Surface histidine mutations for the metal affinity purification of a β-carbonic anhydrase

Metal affinity chromatography using polyhistidine tags is a standard laboratory technique for the general purification of proteins from cellular systems, but there have been no attempts to explore whether the surface character of a protein may be engineered to similar affinity. We present the Arg160His mutation of Haemophilus influenzae carbonic anhydrase (HICA), which mimics the endogenous metal affinity of Escherichia coli carbonic anhydrase (ECCA). The purity and activity of the mutant are reported, and the purification is discussed. This is the first step toward developing a general method to engineer surface metal affinity for use in purification and metal labeling techniques.     

Biomachining: metal etching via microorganisms

The use of microorganisms to remove metal from a workpiece is known as biological machining or biomachining, and it has gained in both importance and scientific relevance over the past decade. Conversely to mechanical methods, the use of readily available microorganisms is low-energy consuming, and no thermal damage is caused during biomachining. The performance of this sustainable process is assessed by the material removal rate, and certain parameters have to be controlled for manufacturing the machined part with the desired surface finish. Although the variety of microorganisms is scarce, cell concentration or density plays an important role in the process. There is a need to control the temperature to maintain microorganism activity at its optimum, and a suitable shaking rate provides an efficient contact between the workpiece and the biological medium. The system’s tolerance to the sharp changes in pH is quite limited, and in many cases, an acid medium has to be maintained for effective performance. This process is highly dependent on the type of metal being removed. Consequently, the operating parameters need to be determined on a case-by-case basis. The biomachining time is another variable with a direct impact on the removal rate. This biological technique can be used for machining simple and complex shapes, such as series of linear, circular, and square micropatterns on different metal surfaces. The optimal biomachining process should be fast enough to ensure high production, a smooth and homogenous surface finish and, in sum, a high-quality piece. As a result of the high global demand for micro-components, biomachining provides an effective and sustainable alternative. However, its industrial-scale implementation is still pending.     

Relationship between Pb/Cd adsorption and metal oxides on surface coatings at different depths in Lake Jingyuetan

Adsorption of heavy metals to metal oxides on surface coatings of sediments in aquatic environments is one of the most important factors governing their toxicity. Many previous studies were carried out to examine relationships between characteristics of surface coatings and heavy metal adsorption. However, a number of uncertainties existed in the related mechanisms. Especially, the effect of depth, at which surface coatings are developed, was not seriously considered. In this study, surface coatings were collected on glass slides at different depths in the Jingyuetan Lake, which is located in the northeast of China, and the related chemical characteristics (Fe and Mn oxides in the surface coatings) were analyzed. Pb and Cd adsorption onto the surface coatings was measured under controlled laboratory conditions. Nonlinear regression analyses and Langmuir adsorption isotherms were used to estimate contributions of Fe and Mn oxides developed at different depths. The results indicated that a strong linear relationship existed between the depth of water and the contents of iron/manganese oxides. The depth of water can also influence the Langmuir parameters (_max) of Pb and Cd adsorbed onto the surface coatings, by reducing values of _max from water surface to the bottom and reaching the lowest level when approaching the sediments. For surface coatings at different depths in the lake, analyses of correlation between the _max and the coating constituents indicated that adsorption of Pb and Cd by Mn and Fe oxides are statistically significant. Based on results of this study, the role of water depth in governing processes of Pb and Cd adsorption to metal oxides in homogeneous surface coatings was verified.     

Heavy metal pollution in aquatic ecosystems and its phytoremediation using wetland plants: an ecosustainable approach

