fe from soil samples, with levels comparable to those obtained using a solution containing 100�μM DTPA. By measuring the amount of ⁵⁹Fe eluted from soil columns pre‐loaded with ⁵⁹Fe‐WEHS it was possible to evaluate the mobility of Fe complexed to the humic molecules. The recovery of ⁵⁹Fe varied from 2% to 25% in respect to the soil type used. The ability of Fe‐WEHS to serve as an Fe source for the phytosiderophore hydroxy‐mugineic acid (HMA) was also analyzed. The removal of ⁵⁹Fe from the Fe‐WEHS complex by HMA was demonstrated by adding the phytosiderophore to a dialysis tube containing the ⁵⁹Fe‐WEHS complex. The observations suggested a ligand exchange between the phytosiderophore and the humic fraction. The results indicate that WEHS is able to increase the amount of Fe present in the soil solution, possibly by forming mobile complexes with the micronutrient. These complexes could act as easily available Fe sources in Fe acquisition processes by both monocot and dicot plants, playing an important role particularly in soils with low available Fe.     

土壤有机矿质复合体研究──X.有机矿质复合体转化的初步研究

本文研究铁键、铝键、钙键复合体与溶液中Ｃａ＾２＋、Ｆｅ＾３＋、Ａｌ＾３＋金属离子的相互作用,结果显示,复合体上键合的Ｆｅ＾３＋,不能被溶液中的Ｃａ＾２＋、Ａｌ＾３＋置换,复合体上键合的Ａｌ＾３＋仅少量被Ｆｅ＾３＋、Ｃａ＾２＋置换进入溶液,复合体上键合的Ｃａ＾２＋则可较多地被Ａｌ＾３＋、Ｆｅ＾３＋置换进入溶液,置换的量与ＰＨ及粘土矿物类型有一定关系,尽管复合体上键合的Ｆｅ＾３＋、Ａｌ＾３＋、Ｃａ＾３     

Isoelectric focusing of β-glucosidase humic-bound activity in semi-arid Mediterranean soils under management practices

Changes in β-glucosidase enzyme–humic complexes and conventional parameters (pH, total organic C, total N, water-soluble C, and bulk density) were studied in an almond-cropped soil prone to erosion under a rehabilitation practice. The experimental plan included three soil slopes (0%, 2%, and 6%) and two type of fertilization (organic and mineral), with sampling of rhizosphere and inter-row soils. The enzyme humic complexes were extracted by pyrophosphate, purified by ultrafiltration of the organic extracts on molecular mass exclusion membranes (mol wt > 10⁴) and fractionated by isoelectric focusing technique (IEF). The IEF on polyacrylamide rod gels with a restricted pH gradient ranging between 6.0 and 4.0 gave five humic bands on the basis of the little differences of their electric charges (pI). Under both organic and mineral fertilization, β-glucosidase activity bound to the fractionated humic substances, especially in the pH range 4.5–4.2 of the rhizosphere soil, was higher than that of the inter-row soil. This also occurred in 6% slope where the enzyme activity was lower than in soil with lower slopes. The higher number of the β-glucosidase active humic bands in rhizosphere than inter-row soil, particularly for the 0% slope, may be due to the presence of humic molecules capable of preserving the enzyme molecules in the active form, other than to the higher microbial activity synthesizing and releasing the tested enzymes.     

Stability constants of complexes of metals with humic and fulvic acids under non-acid-conditions

Stability constants of complexes of four divalent metal ions viz. Cu²⁺, Zn²⁺, Mn²⁺ and Ca²⁺ with humic (HA) and fulvic acids (FA) at pH values of 7 and 8 were determined. The log K (logarithm of the stability constant) ranged from 3.09 to 7.77 and from 2.22 to 5.98 for metal-humic and metal fulvic acid complexes, respectively. Sequentially, the order of stability constants were as follows: Cu> Ca> Mn> Zn and Cu> Ca> Zn> Mn for metal -HA and metal-FA complexes, respectively, indicating a higher degree of complexation with Cu metal ion.     

