蛋白核小球藻对Pb(Ⅱ)和Cd(Ⅱ)的生物吸附及其影响因素

藻类吸附作用影响重金属在水生生态系统中的迁移过程及其环境行为。同时,利用藻类吸附能力是修复重金属污染水体和重金属废水处理的一项清洁、廉价和高效的技术。测定了蛋白核小球藻对Pb2+和Cd2+的吸附和脱附动力学,表明吸附是快速表面过程,吸附4 h后基本达到平衡,不易脱附。研究了蛋白核小球藻对Pb2+和Cd2+的吸附热力学,绘制了吸附等温线,并用Langmuir模型进行拟合,相关系数R2分别为0.9906和0.9827,计算得到最大吸附量分别为0.373 mmol Pb/g和0.249 mmolCd/g。考察了pH值、离子强度和温度等环境因素对蛋白核小球藻吸附Pb2+和Cd2+的影响。结果表明,蛋白核小球藻对Pb2+和Cd2+的吸附量在pH值5.0—6.0之间达到最大值,并随着溶液离子强度的增加而降低,随着溶液温度的升高而增加。温度的影响还表明,蛋白核小球藻对Pb2+和Cd2+的吸附是吸热过程。实验还考察了水体环境中普遍存在的溶解性有机质主要成分-富里酸的影响,表明富里酸会抑制蛋白核小球藻对Pb2+和Cd2+的吸附,重金属离子浓度较低时的抑制效果更明显,最大抑制率分别达到了34.2%和34.9%。由于其对重金属的较高吸附量和吸附本身快速完成的特性,蛋白核小球藻有望成为较理想的生物吸附剂,在重金属污染水体的生物修复及废水处理中发挥重要作用。     

Characterization of Aqueous Pb(II) and Cd(II) Biosorption on Native and Chemically Modified Alstonia macrophylla Saw Dust

This study was conducted in order to understand the mechanism of Cd and Pb adsorption in aqueous solutions by raw and modified saw dust (SD) of Alstonia macrophylla. The biosorbent was characterized by Boehm titration, specific surface area, scanning electron microscopy (SEM), X-ray energy dispersion (EDAX), and Fourier transform infrared (FTIR) analyses. SD was treated using organic acids and bases. Batch studies were conducted for raw and modified SD to determine the effect of initial concentration, pH, ionic strength, and contact time on metal adsorption. The specific surface area and total basic and acidic groups of SD were 77 m²/g and 1521 and 2312 μmol/g, respectively. The adsorption of both metals onto SD was pH dependent. No ionic strength dependency was observed in adsorption of Cd and Pb at pH >6, indicating inner sphere surface complexation. Monolayer adsorption is dominant in both metal sorptions by SD. Furthermore, there is no competition between metals on adsorption and raw SD was found to be suitable for removal of Cd and Pb as compared to organic acid– or base-treated SD. Maximum adsorption capacity of SD for Cd and Pb were 30.6 and 204.2 mg/g, respectively. Results indicate that the A. macrophylla SD can be considered as a potential material for metal ion removal from wastewater.     

The behaviour of lung surfactant in electrolyte solutions

Surface and electrokinetic properties of purified calf lung surfactant in various electrolyte solutions were determined. Surface properties were pH dependent in distilled water and the surfactant performed as a good lung surfactant only below pH 4. In more physiological media it was pH insensitive over the range 2-8.5. In distilled water at pH 6 its surface properties improved when NaCl was added up to 20 mM; above this concentration it had the surface properties required to stabilise alveoli. The surface properties of surfactant in distilled water were also restored by certain cations (Ca2+, Mg2+, Mn2+, Cd2+ and Ni2+) but not others (Na+, K+, La3+ and Fe3+) when added to an ionic strength of 5.6 mM. Cations that restored its surface activity also reduced the surface charge density on the surfactant particles. Aggregation of surfactant by various metal chlorides was studied by light scattering measurements and bore no relation to surface activity or the charge on the particles. Aggregation of surfactant particles by Ca2+, Cd2+ and Mn2+ was instantly reversed by addition of excess EGTA. The influence of electrolytes on the surface properties of lung surfactant is explained in terms of the electrostatic forces operating in the system.     

