Preferential solvation of lysozyme and bovine serum albumin in copper salt solutions. A quantitative chromatographic study

Preferential solvation λ parameters for systems containing water-copper salt-protein (lysozyme or bovine serum albumin) have been determined by gel permeation chromatography. When water is preferentially adsorbed by the protein, good agreement is found between λ values determined by this method and by equilibrium dialysis-differential refractometry. The influence of the concentration and type of anion component of the copper salt, protein concentration and temperature has been investigated. The methodology used also allows direct visualization of the metal ion bound to the protein and to determine binding parameters. Apparent association constants of 2.0 × 10² M⁻¹ and 1.7 × 10² M⁻¹ have been obtained for the binding of copper nitrate to lysozyme at 30 and 4 °C, respectively.     

Synthesis,characterization and nucleic acid binding studies of mononuclear copper(II) complexes derived from azo containing O,O donor ligands

Abstract

Azo linked salicyldehyde and a new 2-hydroxy acetophenone based ligands (HL¹ and HL²) with their copper(II) complexes [Cu(L¹)₂] (1) and [Cu(L²)₂] (2) were synthesized and characterized by spectroscopic methods such as ¹H, 13C NMR, UV–Vis spectroscopy and elemental analyses. Calculation based on Density Functional Theory (DFT), have been performed to obtain optimized structures. Binding studies of these copper (II) complexes with calf thymus DNA (ct-DNA) and torula yeast RNA (t-RNA) were analyzed by absorption spectra, emission spectra and Viscosity studies and Molecular Docking techniques. The absorption spectral study indicated that the copper(II) complexes of 1 and 2 had intrinsic binding constants with DNA or RNA in the range of 7.6?±?0.2?×?10³?M^?1 or 6.5?±?0.3?×?10³M^?1 and 5.7?±?0.4?×?10⁴ M^?1 or 1.8?±?0.5?×?10³ M^?1 respectively. The synthesized compounds and nucleic acids were simulated by molecular docking to explore more details mode of interaction of the complexes and their orientations in the active site of the receptor.     

Oxidation of (hydroxyethyl)ferrocene by [2-pyridylmethylbis(2-ethylthioethyl)/amine]copper(II)

A kinetic study of the oxidation of (hydroxyethyl)ferrocene (HEF) by [2-pyridylmethylbis(2-ethyl-thioethyl)ainine]copper(II) (Cu(pmas)²⁺) is reported, with the objective of documenting the influence of the two thioether sulfur ligands on the electron transfer rate. Both reactants exhibit a first-order dependence at pH 6, I = 0.1 M(NaNO₃); k(25°C) = 1.3 × 10⁴M⁻¹sec⁻¹, ΔH3 = 10.1 kcal/mole, ΔS3 = −6 eu. The apparent Cu(pmas)^2+/+ self-exchange electron transfer rate constant calculated from this reaction on the basis of relative Marcus theory (4.7 × 10¹M⁻¹ sec⁻¹) agrees well with previous findings on ferrocytochrome c, Fe(CN)₆⁴⁻, and Ru(NH₃)₅py²⁺ oxidations. Spectrophotometric titrations of Cu(pmas)²⁺ and Cu(tmpa)²⁺ (tmpa = tris(2-pyridylmethyl)amine) with azide ion showed that both Cu(pmas)N₃)⁺ (K_f1 = 3.1 × 10³M⁻¹) and Cu(pmas)(N₃)₂ (K_f2 = 3.5 × 10¹M⁻¹) but Cu(tmpa)(N₃)⁺ (K_f = 6.6 × 10²M⁻¹) are formed up to 0.15 M N₃⁻ (25°C, pH 6, I = 0.2 M), suggesting that a thioether sulfur atom is displaced in the uptake of a second N₃⁻ ion by Cu(pmas)(N₃)⁺. The effect of thioether sulfur displacement by azide ion on the HEF-Cu(pmas)²⁺ reaction rate may be understood entirely through the tendency of N₃⁻ to shift the position of the redox equilibrium towards the reactant side, without invoking any special role for the sulfur ligand in promoting electron transfer reactivity.     

