Spatially complex distribution of dissolved manganese in a fjord as revealed by high-resolution in situ sensing using the autonomous underwater vehicle Autosub.

Loch Etive is a fjordic system on the west coast of Scotland. The deep waters of the upper basin are periodically isolated, and during these periods oxygen is lost through benthic respiration and concentrations of dissolved manganese increase. In April 2000 the autonomous underwater vehicle (AUV) Autosub was fitted with an in situ dissolved manganese analyzer and was used to study the spatial variability of this element together with oxygen, salinity, and temperature throughout the basin. Six along-loch transects were completed at either constant height above the seafloor or at constant depth below the surface. The ca. 4000 in situ 10-s-average dissolved Mn (Mnd) data points obtained provide a new quasi-synoptic and highly detailed view of the distribution of manganese in this fjordic environment not possible using conventional (water bottle) sampling. There is substantial variability in concentrations (<25 to >600 nM) and distributions of Mnd. Surface waters are characteristically low in Mnd reflecting mixing of riverine and marine end-member waters, both of which are low in Mnd. The deeper waters are enriched in Mnd, and as the water column always contains some oxygen, this must reflect primarily benthic inputs of reduced dissolved Mn. However, this enrichment of Mnd is spatially very variable, presumably as a result of variability in release of Mn coupled with mixing of water in the loch and removal processes. This work demonstrates how AUVs coupled with chemical sensors can reveal substantial small-scale variability of distributions of chemical species in coastal environments that would not be resolved by conventional sampling approaches. Such information is essential if we are to improve our understanding of the nature and significance of the underlying processes leading to this variability.

Complete amino acid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis.

Cadmium-induced metallothioneins from the common sea mussel, Mytilus edulis, were shown to comprise of two groups of isoforms having apparent molecular masses of 10 kDa and 20 kDa. The 10-kDa group was resolved by anion-exchange chromatography into four fractions while the 20-kDa group was resolved into three fractions using this method. After metal removal and S-methylation of the cysteine residues using methyl-p-nitrobenzenesulphonate the complete amino acid sequences were determined. Five isoforms of the 20-kDa group were shown to possess monomeric units consisting of 71 amino acids. These proteins were distinct from the four 72-amino-acid proteins of the 10-kDa group. The FASTA algorithm has been used to compare the degree of similarity between the mussel metallothionein MT-10-IV isoform and other metallothioneins. The mussel MT-10-IV isoform exhibited substantial similarity to other molluscan metallothioneins. Moreover, the mussel metallothionein exhibited more similarity to vertebrate metallothioneins than to those of non-molluscan invertebrates, thus suggesting that the mussel metallothioneins are class I metallothioneins.

The binding of cadmium to crab cadmium metallothionein. A polarographic investigation.

Differential pulse polarography was used to monitor the titration of crab (Cancer pagurus) cadmium metallothionein with additional cadmium. Scatchard analysis was used to calculate an apparent second-order dissociation constant. A mean value of 3.60 x 10(-7) M was calculated at 20 degrees C in 0.016 M-NaClO4, pH 8.0.

Spectroscopic studies on cadmium (II)- and cobalt(II)-substituted metallothionein from the crab Cancer pagurus. Evidence for one additional low-affinity metal-binding site.

The binding of diamagnetic Cd(II) and paramagnetic Co(II) ions to the metal-free form of crab, Cancer pagurus, metallothionein (MT) was studied by various spectroscopic techniques. Both reconstituted and native Cd(II)-MT containing 6 mol Cd(II)/mol protein display electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra which were indistinguishable. The stoichiometric replacement of Cd(II) ions in native Cd(II)6-MT by paramagnetic Co(II) ions enabled the geometry of the metal-binding sites to be probed. The electronic absorption and MCD spectra of Co(II)6-MT revealed features characteristic of distorted tetrahedral tetrathiolate Co(II) coordination for all six metal-binding sites. The stepwise incorporation of Cd(II) and Co(II) ions into this protein was monitored by electronic absorption and CD, and by electronic absorption and EPR spectroscopy, respectively. The results indicate that the metal-thiolate cluster structure is generated when more than four metal ions are bound. Below this titration point separate tetrahedral tetrathiolate complexes exist. This suggests that the cluster formation occurs in a two-step process. Furthermore, the spectroscopic features in both Cd(II)- and Co(II)-metal derivatives above the full metal occupancy of six suggest the existence of one additional metal-binding site. The subsequent loss of one Cd(II) ion from crab Cancer Cd(II)7-MT in the gel filtration studies demonstrate the low metal-binding affinity of the latter site. While the spectroscopic properties indicate an exclusively tetrahedral type of metal-thiolate sulfur coordination for the binding of the first six metal ions, they suggest that the seventh metal ion is coordinated in a different fashion.

