Nitrite binding to metmyoglobin and methemoglobin in comparison to nitric oxide binding

Nitrite binds reversibly to the ferriheme proteins metmyoglobin and methemoglobin in aqueous buffer solution at a physiological pH of 7.4. The spectral changes recorded for the formation of metMb(NO2-) differ significantly from those observed for the nitrosylation of metMb, which can be accounted for in terms of the different reaction products. Nitric oxide binding to metMb produces a nitrosyl product with Fe(II)-NO+ character, whereas the reaction with nitrite produces an Fe(III)-NO2- complex. The kinetics of the binding and release of nitrite by metMb and metHb were investigated by stopped-flow techniques at ambient and high pressure. The kinetic traces recorded for the reaction of nitrite with metMb exhibit excellent single-exponential fits, whereas nitrite binding to metHb is characterized by double-exponential kinetics which were assigned to the reactions of the alpha- and beta-chains of metHb with NO2-. The rate constants for the binding of nitrite to metMb and metHb were found to be much smaller than those reported for the binding of NO, such that nitrite impurities will not affect the latter reaction. The activation parameters (deltaH++,deltaS(ne),deltaV++) obtained from the temperature and pressure dependence of the reactions support the operation of a dissociative mechanism for the binding and release of nitrite, similar to that found for the binding and release of NO in metMb.     

High affinity 3H-cimetidine binding in guinea-pig tissues

Receptor binding studies have been carried out in guinea-pig cerebral cortex and gastric mucosa membrane preparations using ³H-cimetidine as the radioligand. The binding was found to be time dependent and saturable and confined to a single population of binding sites. However, the calculated K_D values were different for the two tissues, did not correlate with those reported from classical pharmacological experimentation and there was either no or limited displacement by known H₂ specific agonists. It was concluded that the observed high affinity binding site was probably related to an imidazole recognition site rather than the histamine H₂ receptor. The need for careful evaluation of the data is stressed.     

Coordination complexes of 4-methylimidazole with Zn<Superscript>II</Superscript> and Cu<Superscript>II</Superscript> in gas phase and in water: a DFT study

A quantum chemistry study of mononuclear metal coordination with four 4-methylimidazole ligands (4-MeIm) was investigated. The four complexes [Cu(4-MeIm)₄]²⁺, [Cu(4-MeIm)₄, H₂O]²⁺, [Zn(4-MeIm)₄]²⁺ and [Zn(4-MeIm)₄, H₂O]²⁺ were studied with particular attention to the Nπ or Nτ possible coordinations of the 4-MeIm ring with the metals, using different DFT methods. The results suggest that the Nτ coordination of 4-MeIm ring to Zn^II or Cu^II is more favorable whatever the level of calculation. In contrast, the addition of one water molecule in the first coordination sphere of the metal ions provides five-coordinated complexes showing no Nπ or Nτ preferences. There is good agreement between the DFT-calculated structure and those available experimentally. When metal ions are four-fold coordinated, they adopt a tetrahedral geometry. When Cu^II and Zn^II are five-fold coordinated, highly symmetric structures or intermediate structures are calculated. Similar energies are calculated for different structures, suggesting flat potential energy surfaces. The addition of implicit solvent modifies the calculated first coordination sphere, especially for [Cu(4-MeIm)₄, H₂O]²⁺ structures. The QTAIM and ELF topological analyses of the interaction between Cu^II and the neutral ligands, clearly indicate a dative bonding with a strong ionic character.     

A comparison of the binding of imidazole to valence hybrid and methemoglobin: an assignment of individual heme reactivity

The ferric hemes of valence hybrid hemoglobins combine with imidazole in a manner analogous with the hemes of methemoglobin. Equilibrium studies show that imidazole binding to methemoglobin is minimally described by the sum of two independent processes (K₁ = 200 M^?1 and K₂ = 37 M^?1), both of which contribute equally to the observed difference spectrum. Using valance hybrid hemoglobins, which show single binding processes under similar conditions, it is possible to identify the high affinity sites in methemoglobin with the α chains and the low affinity sites with the β chains.Kinetic studies show that the valance hybrid hemoglobins react in a single exponential fashion with imidazole in contrast with methemoglobin which shows a biphasic reaction (k₁ = 85 M^?1 sec^?1k₂ = 25 M^?1 sec^?1). A comparison of the rates of reaction of the hybrids allows the assignment of the fast phase in methemoglobin to the β chains and the slow phase to the α chains.The heterogeneity of the imidazole reaction with methemoglobin occurs over the pH range 5.5–9.5 within which two ionization processes are discernable at pH 6.9 and 7.5.     