Spectral characteristics of human fetal adrenal microsomes.

Investigations of human fetal adrenal gland microsomes indicated that a carbon monoxide binding pigment had an absorption maximum of 446 to 448 nm. This pigment, upon heat treatment at 37°C was degraded to the form of cytochrome p-420. NADPH reduced cytochrome p-450 slowly and completely. Typical concentrations of 0.75 and 0.16 nmoles/mg protein cytochrome P-450 and b₅, respectively, were observed. Reduced ethylisocyanide spectra were similar to those of rat hepatic microsomes with absorption maxima at 430 as well as 454 nm. Typical type I spectral changes were observed with progesterone, 17-α-OH-progesterone, pregnenolone and androstenedione when these steroids were added to the sample cuvettes. Androstenedione exhibited an apparent spectral dissociation constant (K_S) of 5×10⁻⁶M pregnenolone and progesterone exhibited higher affinities with apparent dissociation constants of 1.1×10⁻⁷M and 1.8×10⁻⁷M, respectively. The maximal absorbance change induced by androstenedione was lower (Emax = 0.027 per mg protien) than the changes in absorbance maxima induced by pregnenolone or progesterone (Emax = 0.060 and 0.047 per mg protein, respectively) when saturating concentrations of these steroids were added to the sample cuvettes. Ethylmorphine and aminopyrine (10⁻³M final concentrations) did not exhibit observable spectral changes; however, type II spectra could be elicited with aniline and nicotinamide and apparent dissociation constants of 3.5×10⁻²M and 2.5×10⁻²M, respectively, were obtained.     

Reevaluation of the rate constants for the reaction of hypochlorous acid (HOCl) with cysteine,methionine, and peptide derivatives using a new competition kinetic approach

Activated white cells use oxidants generated by the heme enzyme myeloperoxidase to kill invading pathogens. This enzyme utilizes H₂O₂ and Cl⁻, Br⁻, or SCN⁻ to generate the oxidants HOCl, HOBr, and HOSCN, respectively. Whereas controlled production of these species is vital in maintaining good health, their uncontrolled or inappropriate formation (as occurs at sites of inflammation) can cause host tissue damage that has been associated with multiple inflammatory pathologies including cardiovascular diseases and cancer. Previous studies have reported that sulfur-containing species are major targets for HOCl but as the reactions are fast the only physiologically relevant kinetic data available have been extrapolated from data measured at high pH (>10). In this study these values have been determined at pH 7.4 using a newly developed competition kinetic approach that employs a fluorescently tagged methionine derivative as the competitive substrate (k(HOCl + Fmoc-Met), 1.5×10⁸ M⁻¹ s⁻¹). This assay was validated using the known k(HOCl + NADH) value and has allowed revised k values for the reactions of HOCl with Cys, N-acetylcysteine, and glutathione to be determined as 3.6×10⁸, 2.9×10⁷, and 1.24×10⁸ M⁻¹ s⁻¹, respectively. Similar experiments with methionine derivatives yielded k values of 3.4×10⁷ M⁻¹ s⁻¹ for Met and 1.7×10⁸ M⁻¹ s⁻¹ for N-acetylmethionine. The k values determined here for the reaction of HOCl with thiols are up to 10-fold higher than those previously determined and further emphasize the critical importance of reactions of HOCl with thiol targets in biological systems.     

Biological evaluation of a cytotoxic 2-substituted benzimidazole copper(II) complex: DNA damage,antiproliferation and apoptotic induction activity in human cervical cancer cells