Differential Complexation between Zn2+ and Cd2+ with Fulvic Acid: A Computational Chemistry Study

Fulvic (FA) and humic acids (HA) from the solubilization of organic matter are significant agents of the complex of metallic ions in soil and water. Cadmium ions are important natural water pollutants which are discharged along with electroplating and zinc metallurgy liquid wastes, zinc and cadmium being always associated with isomorphic substitution. In this paper we study the complexes formed by Zn²⁺ and Cd²⁺ with fulvic acids, using computational chemistry. We carried out relative free energy calculations using a thermodynamic cycle and Free Energy Pertubation (FEP) and Density Functional Theory (DFT-B3LYP/6-31G**) methods. We also computed the interaction energies between orbitals from Natural Bond Order (NBO) calculations. Cd-FA complexes, octahedral with four water molecules in the coordination sphere are more stable than Zn-FA complexes, tetrahedral with two water molecules. This means that FA should be a better ligand for Cd²⁺ bioavailability and toxicity in natural aquatic environments.     

Hydrolysis of precipitated phytate by three distinct families of phytases

While genetically modified plants that secrete histidine acid phosphatases (HAPs), β-propeller phytases (BPPs) and purple acid phosphatases (PAPs) have been shown to assimilate soluble phytate, little is known about whether these plants have the ability to hydrolyze precipitated phytate. In this study, the ability of representative members of these three classes of phytases to hydrolyze metal-phytate salts and to hydrolyze phytate adsorbed to aluminum precipitates was compared. All three phytases were able to hydrolyze Ca²⁺-, Mg²⁺-, and Mn²⁺-phytates, but were unable to hydrolyze Al³⁺-, Fe²⁺-, Fe³⁺-, Cu²⁺-, and Zn²⁺-phytates. When these ions were present, the hydrolysis of Ca²⁺-phytate was prevented. Citrate was more potent than malate and oxalate in solubilizing some of these phytate salts for enzyme hydrolysis. Phytate adsorbed to aluminum precipitates was resistant to all three enzymes, except when organic acids were added (citrate>oxalate>malate). While increasing concentrations of organic acids were inhibitory to enzyme activity (oxalate >citrate>malate), PAP was more resistant to citrate than HAP. As desorption of phytate from a solid surface by organic acids is essential for phytase activity, the genetic engineering of plants that enhances the secretion of both citrate and phytases from the root may be a feasible approach to improving soil phytate assimilation.     

腐植酸和氮肥用量及其互作对植烟土壤质量的影响

以中烟100为材料,采用腐植酸和氮肥双因子盆栽试验,研究了腐植酸和氮肥用量及其互作对植烟土壤团聚体组成、养分及酶活性的影响。结果表明:干、湿筛法测定的土壤团聚体的平均重量直径均以T3处理(腐植酸900 mg/kg+氮肥40 mg/kg)最高,与其他处理相比提高幅度分别为15.3%~23.2%和6.5%~20.0%;方差分析显示,腐植酸与氮肥互作对土壤pH、有机质影响不显著,但对土壤碱解氮、有效磷、速效钾含量的影响达到极显著水平(P0.01);二者互作对烤烟各生育期土壤脲酶、蔗糖酶活性影响不同。由此,腐植酸和氮肥用量及其互作对提高土壤团聚体稳定性、土壤主要养分含量及酶活性具有显著效果。     

Interactions of allophane with humic acid and cations

Allophanic soils are known to accumulate organic matter, but the underlying mechanism is not well understood. Here we have investigated the sorption of humic acid (HA) by an allophanic clay in the presence of varied concentrations of either CaCl₂ or NaCl as background electrolytes. Both the HA and the clay were separated from New Zealand soils. Much more HA was sorbed in CaCl₂ than in NaCl of the same ionic strength. Apparently Ca²⁺ ions were more effective than Na⁺ ions in screening the negative charge on HA. In CaCl₂ the HA molecule might also assume a more compact configuration than in NaCl. In the presence of CaCl₂ sorption increased, reached a maximum, and then declined as the concentration of HA in solution was increased. This behaviour was not observed in NaCl where sorption showed a gradual and steady increase with HA concentration. We propose that ligand exchange occurs between the surface hydroxyl groups of allophane and the carboxylate groups of HA. As a result, the allophane–HA complex acquires negative charges, requiring the co‐sorption of extraneous cations (Ca²⁺ or Na⁺) for charge balance. The Ca²⁺ co‐sorbed can attract more HA to the complex possibly by a cation‐bridging mechanism, giving rise to a maximum in sorption. The decline in sorption beyond the maximum may be ascribed to a decrease in the concentration of free Ca²⁺ ions through binding to HA molecules in solution. The increase in supernatant pH may be attributed to a ligand exchange reaction between the surface hydroxyls of allophane and the carboxylate groups of HA, and proton binding to the allophane–HA complex.     