Single-cell measurements of two-dimensional binding affinity across cell contacts

The two-dimensional (2D) affinity between protein molecules across contacting cells is a key parameter regulating and initiating several cellular processes. However, measuring 2D affinity can be challenging, and experimental data are limited. In addition, the obtained 2D affinities are typically averaged over the cell population. We here present a method to measure 2D affinity on single cells binding to polyhistidine-tagged fluorescent ligands anchored to a supported lipid bilayer (SLB). By decreasing the density of ligands in the SLB using imidazole, a new steady-state accumulation in the contact is obtained, and from this change, both the 2D affinity and the number of receptors on the cell can be determined. The method was validated on an SLB containing rat CD2 binding to the rat CD48 mutant T92A expressed on Jurkat T cells. The addition of imidazole did not influence the average 2D affinity (1/K_d), and the spread in affinities within the cell population was low, K_d = 4.9 ± 0.9 molecules/μm² (mean ± SD), despite an order of magnitude spread in ligand accumulation because of differences in receptor density. It was also found that cell contact size increased both with ligand density and with the number of receptors per cell but that the contact size stayed approximately constant when lowering the ligand density, above a density of around 10 rat CD2 molecules/μm², after the contact first had formed, indicative of a heterogeneous process. In summary, this method not only allows for single-cell affinities to be measured, but it can also reduce measurement and analysis time and improve measurement accuracy. Because of the low spread in 2D K_d within the cell population, the analysis can further be restricted to the cells showing the strongest binding, paving the way for using this method to study weak binding events.     

Synthesis of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole analogues and their binding affinities for dopamine D2 and D3 receptors

A series of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole derivatives was prepared and their affinity for dopamine D₂ and D₃ receptors was measured using in vitro binding assays. Several oxadiazole analogues were also prepared and tested for their affinity for dopamine D₂ and D₃ receptors. The results of receptor binding studies indicated that the incorporation of an imidazole moiety between the phenyl ring and the basic nitrogen did not significantly increase the selectivity for dopamine D₃ receptors, whereas the incorporation of an oxadiazole at the same region resulted in a total loss of affinity for both dopamine receptor subtype binding sites. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolinomethyl)imidazole (5i), which has a D₃ receptor affinity of 21 nM and a 7-fold selectivity for D₃ versus D₂ receptors. The binding affinity for σ₁ and σ₂ receptors was also measured, and the results showed that several analogues were selective σ₁ receptor ligands.     

Synthetic analogues of the histidine–chlorophyll complex: a NMR study to mimic structural features of the photosynthetic reaction center and the light-harvesting complex

Mg(II)–porphyrin–ligand and (bacterio)chlorophyl–ligand coordination interactions have been studied by solution and solid-state MAS NMR spectroscopy. ¹H, ¹³C and ¹⁵N coordination shifts due to ring currents, electronic perturbations and structural effects are resolved for imidazole (Im) and 1-methylimidazole (1-MeIm) coordinated axially to Mg(II)-OEP and (B)Chl a. As a consequence of a single axial coordination of Im or 1-MeIm to the Mg(II) ion, 0.9–5.2 ppm ¹H, 0.2–5.5 ppm ¹³C and 2.1–27.2 ppm ¹⁵N coordination shifts were measured for selectively labeled [1,3-¹⁵N]-Im, [1,3-¹⁵N,2-¹³C]-Im and [1,3-¹⁵N,1,2-¹³C]-1-MeIm. The coordination shifts depend on the distance of the nuclei to the porphyrin plane and the perturbation of the electronic structure. The signal intensities in the ¹H NMR spectrum reveal a five-coordinated complex, and the isotropic chemical shift analysis shows a close analogy with the electronic structure of the BChl a–histidine in natural light harvesting 2 complexes. The line broadening of the ligand responses support the complementary IR data and provide evidence for a dynamic coordination bond in the complex.Abbreviations (B)Chl a (bacterio)chlorophyll a - HMBC heteronuclear multiple bond correlation - Im imidazole - LH light-harvesting - 1-MeIm 1-methylimidazole - Mg(II)-Por Mg(II)-porphyrin macrocycle - OEP 2,3,7,8,12,13,17,18-octaethylporphyrin     