Exploring novel chemotherapeutic agents is a great challenge in cancer medicine. To that end, 2-substituted benzimidazole copper(II) complex, [Cu(BMA)Cl₂]·(CH₃OH) (1) [BMA = N,N′-bis(benzimidazol-2-yl-methyl)amine], was synthesized and its cytotoxicity was characterized. The interaction between complex 1 and calf thymus DNA was detected by spectroscopy methods. The binding constant (K _b = 1.24 × 10⁴ M^?1) and the apparent binding constant (K _app = 6.67 × 10⁶ M^?1) of 1 indicated its moderate DNA affinity. Complex 1 induced single strand breaks of pUC19 plasmid DNA in the presence of H₂O₂ through an oxidative pathway. Cytotoxicity studies proved that complex 1 could inhibit the proliferation of human cervical carcinoma cell line HeLa in both time- and dose-dependent manners. The results of nuclei staining by Hoechst 33342 and alkaline single-cell gel electrophoresis proved that complex 1 caused cellular DNA damage in HeLa cells. Furthermore, treatment of HeLa cells with 1 resulted in S-phase arrest, loss of mitochondrial potential, and up-regulation of caspase-3 and -9 in HeLa cells, suggesting that complex 1 was capable of inducing apoptosis in cancer cells through the intrinsic mitochondrial pathway.     

Potent inhibition of protein tyrosine phosphatases by copper complexes with multi-benzimidazole derivatives

A series of copper complexes with multi-benzimidazole derivatives, including mono- and di-nuclear, were synthesized and characterized by Fourier transform IR spectroscopy, UV–Vis spectroscopy, elemental analysis, electrospray ionization mass spectrometry. The speciation of Cu/NTB in aqueous solution was investigated by potentiometric pH titrations. Their inhibitory effects against human protein tyrosine phosphatase 1B (PTP1B), T-cell protein tyrosine phosphatase (TCPTP), megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2), srchomology phosphatase 1 (SHP-1) and srchomology phosphatase 2 (SHP-2) were evaluated in vitro. The five copper complexes exhibit potent inhibition against PTP1B, TCPTP and PTP-MEG2 with almost same inhibitory effects with IC₅₀ at submicro molar level and about tenfold weaker inhibition versus SHP-1, but almost no inhibition against SHP-2. Kinetic analysis indicates that they are reversible competitive inhibitors of PTP1B. Fluorescence study on the interaction between PTP1B and complex 2 or 4 suggests that the complexes bind to PTP1B with the formation of a 1:1 complex. The binding constant are about 1.14 × 10⁶ and 1.87 × 10⁶ M⁻¹ at 310 K for 2 and 4, respectively.     

Synthesis,structure, and evaluation of a β-cyclodextrin-artificial carbohydrate conjugate for use as a doxorubicin-carrying molecule

This paper describes the synthesis of a β-cyclodextrin (β-CyD) derivative conjugated with a C,C-glucopyranoside containing a benzene unit. Its doxorubicin-inclusion ability and structure are also discussed. SPR analysis revealed that the β-CyD conjugate had a high inclusion association value of 3.8 × 10⁶ M⁻¹ for immobilized doxorubicin. NMR structural analysis suggested that its high doxorubicin-inclusion ability was due to the formation of the inclusion complex as a result of the π–π stacking interaction between the benzene ring of the conjugate and the A ring of doxorubicin.     

Dual mode of binding of anti cancer drug epirubicin to G-quadruplex [d-(TTAGGGT)]4 containing human telomeric DNA sequence induces thermal stabilization

Epirubicin exerts its anti cancer action by blocking DNA/RNA synthesis and inhibition of topoisomerase-II enzyme. Recent reports on its influence on telomere maintenance, suggest interaction with G-quadruplex DNA leading to multiple strategies of action. The binding of epirubicin with parallel stranded inter molecular G-quadruplex DNA [d-(TTAGGGT)]₄ comprising human telomeric DNA sequence TTAGGG was investigated by absorption, fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy. The epirubicin binds as monomer to G-quadruplex DNA with affinity, K_b1 = 3.8 × 10⁶ M⁻¹ and K_b2 = 2.7 × 10⁶ M⁻¹, at two independent sites externally. The specific interactions induce thermal stabilization of DNA by 13.2–26.3 °C, which is likely to come in the way of telomere association with telomerase enzyme and contribute to epirubicin-induced apoptosis in cancer cell lines. The findings pave the way for drug designing in view of the possibility of altering substituent groups on anthracyclines to enhance efficacy using alternate mechanism of its interaction with G4 DNA, causing interference in telomere maintenance pathway by inducing telomere dysfunction.     

