Coordination properties of Cu(II) and Ni(II) ions towards the C-terminal peptide fragment -ELAKHA- of histone H2B

The coordination properties of the peptide Ac-GluLeuAlaLysHisAla-amide, the C-terminal 102-107 fragment of histone H2B towards Cu(II) and Ni(II) ions were studied by means of potentiometry and spectroscopic techniques (UV/Vis, CD, EPR and NMR). It was found that the peptide has a unique ability to bind Cu(II) ions at physiological pH values at a Cu(II): peptide molar ratio 1:2, which is really surprising for blocked hexapeptides containing one His residue above position 3. At physiological pH values the studied hexapeptide forms a CuL(2) complex {N(Im),2N(-)}, while in acidic and basic pH values the equimolar mode is preferred. In basic solutions Ac-GluLeuAlaLysHisAla-amide may bound through a {4N(-)} mode forming a square-planar complex, in which the imidazole ring is not any more coordinated or it has been removed in an axial position. On the contrary, Ni(II) ions form only equimolar complexes, starting from a distorted octahedral complex at about neutral pH values to a planar complex, where hexapeptide is bound through a {N(Im),3N(-)} mode in equatorial plane. The results may be of importance in order to reveal more information about the toxicity caused by metals and furthermore their influence to the physiologic metabolism of the cell.     

A study on the nickel II-famotidine complexes

Potentiometric studies have shown that Ni(II) forms three pH-dependent complexes with famotidine (L), namely: [NiHL](3+), [NiL](2+) and [NiH(-2)L]. Two of them have been isolated from solution with a Ni/famotidine ratio of 1:1. At pH 6.0, a paramagnetic complex [NiL](2+) with octahedral geometry is formed in which, most likely thiazole N(9) and guanidine N(3) nitrogens are involved in the metal binding. Additionally, two water molecules and two perchlorate anions, ClO(4)(-), fulfil the coordination sphere. The second complex, [NiH(-2)L], that precipitates at pH 8 is diamagnetic and takes square-planar geometry in which four nitrogen donors: N(3), N(9), N(16) and N(20) coordinate to Ni(II). Potentiometric studies, mass spectrometry, FT-IR and Raman spectroscopy are employed to determine and discuss the structure of both complexes. Additionally, 1H, 13C and 15N NMR spectroscopy is used to confirm the binding site in a square-planar complex. The assignment of vibrational bands are made using ab initio HF/CEP-31G method.     

The binding of Ni(II) ions to hexahistidine as a model system of the interaction between nickel and His-tagged proteins

The aim of this work is to study the binding of nickel ions to hexahistidine (His(6)) combining potentiometric titrations and spectroscopic (UV-Vis and circular dichroism) determinations in order to establish the species distribution as a function of the pH, their stoichiometry, stability and geometry. For comparative purposes, the same procedure was applied to the Ni-histidine (His) system. His behaves as a tridentate ligand, coordinating the carboxyl group, the imidazole and the amino nitrogen atoms to Ni(II) ions in an octahedral coordination and a bis(histidine) complex is formed at pH higher than 5. For the Ni-His(6) system, the complex formation starts at pH 4 and five different species (Ni(His(6))H, Ni(His(6)), Ni(n)(His(6))(n), Ni(n)(His(6))(n)H(-n/2), Ni(n)(His(6))(n)H(-n)) are formed as a function of the pH. Ni(His(6))H involves the coordination of the imidazole nitrogen and a deprotonated amide nitrogen (N(Im), N(-)) resulting in an octahedral geometry. In Ni(His(6)), an imidazole nitrogen is deprotonated and coordinated (2N(Im), N(-)) to the metal ion with a square planar geometry. The aggregated forms result from the extra Ni-N(Im) coordination, resulting in a 4N square planar geometry that is stabilized by inter/intramolecular hydrogen bonds. This coordination mode is not altered during the deprotonation steps from Ni(n)(His(6))(n).     

