Speciation of insulin-mimetic VO(IV)-containing drugs in blood serum

The biospeciations of three potential insulin-mimetic VO(IV) compounds, VO(maltolate)2, VO(picolinate)2 and VO(6-Me-picolinate)2, in blood serum were assessed via modelling calculations, using the stability constants reported in the literature for the binary insulin-mimetic complexes and their ternary complexes formed with the most important low molecular mass binders in the serum: oxalic acid, lactic acid, citric acid and phosphate. The binding capabilities of two high molecular mass serum proteins, albumin and transferrin, were also taken into account.     

Metal ion coordination of macromolecular bioligands: formation of zinc(II) complex of hyaluronic acid.

The coordination of zinc(II) ion to hyaluronate (Hya), a natural copolymer, in aqueous solution at pH 6 was investigated by potentiometric and circular dichroism (CD) spectroscopic methods, and by monitoring the changes in macroscopic properties by high-precision measurements. The zinc(II)-selective electrode, and CD measurements proved the binding of zinc(II) by Hya. A number of Hya fragments (Mr approximately 3.3 x 10(3)-1.4 x 10(6)) were studied to estimate the contributions of the polyelectrolyte effect, the solvation and host-guest interactions to the extra stabilization of the macromolecular zinc(II) complexes as compared with the monomeric unit. The zinc(II) ion activity increase reflected a stability decrease for the fragments with Mr < 4 x 10(4). This molecular weight differs from that where cleavage of the Hya skeleton starts (approximately 5 x 10(5), according to the size-exclusion gel, and anion-exchange chromatographic behavior of the Hya fragments) and from that where the polyelectrolyte effect stops (approximately 6 x 10(3)). The excess volumes and Bingham shear yield values of the solutions revealed the transformation of the coherent random coil structure stabilized by intermolecular association in the NaHya to an intramolecular association producing the globular structure of the ZnHya molecule, with a smaller but more strongly bound solvate water sheet.     

Antioxidant effects of the VO(IV) hesperidin complex and its role in cancer chemoprevention

Vanadium compounds are known for a variety of pharmacological properties. Many of them display antitumoral and osteogenic effects in several cell lines. Free radicals induce the development of tumoral processes. Natural polyphenols such as flavonoids have antioxidant properties since they scavenge different free radicals. For these reasons it is interesting to investigate the effects of a new complex generated between the vanadyl(IV) cation and the flavonoid hesperidin. The complex has been synthesized and characterized by physicochemical methods. Spectroscopic analysis revealed a 1:1 stoichiometry of ligand:VO and coordination by deprotonated cis-hydroxyl groups to the disaccharide moiety of the ligand. The complex improves the superoxide dismutase (SOD)-like activity of the ligand, but the scavenging of other radicals tested does not change upon complexation. When tested on two tumoral cell lines in culture (one of them derived from a rat osteosarcoma UMR106 and the other from human colon adenocarcinoma Caco-2), the complex enhanced the antiproliferative effects of the free ligand, and this effect correlated with the morphological alterations toward apoptosis. Also, on the osteoblastic cell line the complex stimulated cell proliferation and collagen type I production at low concentrations. At higher doses the complex behaved as a cytotoxic compound for the osteoblasts.     

Proton magnetic resonance study of complex formation between N6-methyladenosine and polyuridylic acid

The interaction between N₆-methyladenosine and polyuridylic acid in D₂O solution at neutral pD has been studied as a function of temperature and N₆-methyladenosine concentration by proton magnetic resonance spectroscopy. A rigid double-stranded 1:1 complex is formed below ～10°C, involving hydrogen-bonded N₆-methyladenine:uracil base-pairing and stacking of the adenine bases. This complex is less stable than the 1:2 complex formed between adenosine and polyU, and involves a more rapid exchange of the monomer between free and polymer-bound environments.     

Oxovanadium(IV) complex formation by simple sugars in aqueous solution.

