Metal Ion-Biomolecule Interactions. VIII: Methylmercury(II) Complexes of 8-Thioguanosine. Synthesis and Spectroscopic Investigations

The reaction of 8-thioguanosine (8-thioGuo^H₂ with methylmercury(II) has been shown to give rise to 1:1 (neutral and cationic), 1:2 (neutral and cationic), and 1:3 (cationic) complexes of the type [CH₃Hg(8-thioGuoH)], [(CH₃Hg(8-thioGuoH₂)]NO₃, [(CH₃Hg)₂(8-thioGuo)], [(CH₃Hg)₂(8-thioGuoH)]NO₃ and [(CH₃Hg)₃(8-thioGuo)]NO₃, depending upon the reactant stoichiometry and pH. ¹H NMR, ¹³C NMR, and IR, as well as analytical data were used to characterize the complexes. Coupling of methylmercury(II)-protons to mercury-199 has been observed in all compounds. The magnitude of the coupling, ²J(¹H-¹⁹⁹Hg), is strongly dependent on the nature of the ligand bonded to the methylmercury(II) group and correlates with the ¹³C chemical shifts of coordinated CH₃Hg(II) at the different binding sites.     

Metal ion-biomolecule interactions. XII. 1H and 13C NMR evidence for the preferred reaction of thymidine over guanosine in exchange and competition reactions with Mercury(II) and Methylmercury(II)

Mercury(II) bridge complexes of the type [Nuc-Hg-Nuc] (Nuc = thymidine or guanosine), and methylmercury(II) complexes of thymidine and guanosine of the type [CH₃Hg(Nuc)], have been prepared under appropriate conditions of pH and reactant's stochiometry in acqueous soluton. The various complexes have been characterized by ¹H and ¹³C NMR and used as probes, in competition and exchange studies, to establish the relative affinities of Hg(II) and CH₃Hg(II) towards the nucleosides guanosine and thymidine. These studies have confirmed that Hg(II) and CH₃Hg(II) bind to N₃ of thymidine in preference to N₁ of guanosine. The studies further show that reactions of mercury(II) with the nucleosides are thermodynamically controlled; the preperential binding reflects the relative stabilities of the respective complexes.     

Addendum

A number of solid complexes of theophylline with CH₃Hg¹¹, Hg¹¹, as well as both CH₃Hg¹¹ and Hg¹¹, have been isolated from aqueous solution as the result of the reactions of theophylline and mercury-containing species at the appropriate pH and mole ratios of reactants. The complexes have been characterized by elemental analysis, ¹H and ¹³C NMR and infrared spectroscopy, and X-ray crystal structure analysis. The results obtained indicate that the initial site of mercury binding is strongly pH dependent. No evidence could be found for CH₃Hg¹¹ substitution at the C₈ position in theophylline, contrasting with the situation in xanthosine and inosine.     

The heterocytotoxicity of human serum. II. Role of natural antibody and of the classical and alternative complement pathways.

Mouse lymphoid cells and tumor cell lines were employed as target cells for the investigation of the mechanism of heterocytotoxicity in human serum samples. It was shown that the heterocytotoxic effects were due to two differing mechanisms. Cytotoxicity was mediated in part, by activation of the alternative complement pathway on target cell membrane, a process which was antibody-independent. A second mechanism of cytotoxicity was induced by natural antibodies to a target cell, which probably mediated activation of the classical complement pathway. These data may shed light on the frequently observed cytotoxicity in mammalian sera for various target cells.     

The heterocytotoxicity of human serum. I. Activation of the alternative complement pathway by heterologous target cells.

Human serum induces cytolysis of mouse thymus and thymoma cells, and cytostasis of mouse bone marrow and spleen cells, and various methylcholanthrene-induced tumour cells. The latter was manifested by deficient metabolic activity when cultured in the presence of fresh human sera. Decomplementation procedures demonstrated that these heterocytotoxic effects are mediated in part via activation of the alternative complement pathway in human serum samples. The presence of properdin and C3 on the target cell surface was confirmed by immune adherence and indirect immunofluorescent tests. Activation of the alternative complement pathway was elicited by incubation of the human serum with the relevant target cells, resulting in the appearance of the cathodal migrating fragment of the factor B, denoting complement activation. The following publication will present evidence that activation of the alternative complement pathway takes place via an antibody-independent mechanism acting at the cell surface. These and other observations in the literature raise the possibility that activation of the alternative complement pathway by surface cell receptors on tumour cells represents a mechanism of natural immunity versus tumours.     