Proliferation inhibition of novel diphenylamine derivatives

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used drugs in the world but some NSAIDs such as diclofenac and tolfenamic acid display levels of cytotoxicity, an effect which has been attributed to the presence of diphenylamine contained in their structures. A novel series of diphenylamine derivatives were synthetised and evaluated for their cytotoxic activities and proliferation inhibition. The most active compounds in the cytotoxicity tests were derivative 6g with an IC₅₀ value of 2.5 ± 1.1 × 10⁻⁶ M and derivative 6f with an IC₅₀ value of 6.0 ± 3.0 × 10⁻⁶ M (L1210 cell line) after 48 h incubation. The results demonstrate that leukemic L1210 cells were much more sensitive to compounds 6f and 6g than the HEK293T cells (IC₅₀ = 35 × 10⁻⁶ M for 6f and IC₅₀ > 50 × 10⁻⁶ M for 6g) and NIH-3T3 (IC₅₀ > 50 × 10⁻⁶ M for both derivatives). The IC₅₀ values show that these substances may selectively kill leukemic cells over non-cancer cells. Cell cycle analysis revealed that a primary trend of the diphenylamine derivatives was to arrest the cells in the G₁-phase of the cell cycle within the first 24 h. UV–visible, fluorescence spectroscopy and circular dichroism were used in order to study the binding mode of the novel compounds with DNA. The binding constants determined by UV–visible spectroscopy were found to be in the range of 2.1–8.7 × 10⁴ M⁻¹. We suggest that the observed trend for binding constant K is likely to be a result of different binding thermodynamics accompanying the formation of the complexes.     

Differential Reactivity in Epidermal Cells of Begonia rex Excised and Grown in vitro

Thin explants composed of the epidermis and underlying collenchyma excised from leaf veins of Begonia rex and cultured in vitro are capable of neoformation of unicellular hairs, roots and buds. Unicellular hairs were formed over the entire surface of the explant when 10⁻⁶M indole acetic acid or 10⁻⁷M naphthaleneacetic acid (NAA) was added to the basal medium; each epidermal cell was potentially involved. The epidermis was most sensitive to a NAA treatment during the first few days of culture but 30% of the explants could still react after 4 days of culture without NAA. When NAA (5 × 10⁻⁷M) and a cytokinin, zeatin (10⁻⁷M), were added together, roots were formed from epidermal tissue after numerous divisions in the original cells. Their initiation was not related to particular cells. Buds were formed when a cytokinin (10⁻⁶M) was added to the basal medium; bud meristems were formed from small groups of cells surrounding basal cells of glandular hairs. Hair formation was inhibited by either high (32–27°C) or low (12°C) temperatures applied continuously. 32–27°C seemed to inhibit elongation of the hairs specifically, whereas 12°C inhibited earlier phases in hair formation. This hypothesis was supported by short temperature treatments applied at different times during hair formation.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum

A study was made of the time course and kinetics of [³H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [³H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K_m values for GABA neurons for high and low affinity uptake were 0.33 × 10⁻⁶ M and 41.8 × 10⁻⁴ M, respectively. For nonneuronal cells, the apparent K_m for high affinity uptake was 0.29 × 10⁻⁶ M. The apparent V_max values for GABA neurons for high and low affinity uptake were 28.7 × 10⁻⁶ mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V_max for high affinity uptake was 0.06 × 10⁻⁶ mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10⁻⁹ M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [³H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [³H]GABA (4.4 × 10⁻⁶ M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

Biological evaluations of newly-designed Pt(II) and Pd(II) complexes using spectroscopic and molecular docking approaches