This review addresses the global problem of heavymetal pollution originating from increased industrialization and urbanization and its amelioration by using wetland plants both in a microcosm as well as natural/field condition. Heavymetal contamination in aquatic ecosystems due to discharge of industrial effluents may pose a serious threat to human health. Alkaline precipitation, ion exchange columns, electrochemical removal, filtration, and membrane technologies are the currently available technologies for heavy metal removal. These conventional technologies are not economical and may produce adverse impacts on aquatic ecosystems. Phytoremediation of metals is a cost-effective "green" technology based on the use of specially selected metal-accumulating plants to remove toxic metals from soils and water. Wetland plants are important tools for heavy metal removal. The Ramsar convention, one of the earlier modern global conservation treaties, was adopted at Ramsar, Iran, in 1971 and became effective in 1975. This convention emphasized the wise use of wetlands and their resources. This review mentions salient features of wetland ecosystems, their vegetation component, and the pros and cons involved in heavy metal removal. Wetland plants are preferred over other bio-agents due to their low cost, frequent abundance in aquatic ecosystems, and easy handling. The extensive rhizosphere of wetland plants provides an enriched culture zone for the microbes involved in degradation. The wetland sediment zone provides reducing conditions that are conducive to the metal removal pathway. Constructed wetlands proved to be effective for the abatement of heavymetal pollution from acid mine drainage; landfill leachate; thermal power; and municipal, agricultural, refinery, and chlor-alkali effluent. the physicochemical properties of wetlands provide many positive attributes for remediating heavy metals. Typha, Phragmites, Eichhornia, Azolla, Lemna, and other aquatic macrophytes are some of the potent wetland plants for heavy metal removal. Biomass disposal problem and seasonal growth of aquatic macrophytes are some limitations in the transfer of phytoremediation technology from the laboratory to the field. However, the disposed biomass of macrophytes may be used for various fruitful applications. An ecosustainable model has been developed through the author's various works, which may ameliorate some of the limitations. The creation of more areas for phytoremediation may also aid in wetlands conservation. Genetic engineering and biodiversity prospecting of endangered wetland plants are important future prospects in this regard.     

Investigation of the connection between the electrophoretic mobility (EPM) of microorganisms and their capability of metal uptake

The electrophoretic mobility of microorganisms (EPM) as a measure of their electric surface charge was determined as a function of different milieu conditions with the aid of the “Parmoquant 2” cell electrophoresis apparatus manufactured by CARL ZEISS Jena. The object of these researches was to examine the influence of the electric surface charge of microorganisms on their metal loading capacity. The results show a direct correlation between the electric surface charge or the EPM of microorganisms and their maximum metal loading capacity. Cells with a high negative surface also posses a high metal binding capacity. On the other side only a negligible metal uptake can be observed at the isoelectric point of the microorganisms (EPM = 0). The method of cell electrophoresis proved suitable to analyze complex interactions between microorganisms and heavy metal ions.     

Reevaluating the free-ion activity model of trace metal availability to higher plants

The plant uptake and toxicity of many metals show a marked dependence on the aqueous speciation of the metal, and these responses often correlate best with the activity of the free metal ion. Exceptions to this generalization have been observed, however, and we sought to critically reexamine the theoretical foundation of the free-ion activity model (FIAM) of metal bioavailability to higher plants. Binding by an apoplastic functional group is often envisioned as a requisite step in the absorption or toxicity of a metal, and can be modeled in a variety of ways. Typically, however, speciation of the bulk solution is calculated without regard to such surface binding, even though it could influence the pertinent mass balance expressions. A more thorough treatment considers simultaneous formation of both the metal-ligand complex in solution (ML) and the metal-cell surface complex (M-X). Here, empirical conformity to the FIAM can be expected, but only under pivotal assumptions about the relative sizes of the test solution and the root biomass, and about the relative binding strength of L and -X. Moreover, empirical conformity to the FIAM does not preclude cell-surface binding of the complexed metal followed by ligand exchange (ML + -X M-X + L), so that correlations between biological response and free metal-ion activity imply nothing about the molecular species that actually interacts with the cell surface. Computer simulations of Cu (II) binding by a model apoplastic ligand are used to illustrate these and other key features of the FIAM. Departures from the FIAM seem most likely when (i) the quantity of the metal-complexing ligand is limited (as may be the case in soil solution or in the rhizosphere), and/or (ii) the solution ligand is very weak.     