Reversal by nickel(II) of inhibitory effects of some scavengers of active oxygen species upon hydroxylation of 2'-deoxyguanosine in vitro.

Effects of ethanol (EtOH), mannitol (Man), L-histidine (His) and glutathione (GSH) on the oxidation of 2'-deoxyguanosine (dG) to its 8-hydroxy derivative (8-OH-dG) with H2O2 plus L-ascorbic acid (Ascb) in the absence and presence of Ni(II) were investigated in order to unveil the nature of active oxygen species involved in that oxidation. In the absence of Ni(II), production of 8-OH-dG was inhibited by His much greater than GSH greater than or equal to GSSG (oxidized glutathione) much greater than EtOH, but not by Man. The latter tended to enhance the production of 8-OH-dG. In the presence of Ni(II), the inhibition by His, GSH and GSSG, but not EtOH, was prevented. The results indicate involvement of a 'crypto-hydroxyl' radical as the dG oxidizing species in both the absence and presence of Ni(II). Also, the results provide evidence that Ni(II) complexes with His, GSH and GSSG may lack antioxidant capacity. Moreover, the Ni(II) complex with His was found capable of enhancing 8-OH-dG production by the Ascb+H2O2 system to a greater extent than Ni(II) alone. Likewise, although to a lesser extent, the formation of 8-OH-dG was enhanced by the combination of Ni(II) and Man which do not form complexes at pH 7.4. Since His is a major Ni(II) carrier in animal tissues, the dG oxidation enhancing capacity of the Ni(II) complex with His may contribute to the toxic and carcinogenic effects of Ni(II).     

Characterization of pNiXa,a serpin of Xenopus laevis oocytes and embryos,and its histidine-rich,Ni(II)-binding domain

A Ni(II)-binding serpin, pNiXA, is abundant in Xenopus oocytes and embryos. Kinetic assays show that purified pNiXa strongly inhibits bovine α-chymotrypsin (K₁ = 3 mM), weakly inhibits porcine elastase (K₁ = 0.5 μM), and does not inhibit bovine trypsin. The reversible, slow-binding inhibition of α-chymotrypsin by pNiXa is unaffected by Ni(II). Ovochymase in egg exudates is inhibited by pNiXa, but to a limited extent, even at high pNiXa concentrations. An octadecapeptide that models the His-rich domain (-HRHRHEQQGHHDSAKHGH-) of pNiXa forms six-coordinate, octahedral Ni(II)-complexes when the N-terminus is acetylated, and a square-planar Ni(II)-complex when the N-terminus is unblocked. Spectroscopy reveals two distinct types of octahedral Ni(II)-coordination to the N-acetylated octadecapeptide, involving, respectively, 3–4 and 5–6 imidazole nitrogens; the octadecapeptide undergoes partial, reversible precipitation in pH-and Ni(II)-dependent fashion, suggesting an insoluble, Ni(II)-coupled (Hx)_n-dimer. Such (Hx)_n-peptide interaction is confirmed by an enzyme-linked biotin-avidin assay with N-biotin-KHRHRHE-amide and N-acetyl-KHRHRHE-resin beads, which become coupled after adding Ni(II) or Zn(II). H₂O₂ oxidation of 2′-deoxyguanosine to mutagenic 8-hydroxy-2′deoxyguanosine is enhanced by the octahedral Ni(II)-octadecapeptide complex, although the effect is more intense with the square-planar Ni(II) octadecapeptide complex. Immunoperoxidase staining of whole mounts wish pNiXa antibody shows that pNiXa is distributed throughout gastrula-stage embryos and is localized during organogenesis in the brain, eye, spinal cord, myotomes, craniofacial tissues, and other sites of Ni(II) induced anomalies. Patterns of pNiXa staining are similar in controls and Ni(II)-exposed embryos. Binding of Ni(II) to pNiXa may cause embryotoxicity by enhancing oxidative reactions that produce tissue injury and genotoxicity. Although the natural target proteinases for pNiXa inhibition have not been established, pNiXa may be an important regulator of proteolysis during embryonic development. © 1996 Wiley-Liss, Inc.     

Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR: the unstructured N terminus facilitates the coordination of six copper2+ ions at physiological concentrations

The prion protein (PrP) is a Cu2+ binding cell surface glyco-protein. Misfolding of PrP into a beta-sheet rich conformation is associated with transmissible spongiform encephalopathies. Here we use Ni2+ as a diamagnetic probe to further understand Cu2+ binding to PrP. Like Cu2+, Ni2+ preferentially binds to an unstructured region between residues 90 and 126 of PrP, which is a key region for amyloidogenicity and prion propagation. Using both 1H NMR and visible-circular dichroism (CD) spectroscopy, we show that two Ni2+ ions bind to His96 and His111 independently of each other. 1H NMR indicates that both Ni2+ binding sites form square-planar diamagnetic complexes. We have previously shown that Cu2+ forms a paramagnetic square-planar complex in this region, suggesting that Ni2+ could be used as a probe for Cu2+ binding. In addition, competition studies show that two Cu2+ ions can displace Ni2+ from these sites. Upon Ni2+ addition 1H NMR changes in chemical shifts indicate the imidazole ring and amide nitrogen atoms to the N terminus of both His96 and His111 act as coordinating ligands. Use of peptide fragments confirm that PrP(92-96) and PrP(107-111) represent the minimal binding motif for the two Ni2+ binding sites. Analysis of Cu2+ loaded visible-CD spectra show that as with Ni2+, PrP(90-115) binds two Cu2+ ions at His96 and His111 independently of each other. Visible CD studies with PrP(23-231Delta51-90), a construct of PrP(23-231) with the octarepeat region deleted to improve solubility, confirm binding of Ni2+ to His96 and His111 in octarepeat deleted PrP(23-231). The structure of the Cu/Ni complexes is discussed in terms of the implications for prion protein function and disease.     

Sequence-specific Ni(II)-dependent peptide bond hydrolysis in a peptide containing threonine and histidine residues

Previously we demonstrated that Ni(II) complexes of Ac-Thr-Glu-Ser-His-His-Lys-NH2 hexapeptide, representing residues 120-125 of human histone H2A, and some of its analogs undergo E-S peptide bond hydrolysis. In this work we demonstrate a similar coordination and reactivity pattern in Ni(II) complexes of Ac-Thr-Glu-Thr-His-His-Lys-NH2, its threonine analogue, studied using potentiometry, electronic absorption spectroscopy and HPLC. For the first time we present the detailed temperature and pH dependence of such Ni(II)-dependent hydrolysis reactions. The temperature dependence of the rate of hydrolysis yielded activation energy E(a) = 92.0 kJ mol(-1) and activation entropy DeltaS# = 208 J mol(-1) K(-1). The pH profile of the reaction rate coincided with the formation of the four-nitrogen square-planar Ni(II) complex of Ac-Thr-Glu-Thr-His-His-Lys-NH2. These results expand the range of protein sequences susceptible to Ni(II) dependent cleavage by those containing threonine residues and permit predictions of the course of this reaction at various temperatures and pH values.     

Mycobacterium tuberculosis NmtR harbors a nickel sensing site with parallels to Escherichia coli RcnR