The binding of oxovanadium(IV) to simple sugars in neutral or basic aqueous solution, as studied by EPR and electronic absorption spectroscopy, is reported. The complexation is favored in basic media and involves the coordination of the metal ion to couples of adjacent deprotonated hydroxyls of the sugar molecule. However, only the ligands provided with cis couples can adopt this chelating ligand behavior. The ability of the cis hydroxyl couples to yield chelated complexes has been related to the structural rearrangement (decrease of the O-C-C-O torsion angle in the five-membered chelated ring) needed to permit the oxovanadium(IV) coordination by the sugar molecule.     

Semi-synthetic antibiotics: Titanium(IV), zirconium(IV) and mercury(II) complexes of 6-amino penicillinic acid

A number of organometallic derivatives involving 6-amino penicillinic acid (I), of the types η⁵-R)₂M- (Cl)L^?Et₃NH⁺ (II), (η⁵-R)₂M(Cl)L (III) and R′HgL [R = cyclopentadienyl (C₅H₅), indenyl (C₉H₇), R′ = phenyl (C₆H₅), p-acetoxyphenyl (p-CH₃COOC₆H₄), o-hydroxyphenyl (o-HOC₆H₄), p-hydroxyphenyl (p-HOC₆H₄); M = Ti(IV), Zr(IV); LH = 6-amino penicillinic acid] have been synthesized and characterized. Conductance measurements indicate that while the (η⁵-R)₂M(Cl)L^?Et₃NH⁺ complexes are 1:1 electrolytes, the remaining compounds are non-electrolytes. From IR and UV spectral studies it is concluded that the penicillin moiety is bidentate. PMR and CMR studies support the stoichiometry of the complexes. Fluorescence studies have been carried out for o- and p-HOC₆H₄HgL complexes and relevant photochemical parameters have been elucidated. X-ray diffraction studies have been made for the o-HOC₆H₄HgL complex. For the C₆H₅HgL, p-CH₃COOC₆H₄HgL and p-HOC₆H₄HgL complexes, thermal studies (TG and DTA) have been carried out and kinetic parameters for thermal degradation have been enumerated. In addition, the fragmentation pattern of these complexes has been analysed on the basis of mass spectra. The C₆H₅HgL and p-CH₃COOC₆H₄HgL complexes show positive bactericidal activities.     

Host-guest complex formation in cyclotrikis-(1-->6)

The possibility that cyclotrikis-(1-->6)-[alpha-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl] (CGM6) forms inclusion complexes, like cycloamyloses (cyclodextrins), was investigated by means of electrospray mass spectrometry and fluorescence spectroscopy. The complexing ability of both 1-anilinonaphthalene-8-sulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS), which were already used with cyclodextrins, was investigated. The former showed very little or no tendency to be complexed by CGM6, while the latter produced detectable adducts with CGM6. Fixed 90 degree angle light scattering experiments supported the findings obtained by molecular modelling calculations, which indicated a polar character for the CGM6 internal cavity. CGM6-TNS complexes were probably formed throughout interaction of the polar regions of the two molecules.     

Bioconversion of 2,6-dimethylpyridine to 6-methylpicolinic acid by Exophiala dermatitidis (Kano) de Hoog DA5501 cells grown on n-dodecane

Alkane-assimilating microorganisms were isolated from enrichment cultures using n-octane, n-dodecane, n-hexadecane, or pristane (2,6,10,14-tetramethylpentadecane) as a sole carbon source to find microbial catalysts oxidizing methyl groups of 2,6-dimethylpyridine. The cells of Exophiala dermatitidis (Kano) de Hoog DA5501, an n-dodecane-assimilating fungus, oxidized a single methyl group of 2,6-dimethylpyridine to produce 6-methylpicolinic acid (6-methylpyridine-2-carboxylic acid) without the formation of dipicolinic acid (pyridine-2,6-dicarboxylic acid); 67 mM 6-methylpicolinic acid (9.2 g/l) accumulated with a molar conversion yield of 89% by 54-h incubation. The fungus cells also oxidized the methyl group of 2,6-dimethylpyrazine and 2,4,6-trimethylpyridine regioselectively.     