Interaction of metal ions with lupin seed conglutin gamma

Various metal ions were capable of aggregating and precipitating conglutin gamma, an oligomeric glycoprotein purified from Lupinus albus seeds, at neutral pH values. The most effective metal ions, at 60-fold molar excess to the protein, were Zn2+, Hg2+ and Cu2+; a lower influence on the physical status of conglutin gamma was observed with Cr3+, Fe3+, Co2+, Ni2+, Cd2+, Sn2+, and Pb2+, while Mg2+, Ca2+ and Mn2+ had no effect at all. The insolubilisation of the protein with Zn2+, which is fully reversible, strictly depended on both metal concentration and pH. with middle points of the sharp transitions at three-fold molar excess and pH 6.5, respectively. Conglutin gamma is also fully retained on a metal affinity chromatography column at which Zn2+ and Ni2+ were complexed. A drop of pH below 6.0 and the use of chelating agents, such as EDTA and imidazole, fully desorbed the protein. A slightly lower binding to immobilised Cu2+ and Co2+ and no binding with Mg2+, Cd2+ and Mn2+ were observed. The role of the numerous histidine residues of conglutin gamma in the binding of Zn2+ is discussed.     

Modelling complexation and electrostatic attraction in heavy metal biosorption by Sargassum biomass

Biosorption, the passive accumulation of metal ions by biomass, can be used for purifying metal bearing wastewater. Seaweeds represent a readily available source of biosorbent material that possesses a high metal binding capacity. For example, Sargassum can accumulate 2 mequiv of Cd per gram of biomass i.e. 10% of its dry weight. Binding of Cd and Cu by Sargassum is an ion exchange process involving both covalent and ionic bonds. The amount of cations bound covalently or by complexation can be predicted using multi-component sorption isotherms involving 2 types of binding sites, carboxyl and sulphate. A Donnan model was used to account for the effect of ionic strength and electrostatic attraction. The use of a multi-component isotherm that included one term for Na binding was less appropriate than the Donnan model for modelling ionic strength effects. It was possible to predict metal and proton binding as a function of the pH value, metal concentration and ionic strength of the solution. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of heavy metal complexes of a hexapeptide inhibitor of the human immunodeficiency virus integrase protein by using a voltammetric approach

Complexation of the hexapeptide Hys-Cys-Lys-Phe-Trp-Trp, inhibitor of the human immunodeficiency virus integrase protein, with the heavy metal ions Cd2+, Pb2+, and Zn2+ has been investigated using differential pulse polarography. In the case of Pb2+, no significant complexation is detected, whereas in the cases of Cd2+ and Zn2+, strong and electrochemically inert ML2 complexes predominate. In contrast, ML complexes are present in a low proportion or are absent. When possible, the corresponding conditional stability constants have been determined at both pH 7.0 and pH 7.5, showing that Zn2+ complexes are slightly more stable than Cd2+ complexes.     

Activation of yeast enolase by Cd(II)

Activation of yeast enolase by Cd2+ exhibits properties similar to activation by the physiological cofactor Mg2+. The activity is weakly stimulated, then inhibited by increasing ionic strength. The activity increases, then falls with increasing Cd2+ concentration. The effect of pH on activity produced by Cd2+ is very similar to that produced by Mg2+, except that the Cd2+ profile is shifted one pH unit to more alkaline values, and the maximum activity of the Cd2+-enzyme is about 10% of that of the Mg2+-enzyme. The apparent kinetic parameters of Cd2+ activation show little effect of pH except for inhibition by high concentrations of Cd2+: the apparent Ki increases sharply with pH. This is interpreted as the result of Cd2+ being a less effective "catalytic" metal ion, and Cd2+ being more effective in stabilizing the enzyme at alkaline pH's. The similarity of effects of ionic strength, divalent cation, and pH may be due to interaction with the same six sites per mole of enzyme. We also characterized the dependence of what is believed to be the enzyme-catalyzed enolization of a substrate analog, D-tartronate semialdehyde-2-phosphate (TSP) on similar parameters of pH, ionic strength, etc. The putative enolization is dependent on catalytic metal ion, although the TSP binds to the conformational Cd2+-enzyme complex. The reaction is very slow and very pH dependent, increasing with pH with a midpoint of reaction velocity at pH 8.7. There is a strong qualitative correlation between pH dependencies of reaction velocity of substrate conversion and TSP enolization and absorbance of the enzyme-bound TSP enolate, whether with Mg2+ or Cd2+ as cofactor. The slowness of the Cd2+-TSP reaction is not limited by proton release or any reaction involving covalent bonds to hydrogen. The apparent reaction rate constant increases linearly with Cd2+ concentration. Addition of excess ethylenediaminetetraacetic acid reverses the TSP reaction, but again very slowly. The binding of Cd2+ to the catalytic sites is characterized by low association and dissociation rate constants.     

