Charged Chelating Research Articles

Metal ion capillary zone electrophoresis with direct UV detection: Comparison of different migration modes for negatively charged chelates

Metal ion capillary zone electrophoresis with direct UV detection: Comparison of different migration modes for negatively charged chelates

Isolation and Partial Characterization of a Manganese and Chloride Binding Protein Present in Highly Purified Photosystem II Complexes of the Thermophilic Cyanobacterium Synechococcus sp.: The Protein Being Detected by Its L -Arginine Metabolizing Activity

Photosystem II complexes were solubilized with the detergent sulfobetaine 12 from thylakoid membranes of the thermophilic cyanobacterium Synechococcus sp. and purified by two sucrose gradient centrifugations and by chromatography on a Mono Q column. In such photosystem II complexes having a photosynthetic O2, evolving activity of 2938 μmol O2 evolved/mg chlorophyll x h, an ʟ-arginine metabolizing activity leading to ornithine and urea as major products, could be shown to be present. Besides ornithine and urea, a product (or products) of yet unknown structure is formed in addition - especially under aerobic conditions. This activity remained associated with photosystem II complexes even after substantial additional treatments to remove loosely bound proteins. On chlorophyll basis the maximal activity obtained under optimal assay conditions corresponded to 94 μmol ornithine formed/mg chlorophyll x h. This PS II associated, ʟ-arginine metabolizing enzyme was isolated (utilizing a manganese charged chelating Sepharose 6 B column) and partially characterized. It could be shown that this enzyme requires manganese and chloride for its ʟ-arginine metabolizing activity and that manganese becomes totally lost during purification indicating that manganese is bound to a fairly exposed site on the protein. Since it is rather unlikely that two different manganese and chloride binding proteins are present in such highly purified photosystem II complexes, the possibility of this protein being the water oxidizing enzyme will be discussed. Whether the manganese and chloride requiring ʟ-arginine metabolizing activity of this protein which provided a suitable assay for its isolation from photosystem II complexes, has any physiological significance, can not be answered at the present time.

Metal ion capillary zone electrophoresis with direct UV detection: Comparison of different migration modes for negatively charged chelates

Several migration modes suitable for capillary zone electrophoretic (CZE) separation of metal ions in the form of 8-hydroxyquinoline-5-sulphonic acid complexes are described and compared. Superior analysis time, resolution, efficiency and detectability were achieved using reversed movement of anionic metal complexes (in the anode-to-cathode direction) under the action of the electroosmotic flow. This method allows the CZE analysis of multicomponent mixtures of transition metal ions as well as aluminium within about six minutes.

Metal ion capillary zone electrophoresis with direct UV detection: determination of transition metals using an 8-hydroxyquinoline- 5-sulphonic acid chelating system

The application of capillary zone electrophoresis to the separation and determination of metal ions after the precolumn formation of negatively charged chelates is described. Multi-component mixtures of transition metal complexes with 8-hydroxyquinoline-5-sulphonic acid (HQS) were separated in about 10 min in a fused-silica capillary column with a borate buffer of pH 9.2 at an applied voltage of 15 kV followed by direct UV detection. The capillary pretreatment with an electroosmotic flow modifier, namely a tetraalkylammonium salt, is necessary to achieve resonable migration times of these metal complexes. Incorporating the chelating reagent in the electrophoretic buffer markedly improves the detectability of relatively unstable chelates, such as those of Co(II), Zn(II) and Cd(II), and allows the separation of metal ions that form unstable HQS chelates, such as Mn(II) and alkaline earth metals. The effects of electrophoretic buffer parameters affecting the complexation reaction and migration behaviour are discussed. Linearity of calibration graphs is observed for about three orders of magnitude with sub-ppm detection limits. The applicability of the method to the analysis of real samples is demonstrated.

Effect of tetraalkylammonium salt on the retention of metal chelates in reversed phase-high performance liquid chromatography

The retention of metal chelates with azo dye chelating agents was greatly reduced when tetraalkylammonium salts (TAAS) were added to the aqueous organic mobile phase in reversed phase-high performance liquid chromatography (RP-HPLC). TAAS was very effective for decreasing the retention time of the chelates including positively charged chelates strongly retained on the reversed phase stationary support. This was probably due to the preferential adsorption of TAAS on the support by the ion exchange process and the hydrophobic interaction. The use of TAAS was a promising means for improving column selectivity in RP-HPLC without depending upon the properties of a stationary support and of a mobile phase.

