81 Br-NMR studies of carbonic anhydrase

⁸¹Br nmr measurements have been made on high (bovine; BCA) and low (human-B; HCAB) specific activity forms of carbonic anhydrase and on a chemically modified form of the human enzyme (carboxyamidomethyl; CAM-HCAB). The high specific activity form of the enzyme, BCA, exhibits a ⁸¹Br line broadening which is determined by the lifetime of Br^? bound to the zinc ion of the enzyme. The low specific activity form of the enzyme, HCAB, under similar conditions of concentration, pH, etc., does not exhibit a ⁸¹Br nmr line broadening. $\text{Cl}{}^{\mathrm{?}}\text{Br}{}^{\mathrm{?}}$ competitive binding studies, using ³⁵Cl nmr, suggests that the failure to observe ⁸¹Br broadening is due to an increase in the lifetime of a zinc bound Br^?. An increase in this lifetime by a factor of 10–100 over that exhibited by BCA is sufficient to abolish the line broadening. A modified form of HCAB, CAM-HCAB, does, however, exhibit a ⁸¹Br nmr line broadening. Estimates of the lifetime of zinc bound Br^?, τ_M, are 4 × 10^?7 sec. for CAM-HCAB at pH 8 and 1 × 10^?7 sec. for BCA at pH 7. The lifetime for Br^? bound to HCAB is estimated to be ≥10^?6 sec.     

35-Cl nuclear magnetic resonance studies of anion-binding sites in proteins: lactate dehydrogenase.

³⁵Cl nmr relaxation rate measurements have been used to study anion-binding sites in pig heart lactate dehydrogenase. These studies reveal two types of sites, one is intimately associated with the active site, the other is not. The nonactive site has been ascribed to a subunit site in analogy with crystallographic results from the dogfish M₄ enzyme. The binding of either the reduced or the oxidized form of NAD results in an increase in the ³⁵Cl nmr relaxation rate by a factor of 1.8–2. The enhanced nmr relaxation rate of the binary lactate dehydrogenase-NAD complex is reduced on binding of the substrate inhibitor molecules oxamate or oxalate to a value less than that exhibited by lactate dehydrogenase alone. The enhancement of the nmr relaxation rate is attributed to a decrease in the dissociation constant of Cl for the enzyme. The K_p values for Cl binding to the active center site of lactate dehydrogenase is 0.85 m and for lactate dehydrogenase-NADH is 0.25 m. The ratio of these constants, 3.4, agrees well with the measured enhancement value 3.7. The effect of coenzyme analogs on the ³⁵Cl nmr relaxation rate has been examined. 3-Acetylpyridine NAD produces an enhancement of 4.3, thionicotinamide NAD of 2.3, whereas 3-pyridinealdehyde, adenosinediphosphoribose, and adenosine diphosphate do not affect the nmr relaxation state of Cl bound to lactate dehydrogenase.     

113Cd nmr study of the metal cluster structure of human liver metallothionein

Cadmium-113 nuclear magnetic resonance (¹¹³Cd nmr) was used to elucidate the structural properties of the cadmium binding sites in human liver metallothionein. The isotopically labeled ¹¹³Cd-metallothionein was prepared by the in vitro exchange of the native metals (greater than 94% zinc) for ¹¹³CdCl₂ during isolation. The two isoproteins, MT-1 and MT-2, showed ¹¹³Cd nmr resonances in the chemical shift range 610–670 ppm. The multiplet structure of the resonances is due to two bond scalar interactions between adjacent ¹¹³Cd ions linked by cysteine thiolate ligands. Homonuclear ¹¹³Cd decoupling experiments allowed the determination of the metal cluster structure, which, similar to the rabbit liver metallothionein, consists of a four- and a three-metal cluster designated cluster A and cluster B, respectively. Chemical shift similarities in the ¹¹³Cd nmr spectra of the human, rabbit and calf liver MT-1 and MT-2 are observed, especially for cluster A. Small variations in chemical shifts are explained in terms of differences in the primary structure between the two human isoproteins.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

Specificity of zinc binding to myelin basic protein

Z²⁺ appears to stabilize the myelin sheath but the mechanism of this effect is unknown. In a previous report we have shown that zinc binds to CNS myelin basic protein (MBP) in the presence of phosphate and this results in MBP aggregation. For this paper we used a solid phase zinc blotting assay to identify which myelin proteins bind zinc. MBP and a 58 kDa band were found to be the major targets of⁶⁵Zn binding. Moreover, using fluorescence, light scattering and electron microscopy we investigated the binding of zinc and other cations to purified MBP in solution. Among the cations tested for their ability to interfere with the binding of zinc, the most effective were cadmium, mercury and copper, but only cadmium and mercury increased the scattering intensity, whereas MBP aggregation was not inhibited by copper ions. Thus, the effect of zinc on the formation of MBP clusters seems to be specific.     