Galvinoxyl: a new spin probe for detecting phospholipid phase transitions.

Galvinexyl is a stable paramagnetic molecule which can be easily incorporated into phospholipid vesicles (liposomes). The csr spectrum from such labeled vesicles displays striking changes in the region of the phospholipid phase transition. These changes result from an enhancement in the tumbling motion of galvinoxyl molecules as a consequence of the “loosening” of the lipid environment that occurs at the phase transition.     

Monoclonal antibody-directed immunopurification and identification of cytochromes P-450

A 28 amino acid peptide with diuretic and natriuretic activity has been purified from rat atrial muscle. The primary structure of this atrial peptide is H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly- (sequence in text) Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-(Arg)-Tyr-OH. The biological activity of this peptide is identical to that of atrial natriuretic factor and cardionatrin I isolated from rat atria.     

The thermodynamics of lanthanide ion binding to adriamycin

We have examined the thermodynamics of lanthanide ion binding to adriamycin by monitoring the effects of variations in temperature on the dissociation constants of various lanthanide ion complexes of the drug. These constants were obtained by analyzing the extent of quenching of the fluorecence of adriamycin in the presence of lanthanide ions in terms of an equilibrium binding process. Our binding model included the following features, all of which are supported by evidence derived from previous published reports, vide infra. The lanthanides form 1:1 complexes with adriamycin. The binding is dependent on the pH of the solution, indicating that only the nonprotonated amine form of the drug participates in lanthanide ion binding. And finally the drug self-associates in solution to for a dimeric species. Our present results indicate that the binding process is almost completely independent of temperature, indicating that the enthalpy of complex formation is extremely small. The entropy terms are consistent with the formation of a complex in which the adriamycin acts as a bidentate ligand. Our results suggest that the lanthanide complexes are isostructural, at least as far as the adriamycin is concerned, throughout the lanthanide series.     

Quantitation and interaction of glycosaminoglycans with Alcian blue in dimethyl sulfoxide solutions.

An improved method is described for the quantitation of glycosaminoglycans separatedon cellulose acetate, stained with Alcian blue, and dissolved in a dimethyl sulfoxide solution. Standard curves are shown for all eight glycosaminoglycans. It is shown that absorption at the Alcian blue orthochromatic E_max is depressed under conditions which favor formation of dye-glycosaminoglycan complexes. The interaction between Alcian blue and the eight glycosaminoglycans was studied in dimethyl sulfoxide solutions of varying composition. It was shown that the extent of complex formation depends both on the glycosaminoglycan and the composition of the dimethyl sulfoxide solution. A dimethyl sulfoxide solution which contains 0.094 m H₂SO₄ is described which maximizes dye-glycosaminoglycan dissociation and thus the absorbance. Also, an improved staining method is described which improves dye uptake by the glycosaminoglycans and consequently increases the sensitivity of glycosaminoglycan quantitation.     

The binding of copper(II) and zinc(II) to oxidized glutathione

1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained.     

nmr and infrared spectroscopic investigations of the Al(III), Ga(III), and Be(II) complexes of ATP

The interaction of Al(III) with ATP has been examined by ³¹P and ¹H nmr and infrared spectroscopy. At pH 6.2, Al(III) forms a long-lived complex with ATP, in which chemical exchange between free and complexed ATP is slow on the nmr time scale. Infrared spectra of the Al(III)-ATP complex exhibit large perturbations in the band corresponding to the -PO₃^2? antisymmetric stretching mode. At higher pH values, equilibria involving Al(III) and OH^? become favored with the result that Al(III) no longer influences the spectroscopic properties of ATP. Similar spectroscopic results are obtained for the Ga(III) and Be(II) complexes of ATP.     