To perform biological evaluations of newly-designed Pt(II) and Pd(II) complexes, the present study was conducted with targeted protein human serum albumin (HSA) and HCT116 cell line as model of human colorectal carcinoma. The binding of Pt(II) and Pd(II) complexes to HSA was analyzed using fluorescence spectroscopy and molecular docking. The thermal stability and alterations in the secondary structure of HSA in the presence of Pt(II) and Pd(II) complexes were investigated using the thermal denaturation method and circular dichroism (CD) spectroscopy. The cytotoxicity of the Pt(II) and Pd(II) complexes was studied against the HCT116 cell line using MTT assay. The binding analysis revealed that the fluorescence findings were well in agreement with docking results such that there is only one binding site for each complex on HSA. Binding constants of 8.7?×?10³ M^?1, 2.65?×?10³ M^?1, 0.3?×?10³ M^?1, and 4.4?×?10³ M^?1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 25?°C, respectively. Also, binding constants of 1.9?×?10³ M^?1, 15.17?×?10³ M^?1, 1.9?×?10³ M^?1, and 13.1?×?10³ M^?1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 37?°C, respectively. The results of CD and thermal denaturation showed that the molecular structure of HSA affected by interaction with Pt(II) and Pd(II) complexes is stable. Cytotoxicity studies represented the growth suppression effect of the Pt(II) and Pd(II) complexes toward the human colorectal carcinoma cell line. Therefore, the results suggest that the new designed Pt(II) and Pd(II) complexes are well promising candidates for use in cancer treatment, particularly for human colorectal cancer.

Communicated by Ramaswamy H. Sarma     

The uncatalysed and the copper(II) promoted hydrolysis of 4-nitrophenyl L-leucinate

The uncatalysed hydrolysis of 4-nitrophenyl L-leucinate has been studied in detail over a range of pH and temperature at I=0.1 M (KNO₃). Base hydrolysis of the ester is strongly promoted by copper(II) ions. Rate constants have been obtained for the following reactions (where EH⁺ is the N- protonated ester and E is the free base form) EH⁺ + OH⁻ → products E + OH⁻ → products E + H₂O → products CuE²⁺ + OH⁻ → products Base hydrolysis of the copper(II) complex CuE²⁺ is 3.8 × 10⁵ times faster than that of E and 75 times faster than that of EH⁺ at 25 °C and I=0.1 M. Activation parameters for these reactions have been determined and possible mechanisms are considered.     

The interaction of cefotaxime with the serum albumin of several mammalian species

1. The interaction of cefotaxime with the serum albumin of several mammalian species; horses, swine, sheep, dogs and rabbits, was studied comparatively. The technique of ultrafiltration and spectrophotometric determination of the free antibiotic in the filtrate was used.2. Binding percentages, which vary according to the species studied, were found to be higher in swine and rabbit albumins (between 92 and 81%) and lower for sheep, dog and horse albumins (between 67 and 52%).3. The number of binding sites is usually close to 2; in the case of the horse it is 2.43. The apparent binding constants are: swine, 1.61 × 10⁴ M⁻¹; rabbit, 1.19 × 10⁴ M⁻¹; sheep, 2.33 × 10³ M⁻¹; dog, 2.00 × 10³ M⁻¹; horse, 1.42 × 10³ M⁻¹. The Scatchard model was used for data analysis.4. Possible consequences of this interaction regarding clinical use of cefotaxime on different species are discussed.     

Synthesis,Characterization, and Biological Evaluation of Cu(II) Complexes Containing Triflupromazine with Glycine and Histidine

Two novel copper (II) complexes [Cu(TFP)(Gly)Cl] ⋅ 2H₂O complex ( 1 ) and [Cu(TFP)(His)Cl] ⋅ 2H₂O complex ( 2 ) are synthesized, where TFP stands for trifluropromazine, Gly. represents glycine, and His. is histidine. Chemical composition, IR, mass spectra, and magnetic susceptibility tests are performed. Complex binding with macromolecules was investigated using UV-vis, viscosity, gel electrophoresis, and fluorescence quenching. Fluorescence spectroscopy revealed that each complex could replace ethidium bromide (EB). These complexes exhibit grooved, non-covalent, and electrostatic interactions with CT-DNA. Spectroscopy analysis of the BSA interaction showed that complexes bind to protein (K_b values for ( 1 ) is 5.89×10³ M⁻¹ and for ( 2 ) is 9.08×10³ M⁻¹) more strongly than CT-DNA (K_b values for ( 1 ) is 5.43×10³ M⁻¹ and for ( 2 ) is 7.17×10³ M⁻¹). Molecular docking analysis and spectral absorption measurements showed high agreement. Antimicrobial, antioxidant, and anti-inflammatory properties were tested in vitro. The druggability of complex ( 2 ) should be tested in vivo as it is more biologically active.     