重金属胁迫下土壤微生物和微生物过程研究进展

通过对重金属胁迫下土壤微生物和微生物过程研究的进程和研究进展的归纳综述,分析了该研究尚存在的问题,并阐述了其可能原因．认为土壤微生物和微生物学过程的重金属胁迫研究存在如下问题：一是从实验室、田间试验和实地监测得到的结果间无法进行比较,从而使实验室和田间试验的研究丧失了其科学指导意义,并且在实地监测研究中缺乏相应的“精确”对照;二是在重金属的胁迫下土壤微生物不但数量有消长,而且区系结构上也发生了变化,但是用于校园微生物区系结构变化的手段(PLFA、BI-OLOG和DNA等方法)尚处在探索阶段并需要昂贵的设备,难以普及,需发展一些可广泛普及的新方法来代替传统的平板分离法分析土壤微生物结构;三是重金属对土壤微生物和微生物过程产生胁迫的形态、离子效应和根际效应尚未得到有效的研究和探讨;四是土壤微生物和微生物过程重金属胁迫的表征体系尚未建立．     

Reprogramming virus nanoparticles to bind metal ions upon activation with heat

We have reprogrammed the stimulus-responsive conformational change property of a virus nanoparticle (VNP) to enable the surface exposure of metal binding motifs upon activation with heat. The VNP is based on the widely investigated adeno-associated virus (AAV). An intrinsic bioactive functionality of AAV was genetically replaced with a hexahistidine (His) tag. The peptide domain with the inserted His tag is normally inaccessible. Upon external stimulation with heat, the VNP undergoes a conformational change, resulting in externalization of His tag-containing domains and the conferred ability to bind metal. We show that beyond this newfound functionality of the capsid, the VNPs maintain many of the wild-type capsid properties. Our work lays the groundwork for developing stimulus-responsive VNPs that can be used as "smart" building blocks for the creation of higher order structures.     

Microorganisms and heavy metal toxicity

The environmental and microbiological factors that can influence heavy metal toxicity are discussed with a view to understanding the mechanisms of microbial metal tolerance. It is apparent that metal toxicity can be heavily influenced by environmental conditions. Binding of metals to organic materials, precipitation, complexation, and ionic interactions are all important phenomena that must be considered carefully in laboratory and field studies. It is also obvious that microbes possess a range of tolerance mechanisms, most featuring some kind of detoxification. Many of these detoxification mechanisms occur widely in the microbial world and are not only specific to microbes growing in metal-contaminated environments.     

Effects of metal speciation on growth of phytoplankton with special reference to iron

This paper is a compilation of studies concerning the effects of metal speciation on the growth of phytoplankton. Special attention is paid to the speciation and availability of iron in lake Tjeukemeer, The Netherlands. Under laboratory conditions the free ionic metal species generally appear to be most effective in determining metal availability and toxicity. A variety of factors controlling solubility, ion-exchange, complexation or chelation, sorption and electrostatical attraction of metal ions affect the metal speciation, mostly resulting in reduced availabilities. However, some organic metal chelates such as citrates, nitrillotriacetates and the specifically iron chelating siderophores, are sometimes more available than the corresponding free ions. The presence of other metals also influences the availability of a given metal by competing for the same binding sites on the algal cell. This competition leads to antagonism betweene.g. iron nutrition and cadmium toxicity in marine diatoms. In the eutrophic, alkaline, hard and humic lake Tjeukemeer, the free Fe³⁺ concentration is below measurable levels and does not match the iron requirement of the phytoplankton. So most of the algal iron must have been provided by the predominant inorganic iron colloids and particles bye.g. dissolution or photo-degradation (reduction). If the provision rates of available iron are slow in relation to that of iron uptake, the growth of some phytoplankton species may become iron-limited. Continuous culture work indicated that the iron fraction <0.2 m from Tjeukemeer,i.e., the soluble fraction, is about one third as much available as iron from NH₄Fe(SO₄)₂.12H₂O. Different phytoplankton species vary widely in their metal requirements and tolerances. Therefore, metal speciation and availability may affect species composition and succession within phytoplankton communities. So far the assessment of metal availability in natural waters has been complicated by the complex metal chemistry and by methodological limits.     