Molecular dynamics simulation of cationic complexation with natural organic matter

Molecular computer modelling of natural organic matter (NOM) and its interactions with metal cations in aqueous solution is a highly effective tool for helping to understand and quantitatively predict the molecular mechanisms of metal‐NOM complexation. This paper presents the results of molecular dynamics (MD) computations of the interaction of NOM with dissolved Na⁺, Cs⁺, Mg²⁺ and Ca²⁺. They show that Na⁺ forms only very weak outer‐sphere complexes with NOM, whereas Cs⁺ interacts somewhat more strongly, but also mainly via outer‐sphere association. Mg²⁺ interacts little with NOM due to its strongly held hydration shell. Ca²⁺ has the strongest association with NOM and forms inner‐sphere complexes with NOM carboxylate groups. This last result supports the idea of supramolecular, Ca‐mediated NOM aggregation. Cation‐NOM binding occurs principally with carboxylate groups, and to a lesser extent with phenolic and other –R‐OH groups. The contributions of other NOM functional groups are minimal. The diffusional mobility of NOM‐bound cations is ∼20% (NOM‐Na⁺ outer‐sphere complex) to ∼95% (NOM‐Ca²⁺ inner‐sphere complex) less than in aqueous solutions without NOM. The MD simulation results are in good agreement with NMR spectroscopic measurements for Cs‐NOM solutions.     

Aluminum status of synthetic Al–humic substance complexes and their influence on plant root growth

Abstract

Aluminum (Al)–humus complexes are abundant in the A horizons of non-allophanic Andosols and contribute to the unique properties of volcanic ash soils, such as high reactivity with phosphate ions and a low bulk density. Natural non-allophanic Andosols commonly show Al toxicity to plant roots. There have been very few studies examining the contribution of Al–humus complexes to the Al toxicity of plant roots, although the complexes are the probable source of the toxic Al. We extracted humic substances from the A horizon of a non-allophanic Andosol using NaOH solution and reacted the humic substances and partially neutralized the AlCl₃ solution at three pH conditions (pH 4.0, 4.5 and 5.5) to prepare pure Al–humic substance complexes. The Al solubility study (equilibrium study in 10^?2 mol L^?1 CaCl₂) and the Al release study (a stirred-flow method using 10^?3 mol L^?1 acetate buffer solution adjusted to pH 3.5) indicated that all the synthetic complexes easily and rapidly release monomeric Al into the liquid phase with slight changes in pH and ion strength, although the Al contents and their extent of polymerization are considerably different among the complexes. A plant growth test was conducted using a medium containing the Al–humic substance complexes and perlite mixture. Root growth in burdock (Arctium lappa) and barley (Hordeum vulgare L.) was reduced equally by all three complex media, and the roots showed the typical injury symptoms of Al toxicity. These results indicate that in soils dominated by Al–humus complexes the Al released from the Al–humus complexes, as well as the exchangeable Al adsorbed by soil minerals, is definitely toxic to plant roots.     

Ca2+和CaM对苹果果实Ca2+-ATPase,SOD和PEA活性的影响

采用45Ca2+ 示踪等方法研究苹果果肉质膜微囊Ca2+ ATPase与Ca2+ 运输之间的关系 ,在Ca2+ 和CaM激活剂和抑制剂存在条件下培养果实圆片 ,探索Ca2+-ATPase ,SOD和PEA活性受Ca2+和 (或 )CaM调控的可能性。结果表明 ,存在于质膜上的Ca2+-ATPase并受载体A23187刺激而活性增加 ,Ca2+-ATPase活性与Ca2+运输依抑制剂EB浓度增加而下降 ,二者变化趋势十分一致 ,从而证实了Ca2+-ATPase推动苹果果肉质膜微囊Ca2+的主动运输。果肉质膜微囊Ca2+-ATPase同时受到Ca2+和CaM调节 ,而SOD和PEA活性仅受Ca2+ 的调节 ,而与CaM无关。