Mycobacterium tuberculosis NmtR is a Ni(II)/Co(II)-sensing metalloregulatory protein from the extensively studied ArsR/SmtB family. Two Ni(II) ions bind to the NmtR dimer to form octahedral coordination complexes with the following stepwise binding affinities: K(Ni1) = (1.2 ± 0.1) × 10(10) M(-1), and K(Ni2) = (0.7 ± 0.4) × 10(10) M(-1) (pH 7.0). A glutamine scanning mutagenesis approach reveals that Asp91, His93, His104, and His107, all contained within the C-terminal α5 helix, and His3 as part of the conserved α-NH(2)-Gly2-His3-Gly4 motif at the N-terminus make significant contributions to the magnitude of K(Ni). In contrast, substitution of residues from the C-terminal region, His109, Asp114, and His116, previously implicated in Ni(II) binding and metalloregulation in cells, gives rise to wild-type K(Ni) and Ni(II)-dependent allosteric coupling free energies. Interestingly, deletion of residues 112-120 from the C-terminal region (Δ111 NmtR) reduces the Ni(II) binding stoichiometry to one per dimer and greatly reduces Ni(II) responsiveness. H3Q and Δ111 NmtRs also show clear perturbations in the rank order of metal responsiveness to Ni(II), Co(II), and Zn(II) that is distinct from that of wild-type NmtR. (15)N relaxation experiments with apo-NmtR reveal that both N-terminal (residues 2-14) and C- terminal (residues 110-120) regions are unstructured in solution, and this property likely dictates the metal specificity profile characteristic of the Ni(II) sensor NmtR relative to other ArsR family regulators.     

Salivary histatin-5, a physiologically relevant ligand for Ni(II) ions

Histatins are a family of human salivary antimicrobial peptides. Histatin-5 (Hst-5, DSHAKRHHGYKRKFHEKHHSHRGY), a prominent member of this family contains an albumin-like, N-terminal Asp-Ser-His sequence, known to bind a Ni(II) ion in a square-planar geometry. Nickel is a strong allergen, and oral exposure to Ni(II) ions can elicit allergic reaction in sensitized persons. In contrast, prior oral exposure to nickel in non-sensitized persons can prevent sensitization. The fate of Ni(II) ions in saliva is obviously important for these processes, yet little is known about it. Using potentiometry, UV-visible titrations and circular dichroism, we determined stability constants for Ni(II) complexes of Hst-5 and two truncated analogs, 5Hst-5 (DSHAK) and 10Hst-5 (DSHAKRHHGY). The conditional binding constant at pH 7.4 for Hst-5 was 10(7.5±0.2), compared to the corresponding value for albumin, 10(6.8±0.3) (M. Soko?owska, A. Kr??el, M. Dyba, Z. Szewczuk, W. Bal, Eur. J. Biochem. 269 (2002) 1323-1331). These values indicate that Hst-5 binds Ni(II) five times stronger than HSA. The simulated competition for Ni(II) between Hst-5 and albumin shows that significant amounts of Ni(II) ions may be carried by Hst-5 in vivo. Therefore, Hst-5 and other histatins should be considered as factors in nickel allergy and other forms of nickel toxicity.     

The non-specificity of dog serum albumin and the N-terminal model peptide glycylglycyl-L-tyrosine N-methylamide for nickel is due to the lack of histidine in the third position.

Equilibrium dialysis of dog serum albumin (DSA) against Ni(II) in 0.1 M-N-ethylmorpholine/HCl, pH 7.53, demonstrates the absence of a specific Ni(II)-binding site in DSA. To evaluate at the molecular level the influence of the genetic substitution of L-tyrosine for L-histidine at the N-terminal of DSA, a simple model tripeptide of the N-terminal residues, glycylglycyl-L-tyrosine N-methylamide, was synthesized and its Ni(II)-binding properties studied. A comparison of the visible absorption characteristics of Ni(II)-DSA with those of Ni(II)-glycylglycyl-L-tyrosine N-methylamide reveals a similar change from octahedral to planar co-ordination as the pH is increased. Both systems exhibit a low Ni(II)-binding affinity at physiological pH, with DSA binding a greater percentage of Ni(II) owing to the availability of at least two binding sites of similar affinities. The complex equilibria between Ni(II) and glycylglycyl-L-tyrosine N-methylamide were studied by analytical potentiometry (0.15 M-NaCl, 25 degrees C). Four major complex species, MHA, MH-1A2, MH-2A2 and MH-3A [where M and A represent Ni(II) ion and anionic peptide respectively], were detected, MHA being the single species at physiological pH. There is no evidence for the involvement of the phenolic hydroxy group in the octahedral MHA complex, or within the plane of co-ordination in the high-pH species. The results provide direct evidence that the low Ni(II)-binding affinity of DSA is due to the genetic substitution of tyrosine for histidine at the N-terminal region of the protein.     