Ceruloplasmin,extracellular-superoxide dismutase,and scavenging of superoxide anion radicals

Ceruloplasmin and extracellular-superoxide dismutase are similar in physical properties. Both are found in extracellular fluids and both are scavengers of the superoxide radical. The relationship between the two proteins was further explored in the present investigation. Ceruloplasmin preparations were found to be commonly contaminated with extracellular-superoxide dismutase. In one preparation, 80% of the superoxide dismutase activity was due to extracellular-superoxide dismutase. Ceruloplasmin, freed from contaminating superoxide dismutase, was found to catalytically dismute the superoxide anion radical with a rate constant of about 1.0 × 10⁴ M⁻ s⁻¹ per copper atom. Under physiological conditions with a low rate of superoxide production, ceruloplasmin preferentially reacts stoichiometrically with the superoxide radical with a rate constant of about 2 × 10⁵ M⁻¹ s⁻¹ per copper atom. Under such conditions, the reaction does not result in hydrogen peroxide formation. From the kinetic data obtained it was calculated that in normal human plasma, extracellular-superoxide dismutase will scavenge about twice as much superoxide as ceruloplasmin. Using immobilized antibodies toward extracellular superoxide dismutase and ceruloplasmin, no antigenic cross-reactivity between the two proteins could be detected.     

Stepwise Frontal Analysis Coupled with Affinity Chromatography: A Fast and Reliable Method for Potential Ligand Isolation and Evaluation from Mahuang-Fuzi-Xixin Decoction

Mahuang-Fuzi-Xixin Decoction (MFXD) is widely used in the treatment of asthma, however, the functional components in the decoction targeting beta2-adrenoceptor (β₂-AR) remain unclear. Herein, we immobilized the haloalkane dehalogenase (Halo)-tagged β₂-AR on the 6-chlorocaproic acid-modified microspheres. Using the affinity stationary phase, the interactions of four ligands with the receptor were analyzed by stepwise frontal analysis. The association constants were (4.75±0.28)×10⁴ M⁻¹ for salbutamol, (2.93±0.15)×10⁴ M⁻¹ for terbutaline, (1.23±0.03)×10⁴ M⁻¹ for methoxyphenamine, (5.67±0.38)×10⁴ M⁻¹ for clorprenaline at high-affinity binding site, and (2.73±0.05)×10³ M⁻¹ at low-affinity binding site. These association constants showed the same rank order as the radioligand binding assay, demonstrating that immobilized β₂-AR had capacity to screen bioactive compounds binding to the receptor while stepwise frontal analysis could predict their binding affinities. Application of the immobilized receptor in analysis of MFXD by chromatographic method revealed that ephedrine, aconifine, karakoline, and chasmanine were the bioactive compounds targeting β₂-AR. Among them, ephedrine and chasmanine exhibited association constants of (2.94±0.02)×10⁴ M^-1and (4.60±0.15)×10⁴ M⁻¹ to the receptor by stepwise frontal analysis. Molecular docking analysis demonstrated that ephedrine, chasmanine, and the other two compounds interact with β₂-AR through the same pocket involving the key amino acids such as Asn312, Asp113, Phe289, Trp286, Tyr316, and Val114. As such, we reasoned that the four compounds dominate the therapeutic effect of MFXD against asthma through β₂-AR mediating pathway. This work shed light on the potential of immobilized β₂-AR for drug discovery and provided a valuable methodology for rapid screening.