Intramolecular complex formation of poly(N-isopropylacrylamide) with human serum albumin

Complexation of human serum albumin (HSA) with poly(N-isopropylacrylamide) (PNIPA) ranging in molecular weight (M(PNIPA)) from 2.1 x 10(4) to 1.72 x 10(6) was studied in an aqueous system (pH 3) containing NaCl as a supporting salt. Dynamic light scattering, static light scattering, electrophoretic light scattering, and dialyzing techniques were used as the experimental tool in a suitable combination. The measurements were performed mainly at 25 degrees C and at 0.01 M NaCl as a function of mixing ratio (r(m), molar ratio of PNIPA to HSA). The results of DLS and ELS evidently demonstrated the formation of a water-soluble complex through mixing of HSA and PNIPA. A detailed analysis of SLS data with the aid of dialysis data revealed that the resulting complex is an "intramolecular" complex consisting of a PNIPA chain with several of bound HSA molecules. Both hydrodynamic radius (R(h)) and radius gyration (R(g)) of intramolecular complexes decreased as r(m) was increased. This result correlated well to the fact that the number (n) of bound proteins per polymer decreases with increasing r(m). The size and the molar mass of the complex became large depending on M(PNIPA), but the increase of M(PNIPA) led to a decrease in n at r(m) < 1. The increase in NaCl concentration from 0.01 to 0.3 M brought about the increase in the size and the molar mass of an intramolecular HSA-PNIPA complex prepared at r(m) = 1.1. This was found to be due to an increase of n. A similar trend was observed when temperature rose from 25 to 32 degrees C (close to lower critical solution temperature of PNIPA). However, the effect of temperature on the increase of was strong in comparison with that of ionic strength. On the basis of these results obtained, the complexation mechanism was discussed in detail.     

Radioimmunoassay of nafarelin ([ 6-(3-(2-naphthyl)-D-alanine)]-luteinizing hormone-releasing hormone) in plasma or serum

A procedure which is suitable for the radioimmunoassay (RIA) of nafarelin [( 6-(3-(2-naphthyl)-D-alanine)]-luteinizing hormone-releasing hormone) in plasma or serum at concentrations as low as 50 pg/ml is described. Antiserum was prepared by replacing the pyroglutamyl portion of nafarelin with glutaric acid, coupling the product to keyhole limpet hemocyanin, and immunizing rabbits with the resulting conjugate. At a dilution of 1:30,000 the binding was approximately 50%. The antibodies did not cross react with luteinizing hormone-releasing hormone. For RIA, 125I-labeled analyte was used as the tracer and charcoal was used to separate the free and the bound fractions. No purification of samples was required prior to RIA. Accuracy of the method was assessed by adding known quantities of nafarelin to nafarelin-free plasma and determining the ratio of measured to added analyte. Linear regression analysis for the concentration range 0.050-5.00 ng/ml yielded a regression equation of y = 1.01x - 0.066 and a correlation coefficient of 0.997. At 0.050 ng/ml the CV was 11.3% (interassay). Additional validation was obtained from an in vivo study in which [3H]nafarelin was administered to monkeys and plasma profiles were determined by RIA, by high-performance liquid chromatography (HPLC), and by an HPLC-radiochemical method. The results obtained by RIA agreed well with those obtained by the HPLC methods.     

Fluorescence studies on the complex formation between poly(rA) containing 1,N6-ethenoadenosine and poly(rU)

The interaction of the 1,N6-etheno derivatives of poly(rA) (poly(epsilon rA] with poly(rU) has been studied by absorption and fluorescence spectroscopy. The stoichiometry of the interaction is found to be 1 epsilon A:1 rU and 1 epsilon A:2 rU as well as in the case of poly(rA)-poly(rU) interaction. The fluorescence properties, including the intensity and polarization of fluorescence, respond to the conformational transition of poly(epsilon rA)-poly(rU) complexes. The introduction of epsilon A groups into poly(rA) results in a marked decrease in the melting temperature, suggesting that epsilon A may destabilize the helical structure. The three-exponential decay law obtained with poly(epsilon rA)-poly(rU) complexes indicates the existence of at least three different stacked conformational states.     