The binding of Mn2+ and ADP to myosin

The metal ion requirement of myosin-ADP binding was investigated by use of Mn²⁺. Mn²⁺ binds to two sets of noninteracting sites on myosin which are characterized by affinity constants of 10⁶ and 10³, M⁻¹ at 0.016 M KCl concentration. The maximum number of sites is 2 for the high affinity and 20–25 for the low affinity set. Binding of Mn²⁺ to the high affinity sites increases the affinity of ADP binding to myosin. F-actin inhibits ADP binding (Kiely, B., and Martonosi, A., Biochim. Biophys. Acta 172: 158–170 [1969]), but even at F-actin concentrations much higher than that required to saturate the actin binding sites of myosin or its proteolytic fragments, significant ADP binding remained. The actin insensitive portion of ADP binding was inhibited by 10⁻⁴ M inorganic pyrophosphate or ATP. The results are discussed on the basis of a model in which actin and ADP bind to myosin at distinct but interacting sites.     

Synthesis and DNA‐binding study of imidazole linked thiazolidinone derivatives

A novel series of imidazole‐linked thiazolidinone hybrid molecules were designed and synthesized through a feasible synthetic protocol. The molecules were characterized with Fourier transform infrared (FT‐IR), ¹H nuclear magnetic resonance (NMR), ¹³C NMR and high‐resolution mass spectrometry (HRMS) techniques. In vitro susceptibility tests against Gram‐positive (S. aureus and B. subtilis ) and Gram‐negative bacteria (E. coli and P. aeruginosa ) gave highly promising results. The most active molecule (3e) gave a minimal inhibitory concentration (MIC) value of 3.125 μg/mL which is on par with the reference drug streptomycin. Structure–activity relationships revealed activity enhancement by nitro and chloro groups when they occupied meta position of the arylidene ring in 2‐((3‐(imidazol‐1‐yl)propyl)amino)‐5‐benzylidenethiazolidin‐4‐ones. DNA‐binding study of the most potent molecule 3e with salmon milt DNA (sm‐DNA) under simulated physiological pH was probed with UV–visible absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. These studies established that compound 3e has a strong affinity towards DNA and binds at DNA minor groove with a binding constant (K_b) 0.18 × 10² L mol^?1. Molecular docking simulations predicted strong affinity of 3e towards DNA with a binding affinity (ΔG) ‐8.5 kcal/mol. Van der Waals forces, hydrogen bonding and hydrophobic interactions were predicted as the main forces of interaction. The molecule 3e exhibited specific affinity towards adenine–thiamine base pairs. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantum chemical study of silanediols as metal binding groups for metalloprotease inhibitors

DFT (B3LYP and M06L) as well as ab initio (MP2) methods with Dunning cc-pVnZ (n?=?2,3) basis sets are employed for the study of the binding ability of the new class of protease inhibitors, i.e., silanediols, in comparison to the well-known and well-studied class of inhibitors with hydroxamic functionality (HAM). Active sites of metalloproteases are modeled by [R₃M-OH₂]²⁺ complexes, where R stands for ammonia or imidazole molecules and M is a divalent cation, namely zinc, iron or nickel (in their different spin states). The inhibiting activity is estimated by calculating Gibbs free energies of the water displacement by metal binding groups (MBGs) according to: [R₃M-OH₂]²⁺ + MBG → [R₃M-MBG]²⁺ + H₂O. The binding energy of silanediol is only a few kcal mol^?1 inferior to that of HAM for zinc and iron complexes and is even slightly higher for the triplet state of the (NH₃)₃Ni²⁺ complex. For both MBGs studied in the ammonia model the binding ability is nearly the same, i.e., Fe²⁺(t) > Ni²⁺(t) > Fe²⁺(q) > Ni²⁺(s) > Zn²⁺. However, for the imidazole model the order is slightly different, i.e., Ni²⁺(t) > Fe²⁺(t) > Fe²⁺(q) > Ni²⁺(s) ≥ Zn²⁺. Equilibrium structures of the R₃Zn ²⁺ complexes with both HAM and silanediol are characterized by the monodentate binding, but the bidentate character of binding increases on going to iron and nickel complexes. Two types of intermediates of the water displacement reactions for [(NH₃)₃M-OH₂]²⁺ complexes were found which differ by the direction of the attack of the MBG. Hexacoordinated complexes exhibit bidentate bonding of MBGs and are lower in energy for M=Ni and Fe. For Zn penta- and hexacoordinated complexes have nearly the same energy. Intermediate complexes with imidazole ligands have only octahedral structures with bidentate bonding of both HAM and dimethylsilanediol molecules.     

