Metal-protein Complexes Research Articles

Interaction of trivalent Al and Ga cations with proteins of PSII. Cation binding mode and protein conformation by FTIR spectroscopy

The interaction of trivalent Al and Ga cations with proteins of the photosystem II (PSII) enriched membranes is investigated in air-dried film at pH 6–7 with cation concentrations of 0.01–10 mM. Fourier transform infrared (FTIR) difference spectroscopy with self-deconvolution and second derivative methods as well as curve-fitting procedures are applied in order to determine the cation binding mode, the protein conformational changes, and the structural properties of metal-protein complexes. The spectroscopic results showed that at low cation concentration (0.01–0.1 mM), metal-protein interaction is negligible; meanwhile, as the cation concentration is increased, a major metal complexation occurred via protein carbonyl and C-N groups. Similarly, metal coordination to the lipid moieties is observed. The uncomplexed protein secondary structure with α-helix 64%, β-sheet 10%, turn 13%, and β-antiparallel 6% is drastically altered to α-helix 50-20%, β-sheet 10–20%, random 25–35%, turn 10–15%, and β-antiparallel 6–10% in the presence of trivalent Al and Ga at high cation concentration.

The Role of Proteins in the Fixation of Heavy Metals on Oak Leaves

Abstract Dry oak leaves exhibit the property of accumulating metals when exposed to atmospheric pollution and thus can be used as bioindicators of metal pollution. In this paper the role of proteins, one of the components of leaves involved in the metal fixation, has been investigated. Proteins and protein-metal complexes were extracted purified, separated and concentrated by means of ion exchange chromatography and electophoresis. The results revealed that three different types of proteins having different molecular weights are responsible for metal fixation. Dedicated to Professor D. Klockow on the occasion of his 60th birthday.

Computer-Modelling of Metal Speciation in Human Blood Serum

This paper briefly describes two computer programs, BEST and ECCLES, both available on disc, that can generate distribution diagrams for various metal-complexes in human blood plasma. Two heavy metals, lead and mercury, are used as examples. The species distribution vs pH diagrams are based on formation constants obtained from our own potentiometric study of the mercury-glutathione system. The efficacy of a chelating agent for mobilizing a metal ion from the liable metal-protein complex portion of blood, PMI index, is discussed as well. The paper points out that computer modelling of metal speciation in human blood plasma has led to the design of important new therapeutic chelating agents.

The Adsorption of the Adhesive Protein of the Blue Mussel Mytilus edulis L onto Type 304L Stainless Steel

Adsorption measurements of the adhesive protein isolated from the blue mussel Mytilus edulis L onto type 304L stainless steel were performed. The adhesive protein is a high-molecular-weight biopolymer (100 kDa) that contains catecholic functionalities, which in principle are excellent iron chelators. From these measurements, adsorption isotherms were determined, and the maximum number of binding sites/m2 and the affinity constant were calculated. These values were compared to those measured for three other adsorbates onto type 304L stainless steel, namely, bovine serum albumin (BSA), L-3,4 dihydroxybenzoic acid (DHBA), and poly-L-lysine. Studies indicate that the mussel protein exhibits the highest affinity constant for the stainless steel of all the adsorbates and multilayer adsorption occurs at the mussel protein:metal interface; the other adsorbates exhibit Langmuir-type monolayer adsorption. The mussel protein significantly inhibits dissolution of iron and chromium metal from the stainless steel at pH 7.8, whereas the other adsorbates enhance it. These results indicate that the mussel protein forms a stable metal-protein complex at the surface of the metal that does not desorb into solution.

A new technique for quantitation of metal particulates and metal reaction products in tissues near implants.

Tissue specimens retrieved from four regions adjacent to hip implants during revision surgery were subjected to a novel treatment to make possible the quantitative separation of residual metal particulates and metal reaction products (metal ions and metal-protein complexes). The tissues were exposed to sodium hypochlorite solution that degraded and solubilized them, liberating metal reaction product and leaving behind metal wear particles, which were separated by centrifugation. Atomic absorption spectrophotometry was used to analyze the concentrations of the separated metal ions and wear particles. Co ion concentrations were 0.05 to 0.9 mM, Cr ion concentrations were 0.04 to 2.1 mM, and of Ti ion concentrations were 0.30 to 0.60 mM. The weight of Co metal particles was 0.1 to 4.9 mg/100 mg tissue, of Cr metal particles 0.07 to 2.2 mg/100 mg of tissue, and Ti particles 0.09 to 5.2 mg/100 mg tissue; one black tissue sample contained 3333 mg Ti/100 mg tissue. No correlation was found between the concentrations of these two entities in the samples examined, probably due to the complex and varied processes creating them. The procedures discussed here will result in data that can help elucidate the separate contributions of metal reaction products and metal particulates to implant loosening.