First evidences of the formation of a vanadyl(IV) complex of bleomycin

Results of visible/ultraviolet and infrared spectroscopic measurements, as well as chemical evidence are presented which support the formation of a 2:1 bleomycin-VO2+ complex. The information obtained also allows some considerations concerning the probable coordination sphere of the vanadyl ion.     

Comparative studies on the biospeciation of antidiabetic VO(IV) and Zn(II) complexes

The speciation of several insulin-mimetic/enhancing VO(IV) and Zn(II) complexes in human blood serum was studied and a comparison was made concerning the ability of the serum components to interact with the original metal complexes and the distribution of the metal ions between the low and the high molecular fractions of the serum. It was found that the low molecular mass components may play a larger role in transporting Zn(II) than in the case with VO(IV). Among the high molecular mass serum proteins, transferrin is the primary binder of VO(IV), and albumin is that of Zn(II). The results revealed that protein-ligand interactions may influence the metal ion distribution in the serum.     

Electrostatic interaction and complex formation between gum arabic and bovine serum albumin

The interaction of gum arabic (GA) and bovine serum albumin (BSA) has been investigated through turbidity and light scattering intensity measurements and by the use of dynamic light scattering, laser Doppler velocimetry, and isothermal titration calorimetry. It has been shown that GA and BSA can form soluble and insoluble complexes depending on the solution pH and the mixing ratio and is a function of the net charge on the complex. Soluble complexes were obtained when the electrophoretic mobility was greater than ±1. 5 μm s(-1) V(-1) cm(-1). Changes in the value of the isoelectric point of the complexes with mixing ratio and isothermal titration calorimetric data indicated that complexes formed at pHs 3 and 4 consisted of ～60 BSA molecules for every GA molecule, while at pH 5 there were ～10 BSA molecules per GA molecule. Calorimetric studies also indicated that the interaction occurred in two stages at both pH 3 and pH 4, but that the nature of the interaction at these two pH values was significantly different. This was attributed to differences in the relative magnitude of the positive and negative charges on the BSA and GA, respectively, and possibly due to changes in the BSA conformation. The fact that there is an interaction at pH 5, which is above the isoelectric point of the BSA, is due to the interaction of the carboxylate groups on the GA with positive patches on the BSA or to the charge regulation of the protein-polysaccharide system brought about by changes in dissociation equilibria. Complexation is reduced as the ionic strength of the solvent increases and is prevented at a NaCl concentration of 120 mM.     

Complex formation of double-stranded DNA (6-4) photoproducts and anti-(6-4) photoproduct antibody Fabs.

DNA (6-4) photoproducts are major constituents of ultraviolet-damaged DNAs. We prepared double-stranded (ds) (6-4) DNA photoproducts and analyzed formation of their complexes with anti-(6-4) photoproduct antibody Fabs. Elution profiles of the mixtures of ds-(6-4) DNAs and Fabs from anion-exchange and gel-filtration columns indicate that Fab 64M-2 deprives 14mer ds-(6-4) DNA of single-stranded (ss) (6-4) DNA and shows no interaction with 18 mer ds-(6-4) DNA (A18). Fab 64M-5 with an approximately 100-fold higher affinity than Fab 64M-2 forms a complex with the ds-(6-4) DNA (A18), but partly dissociates another 18 mer ds-(6-4) DNA (A18-3), with a lowered G-C content, into ss-DNAs. From these results, antibody 64M-5 possibly accommodates the T(6-4)T photolesion moiety of the ds-(6-4) DNA (A18) by flipping out the moiety from its neighboring segments.