Steroid structural requirements for high affinity binding to human sex steroid binding protein (SBP)

The sex steroid binding protein (SBP) which binds androgens circulating in the blood of man has been examined to determine the structural requirements for high affinity binding. SBP was purified partially and the ability of each of more than 150 steroids to compete with [³H]dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) for binding to SBP was assessed.Binding was enhanced by reduction of the Δ⁴ double bond to 5α-dihydro, addition of a methyl group at C-4 and in one case unsaturation at C-14, 15. Affinity was always reduced by modifications of the C-17β hydroxy. Binding was also severely decreased by deletion of the keto moiety at C-3; however, relatively high affinity was retained by an alcohol or an unsubstituted pyrazole group at C-3. Certain alpha surface substitutions such as 17α-ethinyl had limited effects on binding; whereas, other modifications such as 7α-methyl or 17α-methyl caused significant reduction in binding. Most modifications at C-2, 6, 9 or 11 also impaired affinity, and the 5β steroids had reduced affinity.     

Essential metal ions alter the lactoferrin binding to the erythrocyte plasma membrane receptors

The effect of metal ions at a concentration of l0^-8 to l0^-5 M [using their salts: ZnCl₂, CdCl₂, LiCl, CuSO₄, NiSO₄, A1₂(SO₄)₃, (NH₄)₂MoO₄ on the lactoferrin (Lf) binding to the erythrocyte membrane receptors was studied. In the absence of metal ions, Scatchard’s analysis showed the existence of two kinds of binding site: one with high affinity and low capacity, and the another with low affinity and high capacity. All these metals, excluding Zn²⁺ and Cd²⁺, at a concentration 10^-5 M decreased the affinity of Lf binding (Ka₁) to the high-affinity receptors. In the presence of Zn²⁺ and Cd²⁺, only the lowaffinity binding site was found. Significant inhibition on the affinity (Ka₂) of the low-affinity class of receptors showed Zn²⁺, Al³⁺, and Mo⁶⁺. Depending on their concentration (10^-8-10^-5 M), these ions enhanced to a different extent, the binding capacity of the both types receptors, but the effect did not correspond to the applied doses. Several explanations of the mechanism for influence of the metal ions on the Lf-receptor interaction is discussed.     

Anomalous binding of [3 H] N-methyl-quinuclidinyl benzilate methyl chloride to human lymphocyte muscarinic receptors

Using the muscarinic cholinergic ligand [³ H] N-methyl quinuclidinyl benzilate methyl chloride ([³ H] NM-QNB), we demonstrated that intact, viable human lymphocytes posses specific muscarinic binding sites. Equilibrium binding studies show that muscarinic acethylcholine receptor are devided into two subtype; high affinity (Ms) and low affinity types (Mw) for the ligand.     

Serotonin binding protein:enhancement of binding by Fe2+ and inhibition of binding by drugs.

The binding of serotonin to a soluble, high affinity binding protein, present in synaptosomes and associated with serotonergic tracts, has now been studied for the effects of metallic ions and various drugs. At optimal concentration (10^-4 M) of Fe²⁺ the enhancement of binding was close to 20-fold. A much smaller effect was noted with Cu²⁺. With other ions (Fe³⁺, Mn²⁺, Co²⁺, Ni²⁺, Cr³⁺, Mg²⁺, Ca²⁺) little or no effect was seen. For the effect with Fe²⁺. preincubation was required (10 min, 25°C) and concentrations higher than 10^-4M were inhibitory. Studies based on equilibrium dialysis show that the effect of Fe²⁺ was on the affinity of the binding of serotonin to the protein, rather than on the binding capacity. In polydcrylamide gels at pH 8.6 the migratory properties of thc serotonin-protein complex formed in the presence of Fe²⁺ differ from those of the complex formed without Fe²⁺. Nucleotides (ATP, GTP, ADP, AMP) inhibited thc binding. The effects of several classes of drugs (inhibitors of biogenic amine storage and uptake, psychotomimetics, MAO) inhibitors and drugs binding to contractile proteins) were also studied. The only effective inhibitors of serotonin binding were reserpine, vinblastine and CZ-74, which caused 50% inhibition at 2 × 10^-6 M, 7.5 × 10^-6 M and 0.2 × 10^-6M respectively.     

