In vitro antibacterial activity of meclofenamate metal complexes with Cd(II), Pb(II), Co(II), and Cu(II). Crystal structures of [Cd(C14H10NO2Cl2)2∙(CH3OH)]n and [Cu(C14H10NO2Cl2)2(C5H5N)2].

The synthesis and characterization of five metal complexes derived from sodium meclofenamate (1) are reported: [Cd(C14H10NO2Cl2)2∙(CH3OH)]n∙nCH3OH (6), [Pb(C14H10NO2Cl2)2]n (7), [Co(C14H10NO2Cl2)]n (8), [Cu(C14H10NO2Cl2)]n (9), and [Cu(C14H10NO2Cl2)2(C5H5N)2] (10) (C14H10NO2Cl2=meclofenamate; C5H5N=pyridine). The characterization of the compounds was based on FTIR and UV-visible spectroscopy, mass spectrometry and, in the case of complexes 6 and 10, single crystal X-ray diffraction analysis. For compound 6, the structural analysis revealed a 1-D polymeric chain structure, in which pentagonal planar [Cd(RCOO)2(CH3OH)] units were linked through bridging carboxylate functions of the meclofenamate ligands. The overall coordination environment of the Cd(II) ions was seven-coordinate, since each carboxylate group exhibited a µ3-bridging coordination mode. On the other hand, for complex 10 a discrete mononuclear structure was observed, in which the six-coordinate copper(II) metal atoms were coordinated by two pyridine molecules and the carboxylate functions of two meclofenamate entities, in an anisobidentate coordination mode. The antibacterial activity of compounds 6-9 against four strains of Gram positive (Staphylococcus aureus and Bacillus subtilis) and Gram negative (Escherichia coli and Pseudomonas aeruginosa) bacteria was examined, finding that only complex 6 was active. Additionally, it was found that the Co(II) and Cu(II) complexes 8 and 9 showed peroxidase activity.

Speciation study of the anti-inflammatory drug tenoxicam (Htenox) with Cu(II): X-ray crystal structure of [Cu(tenox)(2)(py)(2)].EtOH.

A speciation study was carried out in aqueous solution of the anti-inflammatory drug tenoxicam (Htenox), under quasi-physiological conditions (temperature of 37 degrees C and ionic strength 0.15 M NaCl) in order to determine the acidity constants from spectrophotometric studies, the pK(a) values found being pK(1)=1.143+/-0.008 and pK(2)=4.970+/-0.004. Subsequently, the spectrophotometrical speciation of the different complexes of Cu(II) with the drug was performed under the same conditions of temperature and ionic strength, observing the formation of Cu(Htenox)(2)(2+) with log beta(212)=20.05+/-0.01, Cu(tenox)(2) with log beta(012)=13.6+/-0.1, Cu(Htenox)(2+) with log beta(111)=10.52+/-0.08, as well as Cu(tenox)(+) with log beta(011)=7.0+/-0.2, all of them in solution, and solid species Cu(tenox)(2)(s) with an estimated value of log beta(012)(s) approximately 18.7. The crystalline structure of the complex [Cu(tenox)(2)(py)(2)]. EtOH, was also determined, and it was observed that tenoxicam employs the oxygen of the amide group and the pyridyl nitrogen to bond to the cation.

Immobilization of conalbumin onto polystyrene/divinylbenzene co-polymers: towards finding the best support for MAMC.

Immediately after the successful immobilization of conalbumin onto CNBr-activated Sepharose, efforts were begun to find a less expensive support and a more benign chemistry of activation. The potential of the Sepharose-conalbumin conjugate for decontamination of several metal-containing waste-waters has been established, and a new method of chromatography has emerged, named metalloprotein affinity metal chromatography (MAMC). Efforts to immobilize conalbumin onto polystyrene/divinylbenzene co-polymers, using the well known and commercially available Merrifield, aminomethyl and plain polystyrene resins are presented here. Immobilizations of conalbumin were carried out on the Merrifield and Aminomethyl resins, but the procedures were time consuming and complicated by polymer aggregation. Because of high cost of these materials, research was directed towards the activation and functionalization of plain polystyrene/divinylbenzene co-polymers. Chlorosulfonation followed by sulfonamide formation was attempted on three commercially available polymers. Successful polysulfonamide formation was achieved with bislysine copper(II) acting as a diamine. Removal of the copper allows the unblocking of the alpha amino group of the immobilized lysine which in turn is treated with glutaraldehyde, affording an activated support for immobilization of proteins. To date, approximately 46 mg transferrin/g dry matrix have been successfully immobilized. The chemical and biological inertness of this support makes it a good candidate to scale up the procedure and continue the optimization of MAMC.

High resolution proton nuclear magnetic resonance spectroscopy of lactoperoxidase.

The first high resolution proton nuclear magnetic resonance spectra are reported for the native ferric and ferric cyano complexes of bovine lactoperoxidase. The spectrum of the native species exhibits broad heme signals in a far downfield region characteristic of the high-spin ferric state. The low-spin cyano complex yields a proton nuclear magnetic resonance spectrum with signals as far as 68.5 ppm downfield and as far as -28 ppm upfield of the tetramethylsilane reference. These peak positions are anomalous with respect to those seen only as far as 35 ppm downfield in other cyano hemoprotein complexes. An extreme asymmetry in the unpaired spin delocalization pattern of the iron porphyrin is suggested. The unusual proton nuclear magnetic resonance properties parallel distinctive optical spectral properties and the exceptional resistance to heme displacement from the enzyme. Lactoperoxidase utilized in these studies was isolated from raw milk and purified by an improved, rapid chromatographic procedure.