Inorganic mercury is transported from muscular nerve terminals to spinal and brainstem motoneurons.

The distribution of mercury within the brainstem and spinal cord of mice was investigated with the autometallographic technique after intramuscular administration of a single dose of mercuric mercury (HgCl2). Deposits of mercury were localized to motor neurons of the spinal cord and to brainstem motor nuclei; i.e., neurons with their peripheral projections outside the blood-brain barrier. Unilateral ligation of the hypoglossal nerve prior to the injection of HgCl2 prevented the accumulation of mercury deposits in the ipsilateral hypoglossal nucleus. The selective accumulation of mercury in spinal and brainstem motoneurons is most probably due to a leakage of metal-protein complexes from capillaries in muscle into myoneural junctions, followed by uptake into nerve terminals and retrograde axonal transport. The possible link between this process and the development of motor neuron degeneration in ALS is discussed.

Regulation of bacterial gene expression by metal–protein complexes

Metal ions are essential cofactors in several transacting bacterial gene regulators. Upon binding of the metal, the receptor proteins act either as repressors of gene expression or, in other systems, as transcriptional activators. Other metal-dependent regulatory proteins may function, directly or indirectly, as sensors of the cellular oxygen status, and may even be mediators in light-responsive gene regulation.

High-performance metal chelate interaction chromatography of proteins with silica-bound ethylenediamine-N,N′-diacetic acid

Wide-pore microparticulate silica gel having surface bound ethylenediamine-N,N′-diacetic acid (EDDA) was developed for the separation of proteins by metal chelate interaction chromatography (MIC). The separation of proteins including glycoproteins was carried out by gradient elution using increasing salt concentration. Different selectivities were obtained when changing the nature of the chelated metal on the surface of the stationary phase, manipulating eluent pH and varying the nature and concentration of the salts in the eluent. The retention—pH dependency of iron-free and holo transferrins suggested that MIC could be used for studying metal—protein complexes provided that the metal binding site in the protein molecule is also involved in the protein intractions with the chelated metal on the surface of the stationary phase. The eluting strength of sodium salts with “hard” metal—EDDA columns increased in the order Cl - ×CH 3COO - ⩽HCOO - ×H 2PO - 4 , whereas with “soft” metal—EDDA columns it increased in the order CH 3COO - ×H 2PO 4 - ×Cl -. Compared to another type of metal interaction column such as silica-bound iminodiacetic acid (IDA)k, the EDDA column exhibited different selectivity and retentivity toward proteins under other wise indentical elution conditions. Metal—EDDA stationary phases can be viewed as contemplementary to metal—IDA ones.

Electrophoretic techniques in element species analysis. Comparison between flow-through and flat-bed techniques

A comparison of electrophoretic techniques for the separation of metal-protein complexes from food extracts is described. A preparative flow-through electrophoresis system with continuous elution of the fractionated substances and an analytical flat-bed gel electrophoresis technique with off-line electro-elution are optimized with regard to this separation problem. The metal-protein complexes are extracted from four flour samples using Tris-glycine buffer (pH 8.3). For the separation, polyacrylamide gels of 14% T and 3% C are prepared and Tris-HCl (pH 8.9) is used as an electrophoresis buffer. For a soy bean flour, not only the separation of protein fractions is achieved but also the metal distribution patterns that are determined by flame atomic absorption spectrometry are given. The results show that the use of the flow-through technique is limited to special fields of application, whereas the flat-bed electrophoresis with subsequent electro-elution of metal-protein complexes is a useful technique in element species analysis.

Structural and functional characterization of human immunodeficiency virus tat protein

Site-directed mutagenesis was used to identify functional domains present within the human immunodeficiency virus (HIV) tat protein. Transient cotransfection experiments showed that derivatives of tat protein with amino acid substitutions either at the amino-terminal end or at cysteine residue 22, 37, 27, or 25 were no longer able to transactivate HIV long terminal repeat-directed gene expression. Incubation of Tat expressed in Escherichia coli with zinc demonstrated that both authentic Tat and cysteine mutation derivatives could form metal-protein complexes. The tat proteins that contained alterations within the cluster of positively charged amino acid residues retained their ability to transactivate gene expression, albeit at markedly reduced levels. Indirect immunofluorescence showed that the authentic tat protein and the amino-terminal and cysteine substitution mutants all localized in the nucleus, with accumulation being most evident in the nucleolus. In contrast, nuclear accumulation was greatly reduced with the basic-substitution mutations. Consistent with this result, a fusion protein that contained amino acids GRKKR, derived from the basic region, fused to the amino-terminal end of beta-galactosidase also accumulated within the nucleus. These results demonstrate that the 14-kilodalton tat protein contains at least three distinct functional domains affecting localization and transactivation.