Synthesis, characterization and biological activity evaluation of Pt(II), Pd(II), Co(III) and Ni(II) complexes with N-heteroaromatic selenosemicarbazones

Five new complexes of Pt(II), Pd(II), Co(III) and Ni(II) with 2-pyridine(quinoline)carboxaldehyde selenosemicarbazones were synthesized and characterized. Crystal structures of Pt(II) complex with the pyridine derivative and Co(III) complex with the quinoline derivative were determined. In all complexes the ligands were coordinated through N₂Se donor atom set forming either square-planar (Pt, Pd) or octahedral (Co, Ni) geometry. All complexes showed biological activity.     

Catalysis by methyl-coenzyme M reductase: a theoretical study for heterodisulfide product formation

Hybrid density functional theory has been used to investigate the catalytic mechanism of methyl-coenzyme M reductase (MCR), an essential enzyme in methanogenesis. In a previous study of methane formation, a scheme was suggested involving oxidation of Ni(I) in the starting square-planar coordination to the high-spin Ni(II) form in the CoM-S-Ni(II)F(430) octahedral intermediate. The methyl radical, concomitantly released by methyl-coenzyme M (CoM), is rapidly quenched by hydrogen atom transfer from the coenzyme B (CoB) thiol group, yielding methane as the first product of the reaction. The present investigation primarily concerns the second and final step of the reaction: oxidation of CoB and CoM to the CoB-S-S-CoM heterodisulfide product and reduction of nickel back to the Ni(I) square-planar form. The activation energy for the second step is found to be around 10 kcal/mol, implying that the first step of methane formation with an activation energy of 20 kcal/mol should be rate-limiting. An oxygen of the Gln147 residue, occupying the rear axial position in the oxidized Ni(II) state, is shown to stabilize the intermediate by 6 kcal/mol, thereby slightly decreasing the barrier for the preceding rate-limiting transition state. The mechanism suggested is discussed in the context of available experimental data. An analysis of the flexibility of the F(430) cofactor during the reaction cycle is also given.     

Cu(II) complexation by "non-coordinating" N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer)

The combined potentiometric and spectroscopic studies of interactions of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) with Cu(II) demonstrated that this popular buffer, commonly labelled as "non-coordinating" forms a CuL+ complex, with the logbeta(CuL) value of 3.22. This complex undergoes alkaline hydrolysis above pH 6, resulting in Cu(OH)2 precipitation. However, the presence of HEPES at a typical concentration of 100 mM at pH 7.4 elevates the apparent binding constant, being determined for a complex of another ligand, by a factor of 80. HEPES does not form ternary complexes with aminoacids Ala, Trp, and His, but may do so with other bioligands, such as nucleotides. Therefore, HEPES can still be recommended for Cu(II) studies in place of other common buffers, such as Tris and phosphate, but appropriate corrections and precautions should be applied in quantitative experiments.     

Metal--glutathione interaction in aqueous solution. Nickel(II), cobalt(II) and copper(II) complexes with oxidized glutathione.

The interaction of copper(II), nickel(II) and cobalt(II) ions with oxidized glutathione in aqueous solutions have been examined by spectroscopic methods. Cu(II) is the only ion which interacts with disulphide bridge and forms dimeric species containing the Cu(II)-S-S-Cu(II) unit. Ni(II) and Co(II) bind mainly with the terminal NH2 and COO- groups of glutamic acid, and the complexes formed are of nearly octahedral symmetry. At high pH, in the Co(II)-GSSG solution Co(II) is oxidized to Co(III) with the concomitant reduction of GSSG to GSH. Considerable differences were observed between the oxidized and reduced form of glutathione in the coordination ability towards metal ions.     