Unique cyanide nitrogen-15 nuclear magnetic resonance chemical shift values for cyano-peroxidase complexes. Relevance to the heme active-site structure and mechanism of peroxide activation.

Cyanide ion has been utilized to probe the heme environment of the ferric states of horseradish peroxidase, lactoperoxidase and chloroperoxidase. The 15N-NMR signal for cyanide bound to these enzymes is located in the downfield region from 578 to 412 ppm (with respect to the nitrate ion reference). The corresponding signal for met-forms of hemoglobin, myoglobin and cytochrome c is much further downfield in the 1047-847 ppm region. The signal position for peroxidases is quite invariant with pH in the physiological ranges. The upfield bias for peroxidase chemical shifts must reflect unique trans iron(III) ligand types and/or proximal-group hydrogen bonding or steric effects. Model compound studies reveal a significant upfield cyanide 15N shift with addition of agents capable of hydrogen-bonding to the coordinated cyanide ion. An even more striking upfield shift of 277 ppm is associated with deprotonation of a trans imidazole residue. The distinctive chemical shifts observed for the cyano ligand in peroxidases support the hypothesis that a distal hydrogen-bonding network and perhaps a polar, basic trans ligand are essential for O-O bond activation by peroxidases.

Proton nuclear magnetic resonance spectroscopy of horseradish peroxidase isoenzymes: correlation of distinctive spectra with isoenzyme specific activities.

High-resolution proton NMR spectra are reported for the paramagnetic ferric native and cyano complexes of the five major horseradish root peroxidase (HRP) isoenzymes (A1, A2, A3, B, and C). Axial imidazole resonances are observed in the native and cyano-complex spectra of all the isoenzymes, thus indicating the presence of a common axial histidine ligand. Proton NMR spectra outside the usual diamagnetic region are identical for sets of A1 and A2 isoenzymes and for the B and C isoenzyme set. Variation in heme residue chemical shift positions may be controlled in part by porphyrin vinyl side chain-protein interactions. Diverse upfield spectra among the isoenzymes reflect amino acid substitutions and/or conformational differences near the prosthetic group, as signals in this region must result from amino acid residues in proximity to the heme center. Acid-base dependence studies reveal an "alkaline" transition that converts the native high-spin iron (III) porphyrin to the low-spin state. The transition occurs at pH 9.3, 9.4, 9.8, and 10.9 for respective HRP A1, A2, A3, and C isoenzymes, respectively. Significantly, this ordering also reflects specific activities for the isoenzymes in the order A1 = A2 greater than A3 greater than B = C. Identical proton NMR spectra for A1/A2 and B/C isoenzyme sets parallel equivalent specific activities for members of a particular set. Proton NMR spectra thus appear to be highly sensitive to protein modifications that affect catalytic activity.

Comparison of heme environments and proximal ligands in peroxidases and other hemoproteins through carbon-13 nuclear magnetic resonance spectroscopy of carbon monoxide complexes.

Carbon-13 nuclear magnetic resonance signals for the carbon monoxide ligand in ferrous complexes of horseradish peroxidase, lactoperoxidase, and chloroperoxidase are located respectively at 209.1, 208.3, and 200.8 parts per million from the tetramethylsilane reference. On the basis of previous hemoprotein and model compound studies these resonance positions are consistent with coordination of a proximal histidine ligand in horseradish peroxidase and lactoperoxidase, and coordination of a cysteinyl mercaptide ligand in chloroperoxidase. Carbonyl chemical shift values for acidic and basic horseradish peroxidase isoenzymes are very similar.

High-resolution proton nuclear magnetic resonance spectroscopy of chloride peroxidase: identification of new forms of the enzyme.

Chloride peroxidase from the mold Caldariomyces fumago in the native high-spin iron(III) and low-spin cyanoiron (III) states has been subjected to high-field proton nuclear magnetic resonance spectroscopic measurements. Signals shifted well outside the diamagnetic envelope by the paramagnetic iron(III) center are surprisingly insensitive to pH changes over the range from pH 3 to pH 7. The previously identified major form of chloride peroxidase (form A) and the minor form (B) show very similar chemical shift patterns. Of greatest significance, however, is the discovery that each of the separable forms of the enzyme exhibits splitting of porphyrin ring methyl resonances. The appearance of two sets of signals in both native and cyanide-complexed enzyme is best explained by the existence of two additional forms of the A and B isoenzymes. Structural differences for the newly identified forms of chloride peroxidase must be located in the vicinity of the heme prosthetic group.

A simple, rapid, high yield isolation and purification procedure for chloroperoxidase isoenzymes.

A simple four-step procedure has been developed for isolation of chloroperoxidase from the mold Caldariomyces fumago. Polyethyleneglycol precipitation of the contaminating pigment in the growth medium, followed by chromatography of the soluble enzyme fraction on a QAE-ZetaPrep-250 cartridge and ammonium sulfate precipitation affords isolation of the chloroperoxidase. Extensive dialysis and chromatography on DE-53 cellulose allows the separation and further purification of chloroperoxidase A and B isoenzymes.