Immobilized metal ion affinity chromatography for the purification of Fel d I, a cat major allergen, from a house-dust extract'

Although the efficient isolation and purification of the major feline allergen have previously been achieved using polyclonal or monoclonal antibody affinity chromatography these methods lead to a relatively low yield of pure allergen. Therefore, attempts were made to establish a process involving ion-exchange chromatography followed by immobilized metal ion affinity chromatography. Although slightly more time consuming than the immunological methods, it gives a higher yield. It consists of four steps. From a crude house-dust exract, precipitation of a Fel d I enriched fraction is obtained by addition of acetone. After dissolution in water, exhaustive dialysis takes place against a citric acid solution to eliminate divalent metal ions .This gives the whole house-dust extract. Then partial isolation and purification are obtained by ammonium sulfate precipitation. The resulting precipitate is dissolved in and dialysed against water to remove ammonium salts. This partially purified house-dust extract can be submitted to chromatography on an anion exchanger and then on a copper ion charged chelating gel. All steps of the isolation and purification were controlled by immunochemical analyses using a crossed immuno-electrophoretic method and enzyme-linked immunoadsorbent assays. The HD75 Q2 Cu2 fraction so obtained can be considered to be a pure cat major allergen Fel d I preparation. Its homogeneity was demonstrated using physico-chemical and immunochemical methods.

Carbon-13 NMR of methylated lysines of fd gene 5 protein: evidence for a conformational change involving lysine 24 upon binding of a negatively charged lanthanide chelate

Helical complexes formed between fd DNA and reductively methylated fd gene 5 protein were indistinguishable by electron microscopy from complexes formed with the nonmethylated protein. 13C NMR spectroscopy of 13C-enriched N epsilon, N epsilon-dimethyllsyl residues of the protein showed that three of these residues (Lys-24, Lys-46, and Lys-69) were selectively perturbed by binding of the oligomer d(pA)7. These were the same lysyl residues that we previously found to be most protected from methylation by binding of the protein to poly[r(U)] [Dick, L. R., Sherry, A. D., Newkirk, M. M., & Gray D. M. (1988) J. Biol. Chem. 263, 18864-18872]. Thus, these lysines are probably directly involved in the nucleic acid binding function of the protein. Negatively charged chelates of lanthanide ions were used to perturb the 13C NMR resonances of labeled lysyl and amino-terminal residues of the gene 5 protein. The terbium chelate was found to bind tightly (Ka approximately 10(5) M-1) to the protein with a stoichiometry of 1 chelate molecule per protein dimer. 13C resonances of Lys-24, Lys-46, and Lys-69 were maximally shifted by the terbium chelate and were maximally relaxed by the gadolinium chelate. Also, the terbium chelate was excluded by the oligomer d(pA)7. Computer fits of the induced chemical shifts of 13C resonances with those expected for various positions of the terbium chelate failed to yield a possible chelate binding site unless the chemical shift for Lys-24 was excluded from the fitting process.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of charge characteristics of Cu-chelates on the Cu uptake from the solution by italian ryegrass and red clover

Most of the studies on the effect of chelation on metal uptake are limited to anionic ligands such as EDTA. These synthetic chelating agents are known to depress the uptake of Cu, Zn, and Cd from nutrient solutions by plants (Harrison et aL 1984; Gregar and Lindberg 1986; Iwasaki et al. 1987b). Moreover, the existence of excess ligand can cause deficiency symptoms (Halvorson and Lindsay 1977). Dekock and Mitchell (1957) have pointed out that the residual charge on the chelate molecule is one of the factors controlling the uptake of metals by the roots. They have concluded that complexes without charge or with a single negative charge are taken up by the roots unlike those with two negative charges. However the effect of positively charged chelates has not yet been compared. The purpose of this study is to compare the effects of Cu-chelates with different charge (negative, neutral, and positive) on the Cu uptake by plants and to analyze the significance of their charges, especially the positive charge. We ...

The regulation of exogenous NAD(P)H oxidation in spinach (Spinacia oleracea) leaf mitochondria by pH and cations.