Using cyclic peptide mixtures as probes for metal ion host-guest interactions

Summary Mixtures of cyclic peptides, formed by head-to-tail cyclizations of side-chain resin-bound linear sequences, have been prepared using solid-phase synthesis. Fast atom bombardment mass spectrometry of cyclic peptides with various metal ions can reveal preferred modes of host-guest patterns, albeit in a nonquantitative manner. This approach could prove useful for more rapid screening of potential peptide ionophores. A cyclic heptapeptide with a dipeptide tail proved to be a particularly effective host for a Ca²⁺ ion; in a small three-component mixture, cyclo[Gly-Asp-d-Pro-Xxx-Asp-d-Pro-Asp(Aca-Phe-NH₂)], binding to Ca²⁺ varied from Xxx=N-MeAla>GlySar. In a 15-component mixture, cyclo[Pro-Xxx-Asn-Pro-Xxx-Asn] where Xxx=Ala, Glu, Leu, Lys or Phe, there were no significant differences with respect to binding to metal ions. We believe this to be the first reported use of cyclic peptide libraries for screening metal ions to discern host-guest relationships.Abbreviations Aca aminocaproic acid - Boc tert-butyloxycarbonyl - BOP benzotriazolyloxy-tris(dimethylamino)phosphonium hexafluorophosphate - DCM dichloromethane - DIEA diisopropylethylamine - DMF N,N-dimethylformamide - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - pMBHA 4-methylbenzhydrylamine - TFA trifluoroacetic acid This paper is based on a presentation given at the Symposium on Peptide Structure and Design as part of the 31st Annual ACS Western Regional Meeting held in San Diego, CA, USA, October 18–21, 1995.     

Apparent and real variability in the presence and metal contents of metallothioneins in the crab Carcinus maenas including the effects of isolation procedure and metal induction

• 1.1. Reducing conditions must be maintained throughout the procedure of isolating metallothioneins from crabs. Dithiothreitol is preferred to 2-mercaptoethanol for long-term protection.
• 2.2. Two metallothioneins (10,100 and 4100 mol. wt, respectively) in the hepatopancreas of the crab Carcinus maenas showed great variability between individual crabs as to their presence and to their contents of Zn, Cu and Cd.
• 3.3. The 10,100 mol. wt metallothionein was induced in the laboratory by exposure to Cu and Cd, and variably by Zn-exposure. Laboratory induction did not raise significantly the total metal content of 0.88 ± 1.13 g atoms/mol protein of this metallothionein in crabs from the Firth of Clyde, Scotland.
• 4.4. The 4100 mol. wt metallothionein was not induced in the laboratory by exposure to Cu, Cd or Zn. This metallothionein in crabs from the Firth of Clyde, Scotland, contained 0.27 ± 0.34 g atoms of total Cu, Cd and Zn per mole of protein.

     

Simplification of rat intubation on inclined metal plate

Small-animal intubation is often necessary during inhalation anesthesia to allow steady-state conditions for large operations and in vivo experiments in all fields of experimental surgery. In rats, placing an orotracheal tube is technically difficult primarily because of the small size of the subject and the lack of equipment specifically designed for this task. We describe a simple rat intubation technique in which the animal is suspended in dorsal recumbency on an inclined metal plate. The animal, anesthetized with ether, is fixed to a 70 degrees-inclined metal plate in a dorsal position by means of a Mersilene ribbon hooked around the upper incisors. This method of positioning the animal is the most important step in the intubation process and further facilitates the technique already described by other authors. A human otoscope was used as a laryngoscope, intubation was performed using the Seldinger technique, and a 14-gauge intravenous catheter served as an endotracheal tube. This inexpensive technique is quickly learned and can be used in any laboratory. Safe and reliable airway management can thus be achieved, permitting in vivo examinations and operations.     

Relationships between metal concentrations and organism size in aquatic insects

SUMMARY.