Are 'light-derepressible' genes controlled by metal-protein complexes?

In plants, fungi and bacteria expression of specific genes is stimulated by light. Analysis of a light-inducible gene in the prokaryote Arthrobacter suggests that metal-protein complexes are involved in the mechanism of control.

Induction of a light-inducible gene in Arthrobacter sp. by exposure of cells to chelating agents and pH 5

Transcription of a light-inducible gene in the prokaryote Arthrobacter sp. is induced in the dark when cells are incubated with chelating agents or in medium at pH 5. However, repletion of metal ions such as Ca 2+, Mn 2+ or Zn 2+ or an increase in pH is required for accumulation of the gene product, an M r 21 000 polypeptide. But such changes in condition restore repression of the gene, and the decay in the rate of synthesis of the polypeptide follows the same time-course as when photodynamically induced cells are transferred to the dark. These results are consistent with regulation of expression of this gene at transcriptional and posttranscriptional steps by mechanisms that involve metal-protein complexes.

A site-specific mRNA-cytoplasmic protein complex in vitro.

In plants, fungi and bacteria expression of specific genes is stimulated by light. Analysis of a light-inducible gene in the prokaryote Arthrobacter suggests that metal-protein complexes are involved in the mechanism of control.

Electrochemical characterization of metallothionein metal—mercaptide complexes: application of cyclic voltammetry to investigation of metalloproteins

Electrochemical studies of metalloproteins have considerable potential in the investigation of metal—complex structures. Due to a lack of information on the precise nature of the results obtained however, application of electrochemistry has been largely neglected by protein chemists. This report discusses the cyclic de voltammetry of a selection of proteins with particular reference to metallothioneins (MTs) and interpretation of waves associated with MT metal—protein complexes. Comparison of voltammograms of proteins in the presence and absence of added metal, together with scans of a selection of native MTs, has allowed for the designation of waves associated with specific metal complexes. The contrast in electrochemical activity between proteins free of thiols or disulfide bonds with those rich in these functionalities was also shown. Evidence from saturation plots, peak current versus scan rate plots and detergent competition studies with MTs, indicated that the major electrochemical process at the mercury electrode was adsorption rather than diffusion controlled. Characterization of native MT metal speciation and assessment of the fidelity of metal reconstitution of laboratory produced thionein was readily feasible using cyclic voltammetry and nanomole quantities of material.

Electrochemical characterization of metallothionein metal-mercaptide complexes: Application of cyclic voltammetry to investigation of metalloproteins

Electrochemical studies of metalloproteins have considerable potential in the investigation of metal-complex structures. Due to a lack of information on the precise nature of the results obtained however, application of electrochemistry has been largely neglected by protein chemists. This report discusses the cyclic dc voltammetry of a selection of proteins with particular reference to metallothioneins (MTs) and interpretation of waves associated with MT metal-protein complexes. Comparison of voltammograms of proteins in the presence and absence of added metal, together with scans of a selection of native MTs, has allowed for the designation of waves associated with specific metal complexes. The contrast in electrochemical activity between proteins free of thiols or disulfide bonds with those rich in these functionalities was also shown. Evidence from saturation plots, peak current versus scan rate plots and detergent competition studies with MTs, indicated that the major electrochemical process at the mercury electrode was adsorption rather than diffusion controlled. Characterization of native MT metal speciation and assessment of the fidelity of metal reconstitution of laboratory produced thionein was readily feasible using cyclic voltammetry and nanomole quantities of material.

“Light-derepressible” genes are regulated by metal-protein complexes: A hypothesis

The hypothesis is presented that genes whose expression is sensitive to light are regulated by metal-protein complexes. This hypothesis is based on induction of a gene for an Mr 21,000 polypeptide in the prokaryote Arthrobacter sp. by photodynamic conditions, chelating agents, or a decrease in pH. It is proposed that these conditions promote dissociation of a regulatory metal-protein complex by photooxidation of the metal-binding site, lowering the concentration of free metal ion, or protonation of amino acid side-chains in the metal-binding site, respectively. Repression of this gene is restored when cells are returned to the dark, metal ions are added in excess of chelating agents or the pH of the medium is returned to neutral. Restoration of repression is inhibited by chloramphenicol and novobiocin, which suggests that this process requires resynthesis of a regulatory protein and reassociation of the complex with DNA. The hypothesis predicts a number of characteristics of the regulatory system, most of which are found among the effects of phytochrome. The well-documented effects of transition metal ions on the Pfr form of phytochrome suggest that changes in gene expression in plants mediated by photoisomerisation of this chromoprotein are initiated by a chelating activity of Pfr.