A spectroscopic and electrochemical study of the interaction between copper (II) glycylglycyl-L-histidine-N-methylamide and iron (III) tetra-p-sulfophenylporphine

A binuclear complex has been produced by the reaction of an iron porphyrin (sodium tetra-p-sulfophenylporphine iron (III)-FeTPPS) with a copper metallo-tripeptide (copper (II) glycylglycyl-L-histidine-N-methylamide-CuGGH) in aqueous solution. The system has been characterized by electron spin resonance (ESR) spectroscopy, optical absorption spectroscopy, and electrochemical methods. Room-temperature ESR spectra of the copper complex and low-temperature ESR spectra of the iron porphine provide evidence for the formation of a binuclear complex. These findings are supported by absorption spectroscopy and electrochemical studies, and lead to a value of ca. 2 X 10(-3) M-1 (at room temperature) for the equilibrium constant for complex formation. The relevance of this system to the enzymic active site of mammalian cytochrome c oxidase is discussed.     

Coordination chemistry of 7,9-disubstituted 6-oxopurine metal compounds. 4. Platinum(II) coordination at N(1). Molecular and crystal structure of [(ethylenediamine)bis(7,9-dimethylhypoxanthine)platinum(II)] hexafluorophosphate

The preparation and molecular and crystal structure of the complex [(ethylenediamine)bis(7,9,-dimethylhypoxanthine)platinum(II)] hexafluorophosphate, [Pt(C₂H₈N₂)(C₇H₈N₄O)₂] (PF₆)₂, are reported. The complex crystallizes in the monoclinic system, space group C2/c, with a = 12.334(2)Å, b = 10.256(2)Å, c = 22.339(3)Å, β = 101.31(1)°, V = 2771.0ų, Z = 4, D_measd = 2.087(3) g cm^?3, D_calc = 2.094 g cm^?3. Intensities for 3992 symmetry-averaged reflections were collected in the θ-2o scan mode on an automated diffractometer employing graphite-monochromatized MoKα radiation. The structure was solved by standard heavy-atom Patterson and Fourier methods. Full matrix least-squares refinement led to a final R value of 0.051. Both the ethylenediamine chelate and the PF₆^? anion are disordered. The primary coordination sphere about the Pt(II) center is approximately square planar with the bidentate ethylenediamine ligand and the N(1) atoms [Pt(II) ? N(1) = 2.020(5)Å] of two 7,9-dimethylhypoxanthine bases (related by a crystallographic twofold axis of symmetry) occupying the four coordination sites. The exocyclic O(6) carbonyl oxygen atoms of the two 7,9-dimethylhypoxanthine ligands participate in intracomplex hydrogen bonding with the amino groups of the ethylenediamine chelate [N(ethylenediamine) ? O(6) = 2.89( )Å]. The observed Pt ? O(6) intramolecular distances of 3.074(6)Å are similar to those found in other Pt(II) N(1)-bound 6-oxopurine complexes and in several Pt(II) N(3)-bound cytosine systems.     

Regulation of humoral immune responses by a bone marrow-derived glycolipid-like molecule

Bone marrow cells have been shown to nonspecifically suppress primary in vitro antibody responses. This suppression appears to be mediated by a low-molecular-weight soluble factor, B-SF which was released from a fraction of cells of similar size to the suppressor as obtained by velocity sedimentation. Like the suppressor cell, B-SF was also shown to be effective very early in the immune response. It was produced by all strains of mice tested and functioned across strain barriers. Characterization of the active suppressor molecule showed it to be a highly heat-stable, nonsialic acid-containing glycolipid of 1000 to 35000 daltons in molecular weight. Recovery of the purified suppressor from thin-layer chromatography plates was achieved indicating that the major glycolipid component visualized on TLC is likely the active suppressor molecule. The characteristics of this suppressor may show it to be a fundamental immune regulatory mechanism.     

The purification of methionine sulfoxide reductase from Escherichia coli

A sensitive assay, based on the acylation of tRNA^Met, has been developed to measure the enzymatic reduction of methionine sulfoxide to methionine. Using this assay, methionine sulfoxide reductase has been purified to near homogeneity from extracts of Escherichia coli.     