• 1 The influence of organism size on whole-body metal concentrations of eight metals was examined in aquatic insects in field and laboratory studies. Information on the partitioning of metals between adsorbed, absorbed and gut content material was also obtained.
• 2 For Co, Cr, Fe, Sb and Sc, an exponential decrease in concentration with increasing organism size was observed, indicating surface adsorption as an important mode of metal accumulation.
• 3 No, or only a slight, concentration-size relationship was found for K, Mn and Na. This is expected for metals with high absorption efficiencies and a low capacity to be adsorbed.
• 4 Clearing of the gut of the mayfly Stenonema modestum (Ephemeroptera: Heptageniidae) did not significantly after the concentration size relationship for any of the metals studied.
• 5 Studies of Cr uptake with Stenacron interpunctatum (Ephemeroptera: Heptageniidae) indicate that the concentration-size relationship is not affected by variability in the concentration of available Cr.
• 6 About 52% of the whole-body burden of Cr in S. interpunctatum was associated with gut material while at least 33% was adsorbed to the organism's exoskeleton and at most 15% was internally absorbed.

     

Variation with depth and season in metal sulfides in salt marsh soils

Seasonal monitoring of metal sulfides was carried out in four soils ofthe Ría de Ortigueira salt marshes. Soils from the high salt marsh (withsuboxic redox conditions at the surface), had low concentrations of ironsulfides (AVS and pyrite fraction) and thus a low degree of trace metalpyritization (DTMP) in surface layers (0–10 cm), butconcentrations of metals associated with the pyrite fraction increasedconsiderably at depth (27.5 cm). In the low salt marsh soils (withanoxic conditions at the surface) maximum concentrations of metal sulfides werefound in the surface layers of soils colonized by Spartina maritima. These results are explained by the double effectexerted by roots in strongly reduced soils. On the one hand, they stimulate theactivity of sulfate-reducing bacteria and on the other, they favour the partialoxidation of the soil, thus generating polysulfides with which Fe²⁺immediately precipitates as pyrite, whereas in the deepest, permanently anoxiclayers, pyrite must be formed in a reaction in which FeS is an intermediate, asfollows: FeS + H₂S FeS₂ + H₂.Concentrations of metal sulfides also varied greatly with the season, with twopatterns being distinguished. In soils colonized by S. maritima in both high and low salt marshes, the lowestconcentrations were found in summer. At this time of the year there is a netloss of metal sulfides throughout the profile, presumably due to physiologicalactivity of plants (evapotranspiration and release of oxygen from roots). Incontrast, maximum concentrations of AVS and pyritic metals were found in thesummer in the low salt marsh soils not colonized by vascular plants (creekbottom). In this case, the higher temperatures increased the activity ofsulfur-reducing bacteria leading to synthesis and accumulation of metalsulfidesin the soil.     

Engineering of microorganisms towards recovery of rare metal ions

The bioadsorption of metal ions using microorganisms is an attractive technology for the recovery of rare metal ions as well as removal of toxic heavy metal ions from aqueous solution. In initial attempts, microorganisms with the ability to accumulate metal ions were isolated from nature and intracellular accumulation was enhanced by the overproduction of metal-binding proteins in the cytoplasm. As an alternative, the cell surface design of microorganisms by cell surface engineering is an emerging strategy for bioadsorption and recovery of metal ions. Cell surface engineering was firstly applied to the construction of a bioadsorbent to adsorb heavy metal ions for bioremediation. Cell surface adsorption of metal ions is rapid and reversible. Therefore, adsorbed metal ions can be easily recovered without cell breakage, and the bioadsorbent can be reused or regenerated. These advantages are suitable for the recovery of rare metal ions. Actually, the cell surface display of a molybdate-binding protein on yeast led to the enhanced adsorption of molybdate, one of the rare metal ions. An additional advantage is that the cell surface display system allows high-throughput screening of protein/peptide libraries owing to the direct evaluation of the displayed protein/peptide without purification and concentration. Therefore, the creation of novel metal-binding protein/peptide and engineering of microorganisms towards the recovery of rare metal ions could be simultaneously achieved.     

