N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) does not modify the angiotensin II-stimulated calcium signal in cultured bovine glomerulosa cells

Studies were performed to determine if the sustained elevation in [Ca2+]c noted previously in glomerulosa cells in response to Ang II resulted from the presence of HEPES in the experimental medium. At confluence, primary cultures of bovine glomerulosa cells were maintained for 24-30 h in the presence of either 14 mM NaHCO3/5% CO2 or 25 mM HEPES/4 mM NaHCO3/air. During subsequent experimental periods, cells were incubated in the presence of the corresponding or reciprocal buffer, and the effects of Ang II on [Ca2+]c were monitored by fura 2 fluorescence. Increases in [Ca2+]c produced by Ang II in cells continuously maintained in either HCO3(-) - or HEPES-buffered media were similar, and with the same monolayer the nature of the Ang II-stimulated Ca2+ signal was independent of the buffer employed. Moreover, the Ang II-stimulated Ca2+ signal was not significantly affected by the removal of HCO3- from the superfusate. These results indicate that the sustained increase in [Ca2+]c is not an artifact introduced by the use of HEPES as an experimental buffer, but rather a normal component of the Ang II-stimulated Ca2+ signal.     

Enhanced growth of T-strain mycoplasmas with N-2-hydroxyethylpiperazone-N'-2-ethanesulfonic acid buffer

The small size of T-strain mycoplasma colonies obtained on agar medium has been a drawback to the study of these organisms. The incorporation of 0.05 mN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), a new hydrogen ion buffer, into agar medium at pH 6.8 led to the production of colonies at least 60% greater in diameter than those obtained on media without HEPES at pH 6.5 or 6.0. In addition, the colonies often had the "fried-egg" appearance typical of "classical" large-colony-forming mycoplasmas. The addition of HEPES to liquid medium in the presence of 0.1% urea resulted in a slightly higher number of viable organisms and a corresponding increase in ammonia production. Rapid death of the mycoplasmas occurred on continued incubation of the liquid medium cultures even in the presence of HEPES. The larger colony size facilitated the study of hemadsorption and tissue-culture cell adsorption. Preliminary results of such tests in which these phenomena have been demonstrated are presented. The advantages and disadvantages of having larger colonies are discussed, and the terminology of T-strain mycoplasmas is considered in the light of the present findings.     

Free metal ion depletion by "Good's" buffers. III. N-(2-acetamido)iminodiacetic acid, 2:1 complexes with zinc(II), cobalt(II), nickel(II), and copper(II); amide deprotonation by Zn(II), Co(II), and Cu(II)

Potentiometric, visible, infrared, electron spin, and nuclear magnetic resonance studies of the complexation of N-(2-acetamido)iminodiacetic acid (H2ADA) by Ca(II), Mg(II), Mn(II), Zn(II), Co(II), Ni(II), and Cu(II) are reported. Ca(II) and Mg(II) were found not to form 2:1 ADA2- to M(II) complexes, while Mn(II), Cu(II), Ni(II), Zn(II), and Co(II) did form 2:1 metal chelates at or below physiological pH values. Co(II) and Zn(II), but not Cu(II), were found to induce stepwise deprotonation of the amide groups to form [M(H-1ADA)4-(2)]. Formation (affinity) constants for the various metal complexes are reported, and the probable structures of the various metal chelates in solution are discussed on the basis of various spectral data.     

Oxidative DNA damage induced by HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer in the presence of Au(III)