The enhanced induction of metallothionein by zinc, its half-life in the marine fish Pleuronectes platessa, and the influence of stress factors on metallothionein levels.

Intraperitoneal injection of zinc raised levels of a hepatic metallothionein-like species. Assuming that this species was metallothionein (MT) then levels were raised from approximately 20 micrograms/g to 300 micrograms/g in 7 days, and levels thereafter remained high for the next 4 weeks. The half-lives of the protein in liver and kidney from starved fish, measured using in vivo incorporation of 35S cysteine at 11 degrees C, were approximately 27 days and 32 days respectively. The following agents failed to stimulate synthesis of MT in plaice: stress (due to catching), endotoxin, dexamethasone, cortisol and turpentine.

Spectroscopic and chemical approaches to the study of metal-thiolate clusters in metallothionein (MT).

Structural properties of the metal-thiolate clusters in mammalian and vertebrate MTs were studied by employing various spectroscopic techniques. The stepwise probing of the metal-binding sites of the four-metal cluster domain (alpha-fragment) of rabbit MT-2 by Co(II) ions monitoring the V2[4A2----4T1(P)] ligand-field transition at 1260 nm and the EPR intensity of the high spin Co(II) complexes revealed spectral changes suggestive of a cluster structure with one metal-binding site of different symmetry. These results are in contrast with our previous findings on rabbit Co(II)7-MT-2 where a tetrahedral tetrathiolate Co(II) coordination at all stages of filling has been demonstrated. Similar titration studies on MT isolated from the crab Cancer pagurus revealed that all six metal-binding sites are bound in tetrahedral type of symmetry as documented by the development of the d-d absorption profile of crab Co(II)-MT as a function of increasing Co(II)/apoMT ratios. Evidence for clusters in the latter species is provided by the manifestation of magnetic exchange coupling in the EPR spectra when the last two Co(II) ions are bound. Unexpected spectral features in the d-d region of Co(II)6-MT and in the CD-profile of the Cd(II)6-MT seen after the addition of an excess of Co(II) or Cd(II), respectively, suggest the existence of a seventh metal-binding site of different symmetry in crab MT. Finally, the isolation of the metal-deficient rabbit Cd(II)6-MT-2 after exposure of the fully metal-occupied Cd(II)7-MT-2 to EDTA is reported. A combination of UV, CD and 113Cd NMR data obtained with both Cd(II)6- and Cd(II)7-MT-2 supports the occurrence of a labile metal-binding site in the three-metal cluster domain of mammalian MT.

NMR studies of crab and plaice metallothioneins.

Metallothioneins isolated from the hepatopancreas of the edible crab (Cancer paqurus) and the plaice (Pleuronectes platessa) after cadmium injection are predominantly cadmium proteins containing only small amounts of zinc and traces of copper. The removal of metal ions from the two metallothioneins by EDTA was studied using proton NMR spectroscopy. The rates of removal of cadmium and zinc were monitored directly from the intensity of the resonances due to the cadmium and zinc-EDTA complexes. Nearly all the zinc present in the protein was extracted by EDTA relatively rapidly, whereas only 10 to 20% of the total cadmium was removed in at least three steps. The total (Cd + Zn) metal removed at equilibrium was 1.2 to 1.8 g-ions/mole protein. Information on conformational changes in the protein were also obtained from studying alterations in the proton resonances of the protein. This was directly correlated with removal of metal from the protein. The coordination environments of the cadmium ions in crab metallothionein were investigated by using 113Cd-NMR, and compared with 113Cd-NMR spectra of rabbit liver MT-II and Scylla serrata MT-I.

Occurrence of cadmium in crabs (Cancer pagurus) and the isolation and properties of cadmium metallothionein.

The occurrence of high cadmium levels in the digestive gland (hepatopancreas) of the crab Cancer pagurus is reviewed and its possible significance to human health is discussed. Previous attempts to isolate this cadmium in its native form are briefly reviewed and the present method used in this laboratory to isolate crab cadmium metallothionein is described. Quantitative amino acid analysis of the purified protein suggests that it has a total of 55 residues, including 19 Cys, and a molecular weight of approximately 5800. This yields an extinction coefficient at 220 nm for the protein in HCl, pH 1.5, of 50,000 and at 250 nm for the native cadmium protein in Tris-HCl, pH 8.4, of 92,000.