35Cl nuclear magnetic resonance study of zinc and phosphate binding of E. coli alkaline phosphatase

³⁵Cl^? quadrupole relaxation was measured in the presence of metal-free alkaline phosphatase and in the presence of Zn²⁺-alkaline phosphatase. The relaxation data show that for an enzyme containing the minimum amount of zinc needed for full activity—2 g atoms of zinc per mole of protein—there appears to be no binding of halide ions to the protein-bound zinc ions. In contrast, when there is a high metal-enzyme ratio, a large relaxation enhancement is observed, demonstrating coordination of halide ions to the metal ions.Addition of inorganic phosphate causes no change in the ³⁵Cl^? relaxation in the presence of metal-free enzyme. However, marked decreases in relaxation are observed upon addition of phosphate to the Zn²⁺-alkaline phosphatase. The relaxation measurements carried out in the presence of phosphate show that substrate binding does prove to be metal-ion dependent. Furthermore, experiments with inorganic phosphate suggest the tight binding of one phosphate to the alkaline phosphatase.     

Complex formation of zinc,cadmium, and mercury with penicillamine

The complex formation of zinc, cadmium, and mercury with D-penicillamine has been studied by pH titrations, using computer evaluation of the most likely complexes, which were found to be of the general formulas ML, MH₂L₂, MHL₂-, ML₂^2?, and ML₃^4?. The formation constants of the complexes were determined at 25 0°C in 0.1 M KNO₃. The magnitude of the respective constants cannot, by itself, account for the lack of effect of penicillamine treatment for mercury and cadmium poisoning.     

Interactions between a soil fungus,Trichoderma harzianum,and IIb metals—adsorption to mycelium and production of complexing metabolites

Fungi are capable of accumulating metals and, in soil, such accumulation may influence metal speciation and transport. The interactions between a common soil fungus, Trichoderma harzianum, and IIb elements were studied in the present investigation. The accumulation of the metals zinc, cadmium and mercury by starved and non-starved mycelium at different pH was determined by a batch technique using radioactive tracers; uptake of the metals was found to be large, with respective distribution coefficients of about 10^3.5, 10^2.5 and 10^4.0 for zinc, cadmium and mercury, respectively. Metal accumulation by a starved system was largely independent of pH in the range 3–9, where in a non-starved system an increased accumulation of zinc (at 10^– m) was observed at low pH (3–5). Potentiometric titrations performed on the two systems revealed significant differences in acid capacities, i.e. values close to zero for the starved system and 500–800 meq kg^– for the non-starved system. The maximum metal uptake was at least 50 mmol kg^– at pH 6.5 (calculated from adsorption isotherms). The present findings suggests that in the non-starved system a metabolite is produced and then released when the pH is within a certain range.     

The relation between activity and zinc and chloride binding of Escherichia coli alkaline phosphatase.

The relation between Zn²⁺ binding of E. coli alkaline phosphatase and enzymatic activity and anion binding (using ³⁵Cl NMR) has been investigated. The results suggest the existence of two forms of the enzyme with different zinc binding properties. The anion binding associated with the enzyme's function appears to be an amino acid residue and not the Zn²⁺ ions; furthermore, there is a rapid internal motion at the anion binding site. ³⁵Cl relaxation studies in the presence of Mg²⁺ ions point to a marked interdependence of Mg²⁺ and Zn²⁺ binding.     

Metal loading and enzymatic degradation of fungal cell walls and chitin

The capacity of chitin (from crab shells) and of fungal cell walls from Trichoderma harzianum to accumulate zinc, cadmium and mercury was studied as well as the effects of adsorbed metals on the enzymatic hydrolysis by Novozym 234 of the two substrates. The total adsorbing capacity with respect to these metals was estimated to be at least 10 mmol kg^–1 chitin (dry weight) and 50 mmol kg^–1 fungal cell walls (dry weight), respectively, at pH 6.1. Enzymatic digestion of fungal cell walls preloaded with mercury and cadmium was significantly reduced, while zinc did not cause any significant inhibition. The effect of metal complexation by chitin on the enzymatic digestion was not as pronounced as for fungal cell walls. This could reflect the fact that chitin sorbed a lower total amount of metals. The inhibitory effect of metals on the enzymatic hydrolysis was caused by the association of the metals with the two substrates and not by the presence of free metals in solution.     

Studies on the mercury volatilizing enzymes in nitrogen-fixing Beijerinckia mobilis

Beijerinckia mobilis KDr₂, a broad-spectrum, mercury-resistant nitrogen-fixing organism, possesses multiple metal-resistance properties. Mercuric reductase and organomercurial lyase activities of this bacterial strain were determined using different mercury compounds and heavy metal salts of copper, nickel, cobalt, cadmium, silver, zinc, lead and arsenate as inducers and substrates. Mercuric reductase was partially purified and the effect of some enzyme inhibitors and heavy metal ions on the enzyme activity was studied. The enzyme activity was completely inhibited by CdCl₂, Bi(NO₃)₃ and KCN at 10^-5 M and by AgNO₃, CoCl₂ and CuSO₄ at 10^-4 M.     