Oxidative DNA damage was investigated by free radicals generated from HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer, which is widely used in biochemical or biological studies, in the presence of Au(III). The effect of free radicals on the DNA damage was ascertained by gel electrophoresis, electron spin resonance (ESR) spectroscopy and circular dichroism (CD) spectroscopy. ESR results indicated the generation of nitrogen-centered cationic free radicals from the HEPES in the presence of Au(III) which cause the DNA damage. No ESR spectra were observed for phosphate, tris(hydroxymethyl)aminomethane (Tris-HCl) and acetate buffers in the presence of Au(III) or for HEPES buffer in the presence of other metal ions such as Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Pd(II) or [Au(III)(TMPyP)](5+) and [Pd(II)(TMPyP)](4+), where [H(2)(TMPyP)](4+) denotes tetrakis(1-methylpyridium-4-yl)porphyrin. Consequently, no DNA damage was observed for these buffer agents (e.g., phosphate, Tris-HCl or acetate) in the presence of Au(III) or for HEPES in the presence of other metal ions or the metalloporphyrins mentioned above. No detectable inhibitory effect on the DNA damage was observed by using the typical scavengers of reactive oxygen species (ROS) ()OH, O(2)(-) and H(2)O(2). This non-inhibitory effect indicated that no reactive oxygen species were generated during the incubation of DNA with HEPES and Au(III). The drastic change in CD spectra from positive ellipticity to negative ellipticity approximately at 270 nm with increasing concentration of Au(III) also indicated the significant damage of DNA. Only HEPES or Au(III) itself did not damage DNA. A mechanism for the damaging of DNA is proposed.     

Metal binding by melanins: studies of colloidal dihydroxyindole-melanin, and its complexation by Cu(II) and Zn(II) ions

Melanins are colloidal pigments known to have a high affinity for metal ions. In this work, the nature of the metal-binding sites are determined and the binding affinities are quantified. Initial potentiometric titrations have been performed on synthetic dihydroxyindole (DHI) melanin solutions to determine the chemical speciation of quinole/quinone subunits. Two types of acidic functionalities are assignable: catechol groups, with pK(a) between 9 and 13, and quinone imines (QI), with pK(a) of 6.3. The presence of the quinone-imine tautomer has, to our knowledge, never been assessed in polymeric melanins. Melanin solutions obtained from N-methylated DHI lack the pK(a) 6.3 buffer, consistent with its inability to form the quinone-imine tautomer. EPR spectroscopy of the DHI-melanin samples demonstrates that the semiquinone radical is in too low a concentration to contribute to the bulk binding of metals. Changes in the titration curves after addition of Cu(II) and Zn(II) ions were analyzed to obtain the binding constants and stoichiometry of the metal-melanin complexes, using the BEST7 program. UV-Vis spectra at neutral and high pH are used to identify absorbances due to Cu-bound quinone imine and catechol groups. The derived binding constants were used to determine speciation of the Cu(II) and Zn(II) ions coordinated to the quinone imine and catechol groups at various pH. The mixed complexes, Zn(QI)(Cat)(-) and Cu(QI)(Cat)(-) are shown to dominate at physiological pH.     

Potentiometric and spectroscopic studies on Cu(II) complexation to aminophosphonic acid, 1-(3-pyridyl)-1-(n-butylamino)-methanephosphonic acid

The results are reported of a potentiometric and spectroscopic study of the copper(II) complexes of aminophosphonic acid containing a pyridyl side chain. The aminophosphonic acid coordinates similarly to carboxyl amino acids, forming chelate MA and MA₂ species. Stable MAH species with only a phosphonic group coordinated to the metal ion exist at lower pH. The pyridyl side chain was found to be noneffective in the interaction with Cu(II) ion.     

Iminodiacetyl-hydroxamate derivatives as metalloproteinase inhibitors: equilibrium complexation studies with Cu(II), Zn(II) and Ni(II)

Two new iminodiacetyl-hydroxamate derivatives, the N-benzyl-N-carboxymethyl-iminoacetohydroxamic acid (H(2)L(1)) and the N-benzyl-N'-hydroxypiperazine-2,6-dione (HL(2)), have been recently reported as very effective inhibitors against a set of zinc-containing matrix metalloproteinases (MMPs). Herein, aimed at understanding that inhibitory function, these compounds are studied in their complex formation equilibria with three biologically relevant first-row transition M(2+) metal ions (M=Cu, Zn, Ni) by using potentiometric and spectroscopic techniques. At physiological conditions, complexation of these metal ions by H(2)L(1) mostly occurs with formation of 1:1 species by tridentate co-ordination (O,N,N) (carboxylate-amino-hydroxamate), whereas complexation with HL(2) mainly involves the formation of 1:2 (M:L) species with normal (O,O) hydroxamate coordination. Moreover, at higher pH, H(2)L(1) is able to form a pentanuclear tetrameric copper complex with an interesting 12-metallacrown-4 structure.     