Synthesis, characterization and antitumor properties of some metal complexes of 3- and 5-substituted salicylaldehyde 2-pyridinylhydrazones

Complexes of Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II), and Pt(II) with 3- and 5-substituted salicylaldehyde 2-pyridinylhydrazones (XSPH, X = H, 3-NO2, 3-CH3O, 5-Br, 5-Cl, 5-CH3, or 5-NO2) have been prepared and characterized by elemental analysis, conductance measurements, magnetic moments (300-78 K), and spectral studies. On the basis of these studies a monomeric, high-spin, distorted octahedral structure for Mn(XSPH)2 and Fe(XSPH)2, a dimeric, high-spin, five-coordinate structure for Co(XSBH)Cl, a dimeric, low-spin, five-coordinate structure for Ni(XSPH)Cl and Zn(XSPH)(OAc), and a square-planar structure for M(XSPH)Cl.H2O (M = Cu(II) or Pt(II] complexes are suggested. The polycrystalline ESR spectra of Cu(II) complexes are isotropic and suggest dx2-y2 ground state in square-planar stereochemistry. M?ssbauer spectral results indicate distorted octahedral structure for iron(II) complexes. All the metal(II) complexes have been screened for their antitumor activity against P388 lymphocytic leukemia test system in mice and have been found to possess no significant activity at the dosages used.     

Role of individual histidines in the pH-dependent global stability of human chloride intracellular channel 1

Chloride intracellular channel proteins exist in both a soluble cytosolic form and a membrane-bound form. The mechanism of conversion between the two forms is not properly understood, although one of the contributing factors is believed to be the variation in pH between the cytosol (~7.4) and the membrane (~5.5). We systematically mutated each of the three histidine residues in CLIC1 to an alanine at position 74 and a phenylalanine at positions 185 and 207. We examined the effect of the histidine-mediated pH dependence on the structure and global stability of CLIC1. None of the mutations were found to alter the global structure of the protein. However, the stability of H74A-CLIC1 and H185F-CLIC1, as calculated from the equilibrium unfolding data, is no longer dependent on pH because similar trends are observed at pH 7.0 and 5.5. The crystal structures show that the mutations result in changes in the local hydrogen bond coordination. Because the mutant total free energy change upon unfolding is not different from that of the wild type at pH 7.0, despite the presence of intermediates that are not seen in the wild type, we propose that it may be the stability of the intermediate state rather than the native state that is dependent on pH. On the basis of the lower stability of the intermediate in the H74A and H185F mutants compared to that of the wild type, we conclude that both His74 and His185 are involved in triggering the pH changes to the conformational stability of wild-type CLIC1 via their protonation, which stabilizes the intermediate state.     

The complex formation of copper(II) with GHL and HSA

The formation constants for complexes of copper(II) with GHL have been determined by means of pH titrations and ESR spectroscopy in aqueous solutions. GHL has an extremely high affinity for copper(II) and forms very stable 1:1 complexes and a comparatively weak 1:2 complex. The ? amino group of GHL seems not to be involved in complex formation as can be deducted from both equilibrium constants and ESR spectroscopy. The ternary system copper(II)-GHL-HSA was investigated by ESR spectroscopy and optical absorption spectroscopy in aqueous solution at physiological pH (7.4). At equimolar concentrations, copper(II), HSA and GHL form a ternary complex.     