Influence of heavy metal ions on the electrophysical properties of Anacystis nidulans and Escherichia coli cells]

The influence of heavy metal ions (Ag+, Cu2+, Cd2+, Pb2+, Mn2+, Zn2+, Gd3+, 1 microM-1 mM) on Anacystis nidulans and Escherichia coli cells has been studied by means of electrophoresis and electro-orientation spectroscopy methods. It has been shown that changes of cell electrophoretic mobility (EM) and low-frequency (20 Hz) electro-orientation effect (EOE) observed with the increase of metal cation concentration characterize the adsorption of these ions on surface layers of cell envelopes. The degree and the character of these changes depend on cation valency and the initial value of cell EM. At the same time different changes of EM and EOE as a result of the multivalent cation adsorption allows to conclude that in that case the anisotropy of the cell surface increases. Cell damages were determined by changes in high-frequency EOE of cells which indicated the disturbance of barrier properties of their cytoplasmic membrane. Toxic effects of Ag+, Cu2+, Cd2+ ions on cells of both species and of Pb2+ on E.coli cells were observed. By toxic effects on the cytoplasmic membrane these ions could be placed in the order: for A.nidulans cells--Ag+ greater than Cu2+ greater than Cd2+; for E.coli cells Ag+ greater than Cu2+ greater than Cd2+ greater than Pb2+. Higher toxicity of heavy metals on E.coli cells seems to be connected with the more negative charge of deep layers of the cell surface.     

Distinct metal ion binding sites on Ca(2+)-activated K+ channels in inside-out patches of human erythrocytes.

Effects of Cd2+, Co2+, Pb2+, Fe2+ and Mg2+ (1-100 microM) on single-channel properties of the intermediate conductance Ca(2+)-activated K+ (CaK) channels were investigated in inside-out patches of human erythrocytes in a physiological K+ gradient. Cd2+, Co2+ and Pb2+, but not Fe2+ and Mg2+, were able to induce CaK channel openings. The potency of the metals to open CaK channels in human erythrocytes follows the sequence Pb2+, Cd2+ > Ca2+ > or = Co2+ > Mg2+, Fe2+. At higher concentrations Pb2+, Cd2+ and Co2+ block the CaK channel by reducing the opening frequency and the single-channel current amplitude. The potency of the metals to reduce CaK channel opening frequency follows the sequence Pb2+ > Cd2+, Co2+ > Ca2+, which differs from the potency sequence Cd2+ > Pb2+, Co2+ > Ca2+ to reduce the unitary single-channel current amplitude. Fe2+ reduced the channel opening frequency and enhanced the two open times of CaK channels activated by Ca2+, whereas up to 100 microM Mg2+ had no effect on any of the measured single-channel parameters. It is concluded that the activation of CaK channels of human erythrocytes by various metal ions occurs through an interaction with the same regulatory site at which Ca2+ activates these channels. The different potency orders for the activating and blocking effects suggest the presence of at least one activation and two blocking sites. A modulatory binding site for Fe2+ exists as well. In addition, the CaK channels in human erythrocytes are distinct from other subtypes of Ca(2+)-activated K+ channels in their sensitivity to the metal ions.     

Alginate as immobilization material: I. Correlation between chemical and physical properties of alginate gel beads

Calcium alginate gel beads were prepared from a range of well characterized alginates. The physical properties of beads depended strongly on the composition, sequential structure, and molecular size of the polymers. Beads with the highest mechanical strength, lowest shrinkage, best stability towards monovalent cations, and highest porosity were made from alginate with a content of L-guluronic acid higher than 70% and an average length of the G-blocks higher than 15. For these "high G" alginates the critical overlap intrinsic viscosities have been determined, and for molecular weight higher than 2.4 x 10(5), the gel strength was independent of the molecular weight.     