Solution properties of Cu(II)-sarcosinehydroxamic acid

Species distribution and relevant stability constants of species present in aqueous solutions of Cu(II) with bidentate sarcosinehydroxamic acid (CH₃NHCH₂CONHOHHl) have been obtained by analytical potentiometry at 25 °C and I = 0.1 M (NaClO₄). Together with ESR of frozen solutions measurements, these results show the existence of four different complex copper(II) species, one of which as a dimeric one. The solution electronic spectra are also reported. The structure of the complexes are discussed and conclusions are drawn based on absorption and ESR spectra.     

Effects of Ca(II) ions on Cu(II) ion-phytic acid interactions

In vitro interactions among phytic acid (PA), Cu(II) ions, and Ca(II) ions were examined as functions of PA:Cu(II):Ca(II) molar ratios and pH. Ca(II) ions competed with Cu(II) ions for binding by the soluble phytate species for PA:Cu(II) molar ratios ranging from 10:1 to 1:6 and pH values in the 2.4-5.9 range. At pH values where precipitation occurred, Ca(II) ions potentiated Cu(II) ion binding by the precipitated phytate species for PA:Cu(II) molar ratios of 10:1 to 1:3. At lower PA:Cu(II) molar ratios, Ca(II) ions competed with Cu(II) ions for binding by the precipitated phytate species. Compositions of the precipitated copper-calcium phytates are reported.     

Oxidative degradation of thymine with O2 promoted by L-ascorbic acid and Cu(II) ion

Oxidation of thymine with O2 was promoted by copper(I) ion generated from reaction of L-ascorbic acid (AA) with copper (II) ion. The main oxidation products were thymine glycol (TG) and N-formyl-N'-pyruvylurea (FPU). At higher concentration of O2, formation of FPU was favored, while TG was dominant at higher Cu(II) ion and lower O2 concentrations. Reaction mechanism involving hydroxy thyminyl radical was proposed.     

Equilibrium and structural studies on Co(II), Ni(II), Cu(II) and Zn(II) complexes with N-(2-benzimidazolyl)methyliminodiacetic acid: crystal structures of Ni(II) and Cu(II) complexes

Combined pH-metric, UV-Vis, ¹H NMR and EPR spectral investigations on the complex formation of M(II) ions (M=Co, Ni, Cu and Zn) with N-(2-benzimidazolyl)methyliminodiacetic acid (H₂bzimida, hereafter H₂L) in aqueous solution at a fixed ionic strength, I=10⁻¹ mol dm⁻³, at 25 ± 1 °C indicate the formation of M(L), M(H₋₁L)⁻ and M₂(H₋₁L)⁺ complexes. Proton-ligand and metal-ligand constants and the complex formation equilibria have been elucidated. Solid complexes, [M(L)(H₂O)₂] · nH₂O (n=1 for M = Co and Zn, n=2 for M = Ni) and {Cu (μ-L) · 4H₂O}_n, have been isolated and characterized by elemental analysis, spectral, conductance and magnetic measurements and thermal studies. Structures of [Ni(L)(H₂O)₂] · 2H₂O and {Cu(μ-L) · 4H₂O}_n have been determined by single crystal X-ray diffraction. The nickel(II) complex exists in a distorted octahedral environment in which the metal ion is coordinated by the two carboxylate O atoms, the amino-N atom of the iminodiacetate moiety and the pyridine type N-atom of the benzimidazole moiety. Two aqua O atoms function as fifth and sixth donor atoms. The copper(II) complex is made up of interpenetrating polymeric chains of antiferromagnetically coupled Cu(II) ions linked by carboxylato bridges in syn-anti (apical-equatorial) bonding mode and stabilized via interchain hydrogen bonds and π-π stacking interactions.     