Ultraviolet difference spectroscopy of bovine carbonic anhydrase substituted with various divalent metals

The ultraviolet (uv) difference spectra of M(II)-apocarbonic anhydrase at pH 5–9 are reported. For Zn(II) at all pH's and Co(II) at pH ? 7.65 identical protein difference spectra are seen and a positive 300 nm feature is interpreted as consistent with interaction of a metal-bound hydroxyl with a Trp chromophore near the active site. Hg(II), Cu(II), and Cd(II) do not provoke a positive 300 nm band even at alkaline pH (although a Cd(II) spectral band at 300 nm becomes less negative, i.e., more like the holoenzyme with increasing pH) and the 280–292 nm spectral region is generally different from that of Zn(II) and high pH Co(II). A specific orientation of M-OH and, hence, an ordered solvent structure in the enzyme site is implied for enzyme activation. Ni(II) appears to bind to the vacated zinc site slowly, at low pH, in a manner similar to zinc. At higher pH's Ni(II) may be displaced toward a Tyr residue in the active site of apocarbonic anhydrase.     

Cadmium(II)-113 NMR studies of the mechanism of metal ion activation of yeast enolase

Yeast enolase binds one mole of 113Cd2+ per subunit at a site that consists of all oxyligands in a distorted octahedral environment. This "conformational" metal ion's environment undergoes further distortion on addition of substrate/product or analogs. At pH's below the optimum value the shifted resonance tends to break up into several, suggesting the existence of several slowly exchanging intermediate forms. At acid pH's, on addition of one additional mole/subunit of 113Cd2+, which greatly increases catalysis, "conformational" resonance(s) further broadens, suggesting that the second, "catalytic" metal ion increases the rates of interconversion between "conformational" species. At more alkaline pH's, near the optimum pH, the "conformational" peak is sharpened, which suggests that very fast interconversion is occurring. The position of the "catalytic" metal ion resonance also suggests all oxyligands in a distorted octahedral geometry. The "catalytic" resonance is often broadened to the point where it cannot be seen, suggesting rapid changes in its geometry due to interconversion of substrate and product.     

Preliminary studies on the inhibitory effect of novel Pd(II) complexes of vitamin B6 on cell divisions

Two novel complexes of Pd(II) involving vitamin B₆ compounds have been synthesized. They are compatible with the compositions Pd(P.H.)₂ C₂(P=pyridoxol) and Pd(PL.H)₂C₂ (PL = pyridoxal). The complexes inhibited the growth as well as the biosynthesis of RNA, DNA, and protein of E. coli B-766. Photoacoustic spectral (PAS) measurements showed that the complexes bound to DNA of the bacteria and were present only in the kidney of treated mice. The complexes inhibited the incorporation of ³H-thymidine as well as ¹⁴C-leucine in the DNA and protein, respectively, of liver cell cultures (BL8L). The inhibition of cell division of Walker-S-cells and human lymphocytes by the complexes was highly significant.     

Equilibrium and structural studies of silicon(IV) and aluminium(III) in aqueous solution. 12. A potentiometric and 29Si-NMR study of silicon tropolonates

Complexation in the H⁺-Si(OH)₄-tropolone (HL) system was studied in 0.6 M (Na)Cl medium at 25° C. Speciation and formation constants were determined from potentiometric (glass electrode) and ²⁹Si-NMR data. Experimental data cover the ranges 1.5 ? - log[H⁺] ? 8.4, 0.002 ? B ? 0.012 M, and 0 ? C ? 0.060 M (B and C stand for the total concentration of Si and tropolone, respectively). In acid solutions (-log[H⁺] ? 3) a hexacoordinated cationic complex, SiL₃⁺, is formed with log K(Si(OH)₄ + 3HL + H⁺ XXX SiL₃⁺ + 4H₂O) = 7.08 ± 0.03. Furthermore, the formation of a disilicic acid was established from ²⁹Si-NMR data. The dimerization constant of Si(OH)₄ was found to be 10 exp (1.2 ± 0.1). In model calculations the solubility of quartz and amorphous SiO₂ in the presence of tropolone is demonstrated. Data were analyzed using the least-squares computer program LETAGROPVRID.     

A Mössbauer study of gold(I) and gold(III) dithiolate complexes related to anti-arthritic drugs

The sensitivity of the ¹⁹⁷Au Mössbauer isomer shift and quadrupole splitting to gold oxidation state has enabled characterization of new stable, water—soluble Au(I) and Au(III) complexes of dimercaptosuccinic acid. The Au(I) complex exhibits a curious asymmetric line—broadening effect. Comparisons are also made with dimercaptopropanol and dithiocarbamate complexes. Relationships to existing and potential gold drugs are discussed.