Isothermal titration calorimetric procedure to determine protein-metal ion binding parameters in the presence of excess metal ion or chelator

Determination of binding parameters for metal ion binding to proteins usually requires preceding steps to remove protein-bound metal ions. Removal of bound metal ions from protein is often associated with decreased stability and inactivation. We present two simple isothermal titration calorimetric procedures that eliminate separate metal ion removal steps and directly monitor the exchange of metal ions between buffer, protein, and chelator. The concept is to add either excess chelator or metal ion to the protein under investigation and subsequently titrate with metal ion or chelator, respectively. It is thereby possible in the same experimental trial to obtain both chelator-metal ion and protein-metal ion binding parameters due to the different thermodynamic "fingerprints" of chelator and protein. The binding models and regression routines necessary to analyze the corresponding binding isotherms have been constructed. Verifications of the models have been done by titrations of mixtures of calcium chelators (BAPTA, HEDTA, and EGTA) and calcium ions and they were both able to account satisfactorily for the observed binding isotherms. Therefore, it was possible to determine stoichiometric and thermodynamic binding parameters. In addition, the concept has been tested on a recombinant alpha-amylase from Bacillus halmapalus where it proved to be a consistent procedure to obtain calcium binding parameters.     

Characterization of cadmium- and lead- phytochelatin complexes formed in a marine microalga in response to metal exposure

Phytochelatins (PC_n) are thiol-containing peptides with general structure (-Glu-Cys)_n-Gly enzymatically synthesized by plants and algae in response to metal exposure. They are involved in the cellular detoxification mechanism for their capability to form stable metal-phytochelatin complexes. The speciation of Cd and Pb complexes with phytochelatins has been studied in laboratory cultures of the marine diatom Phaeodactylum tricornutum. An approach based on size-exclusion chromatography (SEC) with off-line detection of phytochelatins, by reverse-phase HPLC, and metal ion, by atomic absorption spectrometry, has been used. The formation of Cd- and Pb-PC_n complexes with n-value from 3 to 6 was demonstrated. The metal-PC_n complexes formed with Cd appear to be different from those formed with Pb for the number of molecules of peptide involved in the complex and for the amount of the metal ion bound. The chromatographic behaviour of metal-PC_n complexes is consistent with Pb-PC_n complexes in which only a molecule of peptide binds the metal ion, and with Cd-PC_n complexes containing two or more molecules of peptide. The metal/peptide molar ratio in Cd-PC_n complexes was higher that in Pb-PC_n complexes. The formation of Cd- or Pb-PC₂ complexes was not demonstrated, probably for a dissociation during the cellular extract preparation. The effectiveness of phytochelatins in the detoxification of these two metal ions in this alga is discussed.     

Validation of a Laboratory Method for Evaluating Dynamic Properties of Reconstructed Equine Racetrack Surfaces

Background

Racetrack surface is a risk factor for racehorse injuries and fatalities. Current research indicates that race surface mechanical properties may be influenced by material composition, moisture content, temperature, and maintenance. Race surface mechanical testing in a controlled laboratory setting would allow for objective evaluation of dynamic properties of surface and factors that affect surface behavior.

Objective

To develop a method for reconstruction of race surfaces in the laboratory and validate the method by comparison with racetrack measurements of dynamic surface properties.

Methods

Track-testing device (TTD) impact tests were conducted to simulate equine hoof impact on dirt and synthetic race surfaces; tests were performed both in situ (racetrack) and using laboratory reconstructions of harvested surface materials. Clegg Hammer in situ measurements were used to guide surface reconstruction in the laboratory. Dynamic surface properties were compared between in situ and laboratory settings. Relationships between racetrack TTD and Clegg Hammer measurements were analyzed using stepwise multiple linear regression.

Results

Most dynamic surface property setting differences (racetrack-laboratory) were small relative to surface material type differences (dirt-synthetic). Clegg Hammer measurements were more strongly correlated with TTD measurements on the synthetic surface than the dirt surface. On the dirt surface, Clegg Hammer decelerations were negatively correlated with TTD forces.

Conclusions

Laboratory reconstruction of racetrack surfaces guided by Clegg Hammer measurements yielded TTD impact measurements similar to in situ values. The negative correlation between TTD and Clegg Hammer measurements confirms the importance of instrument mass when drawing conclusions from testing results. Lighter impact devices may be less appropriate for assessing dynamic surface properties compared to testing equipment designed to simulate hoof impact (TTD).

Potential Relevance

Dynamic impact properties of race surfaces can be evaluated in a laboratory setting, allowing for further study of factors affecting surface behavior under controlled conditions.