Zinc,cadmium, mercury and selenium in minke whales,belugas and narwhals from West Greenland

Summary Samples of muscle, liver and kidney from 24 minke whales (Balaenoptera acutorostrata), 43 belugas (Delphinapterus leucas), and 98 narwhals (Monodon monoceros) were analyzed for zinc, cadmium, mercury, and selenium. Highly significant age accumulation of mercury was found. A lower level of significance of age accumulation of cadmium in belugas and narwhals is probably due to the fact that some of the highest cadmium concentrations are in subadults and young adults. The maximum concentrations of cadmium and mercury are very high: 1.68, 73.7, and 125 g cadmium, and 9.88, 42.8, and 4.61 g mercury per g wet weight of narwhal muscle, liver and kidney, respectively. The cadmium concentrations are correlated in the three organs, as are mercury and to a lesser extent selenium concentrations. The concentrations of mercury and selenium in liver are highly correlated.     

The structure of the antitumor complex cis-(diammino) (1,1-cyclobutanedicarboxylato)-Pt(II): X ray and nmr studies

X-ray crystallography shows that Pt(NH₃)₂(CBDCA) is a square-planar complex with the dicarboxylate chelate ring in the boat conformation and a planar cyclobutane ring. ¹H and ¹³C nmr studies suggest that rapid chelate ring flipping occurs in solution. The value of ¹⁹⁵Pt nmr combined with ¹⁵N labeling as an informative new method of studying carboxylate coordination is illustrated. nmr results are also reported for the analogous ethylmalonate complex.     

Identification of a cadmium-binding protein from a cadmium-resistant variant of human lymphoblastoid cells (WI-L2)

Cadmium-binding protein synthesis and induction by cadmium chloride were studied in the human lymphoblastoid cell line WI-L2. Lymphoblasts were adapted to growth in 5 μM cadmium chloride (Cd^r) and these cells were 2.5-fold more resistant to cadmium than the parental line. There was no difference in the cellular protein profile between the parental line and lymphoblasts grown for a short period, less than 10 days, in cadmium chloride as measured by [³⁵S]cysteine labelling and SDS-polyacrylamide gel electrophoresis. A basal level of cadmium binding protein was apparent, however, by gel filtration. The Cd^r lymphoblasts were found to synthesize a substantial amount of cadmium-binding protein, approximately 25-fold more than the parental line. The cadmium-binding protein has the following properties which are consistent with its being a metallothionein: (1) [³⁵S]Cysteine-labelled protein eluted at a on a Sephadex G-75 column; (2) the molecular weight was estimated as 11 kDa on 7–17% SDS polyacrylamide gels; (3) the protein was heat-stable; (4) the unlabelled protein bound ¹⁰⁹Cd²⁺.     

Determination of the relaxation time of the helix–coil transition of poly(γ-benzyl-L-glutamate) by the nmr technique

NMR measurements of poly(γ-benzyl-L -glutamate) are reported in several different strengths of magnetic field to determine the relaxation time of the helix–coil transition. Nmr spectra of various samples had line shapes varying from the double to single, depending on the extent of the polydispersity of the sample. This result indicated that the correct line shape of a polypeptide is obscured in the overlapping of multipeaks, which are due to the heterogeneity of the molecular weight in the sample. Thus, the conventional line-shape analysis could not be applied to the kinetic study of the helix–coil transition of polypeptides without consideration of this polydispersity effect on the line shape. To overcome this difficulty, we measured linewidths of nmr spectra for fairly monodisperse samples, using various nmr spectrometers, having field strengths from 60 to 220 MHz. The results were analyzed by a quadratic equation, which involves an additional term proportional to the frequency difference of two sites. The equation differs from the conventional quadratic equation, usually utilized in the case of the fast-exchange limit, only in this additional term. This modification is required to evaluate correctly the unusual broadening of the linewidth resulting from the polydispersity effect and to determine the relaxation time reflected in nmr. Nmr spectra of three samples (DP-35, 85, and 250) were measured by 220-, 100-, and 60-MHz spectrometers in trifluoroacetic acid/chloroform at 28°C and linewidths were analyzed. Relaxation times of the helix–coil transition obtained at the transition midpoint are 2.5 × 10^?4, 7 × 10^?4, and 1.1 × 10^?3 sec, for DP-35, 85, and 250, respectively.     

A 13C nuclear magnetic resonance study of the interaction of ligands with arginine residues in dihydrofolate reductase.