Cadmium-binding proteins in the scallop Pecten maximus.

Scallops, Pecten maximus, accumulate cadmium naturally in the digestive gland to a level of approximately 100 ppm wet weight. Of this cadmium, 60% was soluble and was composed of three weight classes as judged by Sephadex G-100 chromatography. Of the soluble cadmium, 60% was in the 55,000 molecular weight range and 20% each in an excluded fraction and a 10,000 molecular weight fraction. The 55,000 molecular weight fraction, after further purification, showed a maximum cadmium concentration of 1.4% by weight. The cadmium was thiolate bound but not as strongly bound as in the case of metallothionein. The 10,000 molecular weight fraction was a metallothionein-like protein.

Characterization of two molecular weight classes of cadmium binding proteins from the mussel, Mytilus edulis (L.).

Inducible cadmium binding proteins (Cd-BP) in the mussel, Mytilus edulis, were resolved into two molecular weight components by gel permeation chromatography on Sephadex G-75. Each of these two molecular weight components was further resolved into four subcomponents by DEAE ion exchange chromatography. All eight subcomponents bound cadmium and exhibited significant u.v. absorption at 254 and little absorption at 280 nm. Based on amino acid composition analysis two classes of proteins were identified, one having higher cysteine (approximately 25 mole %) and lower serine and glutamic acid contents compared to the other class.

Non-metallothionein cadmium binding proteins.

In this review we discuss cadmium binding proteins which are similar to equine metallothionein but have significant differences and also binding proteins which appear to be totally unrelated.

Use of 2-mercaptoethanol during chromatography of crab (Cancer pagurus) metallothionein on DEAE cellulose.

The chromatographic properties of crab metallothionein appear particularly sensitive to oxidation, and this oxidation does not take place when 2-mercaptoethanol is present in the buffers. The chromatographic changes brought about by oxidation can be reversed by subsequent addition of 2-mercaptoethanol.

The partition of copper and cadmium between different charge-forms of metallothionein in the digestive tubules of the crab, Cancer pagurus.

In the presence of 2-mercaptoethanol DEAE cellulose chromatography of desalted hepatopancreas cytosols of crabs rich in both copper and cadmium yielded three peaks, probably all due to metallothioneins, with very different copper to cadmium ratios. Addition of a copper glycine complex to a desalted crab cytosol containing cadmium metallothionein gave a chromatogram similar to that obtained by in vivo administration of copper and cadmium. The chromatographic distribution of the metals between the different charge-forms in the distal and proximal portions of the tubules was basically similar although the concentrations of the metals were different.

Purification and properties of plaice metallothionein, a cadmium-binding protein from the liver of the plaice (Pleuronectes platessa).

A low-molecular-weight protein induced in the liver of the plaice (Pleuronectes platessa) by exposure to cadmium was purified and characterized. It is closely similar to mammalian metallothioneins in all of its properties in that it is a single-chain cadmium-binding protein of approx. 7000 mol.wt. with a high cysteine content (31 mol%) and no aromatic amino acid residues. The thiol groups of the cysteine residues complex with the cadmium in a SH/Cd molar ratio of 3:1 and produce a characteristic absorption maximum at 250 nm. Unlike the mammalian metallothioneins, however, metal analyses reveal only traces of zinc and copper in addition to cadmium. The presence of carbohydrate previously assumed from a positive reaction with periodic acid/Schiff reagent has now been disproved, and the positive reaction attributed to interaction with the thiol groups in the protein.

Characterisation of cadmium-binding proteins from mussels, Mytilus edulis (L), exposed to cadmium.

Three Cd2+-binding proteins have been purified and partially characterised from the digestive gland of the bivalve mollusc, Mytilus edulis, after exposure to Cd2+. The major protein, which was judged to be pure on polyacrylamide gel electrophoresis, showed many of the characteristics of mammalian metallothionein; having a high -SH content, few aromatic amino acids and a high A250/A280 nm ratio which disappears on acidification. It also contains Zn and Cu, but differs in its higher apparent molecular weight of about 25 000 and high glycine content (12-19%). The two additional Cd2+-binding proteins had lower cysteine contents and different molar proportions of Cd2+, Zn2+ and Cu2+.