Mutation of surface-exposed histidine residues of recombinant human granulocyte-colony stimulating factor (Cys17Ser) impacts on interaction with chelated metal ions and refolding in aqueous two-phase systems

Site directed mutagenesis of Cys17-->Ser17 form of recombinant human granulocyte colony stimulating factor (rhG-CSF C17S) for sequential replacing of surface His(43) and His(52) with alanine was used to identify residues critical for the protein interaction with metal ions, in particular Ni(2+) chelated by dye Light Resistant Yellow 2 KT (LR Yellow 2KT)-polyethyleneglycol (PEG), and refolding after partitioning of inclusion bodies in aqueous two-phase systems. Strong binding of rhG-CSF (C17S) to PEG-LR Yellow 2KT-Cu(II) complex allowed for the adoption of affinity chromatography on Sepharose-LR Yellow 2KT-Cu(II) that appeared to be essential for the rapid isolation of mutated forms of rhG-CSF. Efficiency of that purification stage is exemplified by isolation of rhG-CSF (C17S, H43A) and rhG-CSF (C17S, H43A, H52A) mutants in correctly folded and highly purified state. Affinity partitioning of rhG-CSF histidine mutants was studied in aqueous two-phase systems containing Cu(II), Ni(II) and Hg(II) chelated by LR Yellow 2KT-PEG at pH 7.0 and Cu(II)-at pH 5.0. It was determined, that affinity of rhG-CSF mutants for metal ions decreased in the order of C17S>C17S, H43A>C17S, H43A, H52A for Cu(II), and C17S=C17S, H43A>C17S, H43A, H52A for Ni(II) ions, while affinity of all rhG-CSF mutants for Hg(II) ions was of the same order of magnitude. Influence of His(43) and His(52) mutation on protein refolding was studied by partitioning of the respective inclusion body extract in aqueous two-phase systems containing Ni(II) and Hg(II) ions. Data on rhG-CSF histidine mutant partitioning and refolding indicated, that His(52) mutation is crucial for the strength of protein interaction with chelated Ni(II) ions and refolding efficiency.     

Binding of diclofenac sodium with bovine serum albumin at different temperatures, pH and ionic strengths

The study was designed to examine the binding of diclofenac sodium with bovine serum albumin (BSA) at different temperatures (20 degrees, 30 degrees and 40 degrees C), pH (6.4, 7.4 and 8.4) and ionic strengths (micro = 0.1, 0.2 and 0.3) by means of equilibrium dialysis method. The concentration of diclofenac sodium was maintained at wider range from 15 to 900 micromole/l and BSA concentration was maintained at 61.5 micromole/l. The data obtained were interpreted by nonlinear regression method using Graphpad prism software. The analysis showed that the interaction between diclofenac sodium with BSA results in two-site saturable binding. A decrease in association constant was observed with increasing temperature. The average standard free energy change (deltaGdegrees) value was -7.07 (site I) and -4.2 (site II) Kcal/mol. The standard enthalpy change (deltaHdegrees) and the standard entropy change (deltaSdegrees) were -7.8 Kcal/mole, -2.35 cal/mole (site I) and -7.4 Kcal/mole, -10.5 cal/mole (site II), respectively. The negative enthalpy change suggested the binding between diclofenac sodium and the binding sites of BSA were spontaneous and exothermic. The negative value of deltaHdegrees and deltaSdegrees indicated hydrogen bonding and van der Waal's force was the major mechanism for diclofenac sodium and BSA interaction. Increase in pH and ionic strength also caused decrease in association constant of diclofenac sodium and BSA binding.     

N-(2-Pyridyl)acetamide complexes of palladium(II), cobalt(II), nickel(II), and copper(II)

N-(2-Pyridyl)acetamide (aapH) complexes of palladium(II), cobalt(II), nickel(II), and copper(II) have been studied by means of magnetic susceptibilities, and infrared, electronic, and PMR spectra. In the octahedral complexes M(aapH)₂X₂(M = Co, Ni, Cu; X = Cl, Br, NCS, NO₃), bidentate aapH is chelated through the pyridine-N and amid-O atomes, whereas in the square-planar Pd(aapH)₂X₂ (X = Cl, Br) unidentate aapH is coordinated through the pyridine-N atom alone. Under alkaline conditions aapH is deprotonated in the presence of palladium(II) to form Pd(aap)₂·4H₂O, aap being an anionic bidentate ligand and chelating through the pyridine-N and amide-O atoms.