DECREASE IN DYNAMIC VISCOSITY AND AVERAGE MOLECULAR WEIGHT OF ALGINATE FROM LAMINARIA DIGITATA DURING ALKALINE EXTRACTION1

Alginates are natural polysaccharides that are extracted from brown seaweeds and widely used for their rheological properties. The central step in the extraction protocol used in the alginate industry is the alkaline extraction, which requires several hours. In this study, a significant decrease in alginate dynamic viscosity was observed after 2 h of alkaline treatment. Intrinsic viscosity and average molecular weight of alginates from alkaline extractions 1–4 h in duration were determined, indicating depolymerization of alginates: average molecular weight decreased significantly during the extraction, falling by a factor of 5 between 1 and 4 h of extraction. These results suggested that reducing extraction time could enable preserving the rheological properties of the extracted alginates.     

Structurally colored thin films of Ca2+-cross-linked alginate

Alginate, or alginic acid, is an unbranched binary copolymer of (1-->4)-linked beta-D-mannuronic acid and alpha-L-guluronic acid. Alginate readily forms binding interactions with a variety of divalent metal ions, such as calcium. This binding has been used to cross-link bulk alginates for a wide variety of applications, particularly in areas of tissue engineering, medical devices, and wound-healing dressings. A new method is identified here for producing Ca2+-cross-linked thin films of sodium alginate, using an aerosolized spray of CaCl2 solution. These thin films exhibit structural color that varies with film thickness. It is demonstrated that this structural color is highly reproducible and can also be tuned to produce a wide range of colored films. The noted ability of alginates to bind metal ions is used in combination with the structural coloration afforded by the thin film structure as a basis for color-based optical sensing of metal ions in aqueous solutions. Changes in film thickness, refractive index, and reflectivity in response to metal ions have been measured and reported. For certain ions such as Cr(III) and Cr(VI), changes in film thickness are the predominate factors in shifting the reflected film color. In the case of other ions such as Pb(II), a change in film refractive index plays a significant role in the reflectance properties of films.     

The role of divalent cations in structure and function of murine adenosine deaminase.

For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6- methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (Ki > or = 1000 microM) was noted for Ca2+, Fe2+, and Fe3+.     

Ordered association of tobacco mosaic virus in the presence of divalent metal ions

The formation of ordered aggregates of tobacco mosaic virus (TMV) in the presence of divalent metal ions has been studied in concentrated (1-25 mg/ml) solutions of the virus. The divalent metal cations Cd2+, Zn2+, Pb2+, Cu2+, and Ni2+ have been found to promote TMV precipitation from solution at a critical concentration Ccrit, which for a given metal depends on the pH and the ionic strength of the solution, but is largely independent of the virus concentration. The TMV precipitate behaves as a nematic liquid crystal and on drying at a glass surface produces highly ordered, optically birefringent films. However, precipitation is not observed with alkali-earth metals such as Ca2+ and Mg2+. The experimental data suggest that, apart from two 'internal' metal-binding sites in each TMV subunit, the virus contains metal-binding sites of a lower affinity which promote cross-linking of TMV rods via metal bridges. The latter seem to be responsible for the precipitation of TMV in the presence of divalent cations at neutral pH. We propose that the metal-induced cross-linking may be the predominant mechanism to account for the limited solubility of a variety of proteins in solution containing metal cations with valence 2 and higher.     

Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity

Glutathione reductase (E C: 1.8.1.7; GR) was purified from rainbow trout (Oncorhynchus mykiss) liver, and some characteristics of the enzyme were investigated. The purification procedure consisted of four steps: preparation of homogenate, ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose-4B and gel filtration chromatography on Sephadex G-200. The enzyme, with a specific activity of 27.45 U/mg protein, was purified 1,654-fold with a yield of 41%. Optimal pH, stable pH, optimal temperature, optimum ionic strength, molecular mass, KM and Vmax values for GSSG and NADPH were also determined for the enzyme. In addition, Ki values and inhibition types were determined for GSH and NADP+. Additionally, inhibitory effects of metal ions (Cd+2, Cu+2, Pb+2, Hg+2, Fe+3 and Al+3) on glutathione reductase were investigated. Ki constants and IC50 values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I], respectively. IC50 values of Cd+2,Cu+2, Pb+2, Hg+2, Fe+3 and Al+3 were 0.0655, 0.082, 0.122, 0.509, 0.797 and 0.804 mM, and the Ki constants for Cd+2 and Cu+2 were 0.104+/-0.001, 0.117+/-0.001, respectively.     