Protection against cisplatin-induced nephrotoxicity by Cu(II)2(3,5-diisopropylsalicylate)4

The ability of Cu(II)(2)(3,5-diisopropylsalicylate)(4), CuDIPS, which exhibits superoxide dismutase (SOD)-like activity, to prevent cisplatin-induced nephrotoxicity was examined in rats. Rats were divided into four groups and treated as follows: (i) vehicle control; (ii) cisplatin (16 mg/kg, intraperitoneally); (iii) CuDIPS (10 mg/kg, intraperitoneally); and (iv) cisplatin plus CuDIPS. Rats were sacrificed 3 days post-treatment. Cisplatin alone resulted in significantly increased plasma creatinine and urea. Administration of 10 mg/kg CuDIPS prevented the cisplatin-induced elevation of plasma creatinine and urea and protected against kidney damage. Relative to controls, rats that received cisplatin treatment displayed a decrease of reduced glutathione (GSH) and elevated platinum and thiobarbituric acid reactive substances (TBARS) levels in the kidney. In comparison with controls, activities of antioxidant enzymes (SOD, CAT, GSH-Px and GSH-Rd) were also reduced in the kidney of rats treated with cisplatin. Administration of 10 mg/kg CuDIPS prevented cisplatin-induced alterations in renal platinum, GSH, TBARS, and antioxidant enzyme activities. This study suggests that the protection offered by CuDIPS against cisplatin-induced nephrotoxicity is partly related to maintenance of renal antioxidant systems.     

Zinc(II) complexation by some biologically relevant pH buffers

The isothermal titration calorimetry (ITC) technique supported by potentiometric titration data was used to study the interaction of zinc ions with pH buffer substances, namely 2‐(N‐morpholino)ethanesulfonic acid (Mes), piperazine‐N,N′‐bis(2‐ethanesulfonic acid) (Pipes), and dimethylarsenic acid (Caco). The displacement ITC titration method with nitrilotriacetic acid as a strong, competitive ligand was applied to determine conditional–independent thermodynamic parameters for the binding of Zn(II) to Mes, Pipes, and Caco. Furthermore, the relationship between the proposed coordination mode of the buffers and the binding enthalpy has been discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Free metal ion depletion by "Good's" buffers. I. N-(2-acetamido)iminodiacetic acid 1:1 complexes with calcium(ii). magnesium(II), zinc(II), manganese(II), cobalt(II), nickel(II), and copper(II).

Formation (affinity) constants for 1:1 complexes of N-(2-acetamido)iminodiacetic acid (ADAH₂) with Ca(II), Mg(II), Mn(II), Zn(II), Co(II), Ni(II), and Cu(II) have been determined. Probable structures of the various metal chelates existing in solution are discussed. Values for the deprotonation of the amide group in [Cu(ADA)] and subsequent hydroxo complex formation are also reported. The use of ADA as a buffer is considered in terms of metal buffers complexes which can be formed at physiological pH, i.e., at pH 7.0 there is essentially no free metal ion in 1:1 M²⁺ to ADA solutions.     

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.     

Copper(II) complexation by D-glucosamine. Spectroscopic and potentiometric studies

The Cu(II) complex formation equilibria of D- glucosamine were studied in aqueous solution by potentiometric and spectroscopic (ESR, CD, absorption spectra) techniques. All data agree that two major species are formed in the pH region 6–9 involving two D-glucosamine ligand molecules bound to the cupric ion via NH₂(CuL₂) or NH₂ and O^? (CuH_?2L₂). In the latter case deprotonated hydroxyls were found to be very effective coordination sites for Cu(II) giving rise to chelate complexes. On the contrary, no complex formation was observed for the Cu(II) N-acetyl-D-glucosamine system.