Metal ion activities in oscillating temperatures

Experiments with ion specific electrodes and copper-histidine mixtures show that there is an increase in [Cu 2+] with a rise in temperature and a decrease in [Cu 2+] with a fall in temperature. As a consequence it is suggested that cells that are exposed to oscillating temperatures will experience intermittent ‘metal pollution’ through dissociation of metal-protein complexes in their cytoplasm even though they are in clean environments. Analyses of the digestive gland of Mytilus edulis exposed to oscillating temperatures show a change in the subcellular distribution of 65Zn which is associated with changes in temperature rather than absolute temperatures. The cytosol metal proteins also show changes under these conditions with a decrease in high molecular weight (c. 70 kDa) and an increase in low molecular weight (c. 3 kDa) fractions. As a consequence oscillating temperatures induce metal fluxes and enhance bioaccumulation.

Unfolding of iron and copper complexes of human lactoferrin and transferrin

1. 1. Human lactoferrin and transferrin are capable of binding two iron or copper ions into specific binding sites in the presence of bicarbonate. 2. 2. Urea and several alkyl ureas have been effective in unfolding these metal-protein complexes. 3. 3. Biphasic transitions are observed for the unfolding of each of the metal complexes of these proteins as determined by direct visible spectroscopy suggesting the release of iron(III) and Cu(II) ions from both of these metal-binding proteins during the unfolding process. 4. 4. Greater stabilization and increased resistance to protein unfolding is observed for all iron(III) complexes compared to Cu(II) complexes of lactoferrin and transferrin as determined by isothermal unfolding and thermal denaturation. 5. 5. Relative stabilization of the different metal-protein complexes investigated within this study were determined to be as follows: Lf-Fe(III)⪢Lf-Cu(II); Tf-Fe(III)⪢Tf-Cu(II), and Lf-Fe(III)⪢Tf-Fe(III); Lf-Cu(II)⪢Tf-Cu(II).

Vanadium complexes of transferrin and ferritin in the rat

Vanadium associates with serum transferrin of rats administered vanadyl(IV) sulfate or ammonium metavanadate(V) by gastric intubation. Low molecular weight species account for only 3% of the vanadium present in plasma. The element distributes between the two major isotransferrins in proportion to their concentrations. Rat apotransferrin binds both vanadium(IV) and vanadium(V), forming 2:1 metal-protein complexes in both instances. Although the two isotransferrins apparently differ in their physiological properties, they exhibit identical vanadyl(IV) (VO 2+ EPR spectra, indicating identical or very similar metal binding sites for both proteins. In contrast to other transferrins, the two sites of the rat protein are spectroscopically indistinguishable and exhibit a VO 2+ EPR spectrum similar to that of the C-terminal metal binding site of human serum transferrin. VO 2+ EPR signals are observed with liver, spleen, and kidney tissue samples form animals maintained on a vanadium-supplemented diet. These signals arise from a specific intracellular VO 2+ complex with iron storage protein ferritin.

Binding of luminescent metal probes to proteins: the case of terbium

Some lanthanides show room temperature phosphorescence related to f–f transitions. The excitation to the emitting state can be accomplished by either direct or indirect absorption of light by the rare earth ions. However, the low oscillatory strength of f–f transitions makes the direct excitation not very useful to probe dilute protein-metal complexes. Indirect (sensitized) emission occurs than a ligand or a nearest chromophore is able to transfer non radiatively the energy absorbed on the lanthanide. This process is particularly effective with protein Tb 3+ complexes. The green Tb 3+ phosphorescence can be increased more than 10 4 times upon binding of the metal to binding sites of proteins. Terbium may exchange with ligands 69 bonds, which preferentially involve charged or uncharged oxygen atoms. Up to date Tb 3+ has been shown to substitute more or less conservatively in proteins calcium, iron or manganese. The former substitution is particularly relevant since calcium is spectroscopically silent. The substitution of iron and manganese by Tb 3+ may give very useful information on the environment of the metal site. The Tb 3+ substitution for Ca 2+ in hemocyanin, a copper protein where Ca 2+ plays an important regulatory role, and in concanavalin, a plant agglutinin containing both Mn 2+, and for Fe 2+ or Fe 3+ in ferritin, a metal storage protein, will be discussed in detail.