Preparation and properties of metal ion derivatives of the lentil and pea lectins

Lentil lectin (LcH) and pea lectin (PSA) belong to the class of D-glucose/D-mannose binding lectins and resemble concanavalin A (Con A) closely in physicochemical, structural, and biological properties. LcH and PSA, like Con A, are Ca2+-Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. Studies of the relationship between the metal ions binding and saccharide binding activity in LcH and PSA have been difficult due to the problem of metal ion replacement in these proteins. We now report a method of metal ion replacement in both lectins that allows substitution of the Mn2+ in the native proteins with a variety of transition metal ions, as well as substitution of the Ca2+ with Cd2+ in a particular complex. The following metal ion derivatives of both LcH and PSA have been prepared: Ca2+-Zn2+, Ca2+-Co2+, Ca2+-Ni2+, and Cd2+-Cd2+. All of these derivatives are as active as the native lectins, as demonstrated by precipitation with specific polysaccharides, saccharide inhibition of precipitation, and hemagglutination assays. The yields of these derivatives are good (generally greater than 70%), and the degree of metal ion incorporation is high (generally greater than 90%). The method of preparation is quite different from that for metal ion substitution in Con A, which proceeds via the apoprotein. In contrast, the apoproteins of LcH and PSA are unstable, aggregate above pH 4.0, and cannot be remetallized once formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of acetylation on metal induced precipitation of alginates

Acetylation dramatically effects both the solution properties and the metal induced precipitation of alginates. The presence of acetyl groups on both bacterial and seaweed alginate polymers marginally increased the weight average molecular weight (M_w) of each polymer by 7% and 11%, respectively. Acetylated bacterial alginate showed a significant increase in solution viscosity compared to its deacetylated counterpart. However microbial acetylation of seaweed alginate did not change its solution viscosity. Acetylation altered the calcium induced precipitation of both alginates. The presence of acetyl groups decreased the ability of each polymer to bind with calcium but increased their ability to bind with ferric Ion (Fe³⁺). By controlling the degree of acetylation on the alginate chains, it was possible to modify solution viscosity and cation induced precipitation of these polymers.     

Binding of horse heart cytochrome c to yeast porphyrin cytochrome c peroxidase: a fluorescence quenching study on the ionic strength dependence of the interaction

The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c peroxidase fluorescence by ferricytochrome c was observed at 0.1 M ionic strength and below. The quenching could be described by 1:1 complex formation between the two proteins. Values of the equilibrium dissociation constant determined from the fluorescence quenching data are in excellent agreement with those determined previously for the native enzyme-ferricytochrome c complex at pH 6.0 by difference spectrophotometry (J. E. Erman and L. B. Vitello (1980) J. Biol. Chem. 225, 6224-6227). The binding of both ferri- and ferrocytochrome c to cytochrome c peroxidase was investigated at pH 7.5 as functions of ionic strength in phosphate/KNO3 buffers using the fluorescence quenching technique. The binding in independent of the redox state of cytochrome c between 10 and 20 mM ionic strength, but ferricytochrome c binds with greater affinity at 30 mM ionic strength and above.     

Binding of bivalent cations by hyaluronate in aqueous solution

The interaction between sodium hyaluronate and bivalent cations was investigated by conductometry, viscosimetry, circular dichroism and nuclear magnetic resonance spectroscopy. It is shown that the hyaluronate chains (Mn approximately 4.0 x 10(5)-1.7 x 10(6)g/mol) bind various bivalent cations (Ca2+, Mg2+, Mn2+, Fe2+, Cu2+, Zn2+, Cd2+ and Pb2+) at pH 6 in aqueous solutions. Hyaluronate deriving from Streptococcus equi was studied in comparison with dextran from Leuconostoc mesenteroides which was shown to develop no specific interactions with the bivalent cations. The molar relation between the bivalent cations and the disaccharide units of the resulting complex was determined with the result that one bivalent cation is bound by approximately five disaccharide units. Heavy metal ions (Cd2+, Pb2+) seem to bind stronger to the hyaluronate chain than their lighter counterparts (Ca2+, Mg2+). Circular dichroism spectra of the hyaluronate exhibit a cation-induced change in the n-pi* transition, indicating that the acetamide group of the aminoglucane unit is involved during the complexation. NMR spectra of hyaluronic acid in presence of paramagnetic manganese cations show strong interactions between the acetamide as well as the carboxylate groups and the cations. Based on these data, a structure of the binding complex is proposed which involves two disaccharide units.