Essentially chlorophyll-free mitochondria were isolated from green leaves of spinach (Spinacia oleracea L. cv. Viking II). Uncoupled oxidation of exogenous NADPH (1 mM) to oxygen had an optimum at pH 6.0, and activity was relatively low at pH 7.0, even in the presence of 1 mM-CaCl2. There was a proportional increase in the apparent Km for NADPH with decreasing H+ concentrations, suggesting that NADPH protonated on the 2'-phosphate group was the true substrate. Exogenous NADH was oxidized by oxygen with an optimum at pH 6.9. Under low-cation conditions, EGTA or EDTA (both 1 mM) had no effect on the Vmax. of NADH oxidation, although the removal of bivalent cations from the membrane surface by the chelators could be observed by use of 9-aminoacridine fluorescence. In contrast, under high-cation conditions, chelators lowered the Vmax. by about 50%, probably due to a better approach of the negatively charged chelators to the negative membrane surface than under low-cation conditions. In a low-cation medium, the Vmax. of NADH oxidation was increased by about 50% by the addition of cations. This was caused by a lowering of the size of the negative surface potential through charge screening. In contrast with other cations, La3+ inhibited NADH oxidation, possibly through binding to lipids essential for NADH oxidation. The apparent Km for NADH varied 6-fold in response to changes in the size of the surface potential, suggesting that the approach of the negatively charged NADH to the active site is hampered by the negative surface potential. The results demonstrate that the spinach leaf cell can regulate the mitochondrial NAD(P)H oxidation through several mechanisms: the pH; the cation concentration in general; and the concentration of Ca2+ in particular. The results also emphasize the importance of electrostatic considerations when investigating the kinetic behaviour of membrane-bound enzymes.

Role of the spoT gene product and manganese ion in the metabolism of guanosine 5‘-diphosphate 3‘-diphosphate in Escherichia coli.

Addition of divalent ion chelating agents picolinic acid, 1,10-phenanthroline, or quinoline-2-carboxylic acid to wild type, relA, or relX, but not spoT strains of Escherichia coli increases the levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). Poorly chelating analogs of these agents and a larger and more highly charged chelating agent, ethylene glycol bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid are ineffective. Mn2+ reverses the increase in ppGpp. The increase in ppGpp in wild type cells can be explained by an inhibition of degradation. In spoT cells the response is more complex; ppGpp does not increase although degradation is completely inhibited. The lack of increase in spoT cells suggests a role for spoT in synthesis of ppGpp in addition to its known role in degradation. Growth of both spoT+ and spoT cells is inhibited following chelator addition. This suggests that growth inhibition is through a mechanism not directly involving ppGpp. The results of this study provide evidence in intact cells for a role for Mn2+ and the spoT gene product in ppGpp degradation, and provide further evidence for an involvement of spoT and possibly divalent ions in ppGpp synthesis.

Stability of fused rings in metal chelates. VI. Structures and stability constants of the copper(II) chelates of dipeptides containing glycine and/or β-alanine

The reactions of the dipeptides containing glycine and/or β-alanine with an equimolar amount of copper(II) nitrate in acid solution were investigated potentiometrically, and the stability constants for the copper(II) chelates formed in acid solution were determined at 25 °C by the method of Datta and Rabin. The chelates with the composition [Cu.peptide(OH2)] were isolated in the pure state. The dipeptides exhibited analogous reactivities, and the stability constants, log K1, for the charged chelates, CuHL+, were found to be between 5.5–5.7. Comparison of the values for glycylglycine and β-alanyl-glycine with those for glycine and β-alanine suggests that these dipeptides act as terdentate ligands forming fused-ring chelates, where the coordinating sites are probably the amino nitrogen, amide nitrogen, and carboxyl oxygen, although the complexing ability of the amide NH group may be weak. The constants, Kc, for the dissociation of the amide hydrogen from the charged chelates to form the deprotonated chelates, CuL, with the rigid 5-5-, 5-6-, or 6-6-membered ring system decrease in the order glycylglycine > β-alanylglycine > glycyl-β-alanine > β-alanyl-β-alanine. The log K1Kc values indicate that the tendency of forming the deprotonated chelates also decreases in this order.