¹³C nmr spectra of $\text{Streptococcus faecium}$ dihydrofolate reductase containing [¹³C-guanidino] arginine and ligand complexes with the labeled enzyme are reported. The spectrum of the native enzyme shows 5 well-resolved resonances (the enzyme contains 8 Arg) with a chemical shift range of 1.2 ppm. Addition of ligands to the enzyme produces distinct changes in the enzyme spectrum, and demonstrates that ¹³C nmr of labeled protein can be used in studies of protein-ligand interactions. An example of the use of ¹³C-depleted material is also presented.     

Levels of toxic and essential elements in arctic fox in Svalbard

Concentrations of cadmium, mercury, lead, arsenic, selenium, copper, zinc, manganese and iron in liver, and cadmium in kidneys, were analysed in 95 carcasses of arctic fox (Alopex lagopus) caught in Svalbard during three winter seasons from 1984 through 1986. The hepatic concentration ranges of cadmium, mercury, lead and arsenic were 0.1–2.4, 0.01–2.2, < 0.5–2.9 and 0.01–1.3 g·g^–1 WW, respectively. The range of cadmium concentration in the kidneys was from 0.2 to 13 g·g^–1 WW. Cadmium and mercury concentrations were higher in adult animals than in juveniles. The average concentrations of cadmium and lead were similar to recently published levels in polar bear from Svalbard, but the mercury concentrations were lower. Significant geographical differences were observed between trapping areas. Foxes caught north of Isfjorden had lower levels of liver iron and higher levels of all other elements analysed than those caught south of Isfjorden. The recorded concentrations of heavy metals indicate a moderate degree of exposure, which most likely is of natural origin.Gunnar Norheim died January 9, 1991     

Synthesis of coordination compounds via dehalogenation of zinc bromoacetate in presence of some amines

The dehalogenation reactions of pure zinc bromoacetate and its mixtures with hexamethylenetetramine, 2,2′-bipyridine, 1,10-phenanthroline were studied in water solutions. Due to the decomposition of hexamethylenetetramine during reaction, the cadmium bromoacetate-hexamethylenetetramine system was also studied. The four new coordination compounds, catena-[bis(μ₂-α-hydroxyacetato-κ³O¹,O²:O^1′)-zinc], catena[μ₂-1-(8-carboxylateethyl)-1,3,5,7-tetra-aza-adamantan-1-ium-N,O′]-dibromo-cadmium, dibromo-(2,2′-bipyridine-N,N′)-zinc, dibromo-(1,10-phenanthroline-N,N′)-zinc were synthesised and characterised by elemental and thermal analysis, IR and UV-vis spectroscopy, and X-ray crystallography. All compounds are air stable and well soluble in water. The zinc hydroxyacetate creates two-dimensional 4-c uninodal net. The zinc atoms are four and six coordinated and the cadmium atom is five coordinated. The coordination polyhedra of central atoms can be described as octahedron and trapezoidal pyramid for Zn and Cd polymers, respectively, and as trigonal pyramid for ZnBr₂ complexes. The bond valences considerations show that the chelating amines are bonded almost two times stronger to the central atoms than the chelating carboxylate groups. In the structures of polymeric compounds exist O-H?O, C-H?O, C-H?N and C-H?Br hydrogen bonds. The IR spectra show typical vibrations for chelating amine molecules. The thermal decomposition of studied compounds proceeds via multiple steps with gradual evolution of ligands.     

Effect of zinc on cadmium toxicity to the amphipod Gammarus pulex

The freshwater amphipod Gammarus pulex was sampled from several sites in Northumberland, England and analysed for total body zinc and cadmium concentrations.The toxicity of cadmium to animals from the different sites was determined and a positive relationship was found between the initial total body zinc concentration and the ability to survive in an artificial medium containing 0.5 mg 1^–1 cadmium as cadmium sulphate.Exposure in the laboratory to a low concentration of zinc (0.01 mg 1^–1) as zinc sulphate for two weeks resulted in a decreased susceptibility to cadmium poisoning.     

Concentrations of heavy metals in maternal and umbilical cord blood

Concentrations of lead, cadmium, methylmercury and total mercury were measured in maternal and umbilical cord blood using graphite atomic absorption spectrometry. Two essential metals, copper and zinc, were also determined using ion chromatography. Lead, copper and zinc were found to be lower in the cord blood, whereas methylmercury and total mercury were higher in cord blood than in maternal blood. Little differences were noted for cadmium in maternal and cord blood. Significant positive correlations were observed between the concentrations in maternal and cord blood with regard to lead (correlation coefficient, r = 0.44), copper (r = 0.34), zinc (r = 0.29), methylmercury (r = 0.44) and total mercury (r = 0.58). These results suggest that, like essential metals, most heavy metals can move rather freely across the human placenta. The potential health effects of heavy metal transfer from mothers to young infants cannot be discounted.