Naloxazone irreversibly inhibits the high affinity binding of [125I]D-ala2-D-leu5-enkephalin

Scatchard analyses of [¹²⁵I]D-ala²-D-leu⁵-enkephalin binding in rat brain membranes are curvilinear, suggesting low and high affinity sites. Treating the membranes with naloxazone abolishes the high affinity binding with slight effect on low affinity binding. Displacement of [¹²⁵I]-D-ala²-D-leu⁵-enkephalin binding by morphine in untreated membranes is biphasic. Displacement by morphine in naloxazone-treated tissue is monophasic, with no inhibition by low concentrations of morphine. Naloxazone treatment has little effect on displacements by unlabeled D-ala²-D-leu⁵-enkephalin. Binding in N4TG1 neuroblastoma cells, which demonstrates a linear Scatchard plot with single affinity constant similar to that of the low affinity binding in brain, is less sensitive to naloxazone's actions. Naloxazone treatment $\text{in vivo}$ inhibits D-ala²-D-leu⁵-enkephalin analgesia.     

Synthesis of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole analogues and their binding affinities for dopamine D(2) and D(3) receptors.

A series of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole derivatives was prepared and their affinity for dopamine D₂ and D₃ receptors was measured using in vitro binding assays. Several oxadiazole analogues were also prepared and tested for their affinity for dopamine D₂ and D₃ receptors. The results of receptor binding studies indicated that the incorporation of an imidazole moiety between the phenyl ring and the basic nitrogen did not significantly increase the selectivity for dopamine D₃ receptors, whereas the incorporation of an oxadiazole at the same region resulted in a total loss of affinity for both dopamine receptor subtype binding sites. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolinomethyl)imidazole (5i), which has a D₃ receptor affinity of 21 nM and a 7-fold selectivity for D₃ versus D₂ receptors. The binding affinity for σ₁ and σ₂ receptors was also measured, and the results showed that several analogues were selective σ₁ receptor ligands.     

Effect of methyl substitution in a ligand on the selectivity and binding affinity for a nucleobase: A case study with isoxanthopterin and its derivatives

Isoxanthopterin (IX) has two edges with hydrogen bond-forming sites suitable for binding to thymine (T) and cytosine (C). The binding affinity of IX for T or C is stronger than for adenine (A) and guanine (G), whereas the base selectivity of IX for T over C (and vice versa) is moderate. In order to improve both the binding affinity and base selectivity for T over C or C over T, a methyl group is introduced respectively at the N-3 or N-8 position of IX. This leads to the known ligands 3-methyl isoxanthopterin (3-MIX) and 8-methyl isoxanthopterin (8-MIX), and the binding affinity for C or T is expected to be tuned and improved by methyl substitution. Indeed, 3-MIX selectively binds to T more strongly than IX with a binding constant of 1.5 × 10⁶ M^?1 and it loses its binding affinity for C. In contrast, 8-MIX selectively binds to C over T with a binding constant of 1.0 × 10⁶ M^?1 and the binding affinity is greatly improved compared to the parent ligand IX. The thermodynamics of the ligand–nucleotide interaction is analyzed by isothermal calorimetric titrations, and the results show that the interaction follows a 1:1 stoichiometry and is enthalpy-driven. The introduction of methyl groups at both N-3 and N-8 positions results in an increase in enthalpy of the ligand–nucleotide interaction, which leads to the improved binding affinity.     

Interaction of human serum albumin with novel imidazole derivatives studied by spectroscopy and molecular docking

This study was a detailed characterization of the interaction of a series of imidazole derivatives with a model transport protein, human serum albumin (HSA). Fluorescence and time‐resolved fluorescence results showed the existence of a static quenching mode for the HSA–imidazole derivative interaction. The binding constant at 296 K was in the order of 10⁴ M^–1, showing high affinity between the imidazole derivatives and HSA. A site marker competition study combined with molecular docking revealed that the imidazole derivatives bound to subdomain IIA of HSA (Sudlow's site I). Furthermore, the results of synchronous, 3D, Fourier transform infrared, circular dichroism and UV–vis spectroscopy demonstrated that the secondary structure of HSA was altered in the presence of the imidazole derivatives. The specific binding distance, r, between the donor and acceptor was obtained according to fluorescence resonance energy transfer. Copyright © 2015 John Wiley & Sons, Ltd.     

Allosteric effects of the antipsychotic drug trifluoperazine on the energetics of calcium binding by calmodulin

Trifluoperazine (TFP; Stelazine?) is an antagonist of calmodulin (CaM), an essential regulator of calcium‐dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, and 1LIN) show TFP bound to (Ca²⁺)₄‐CaM in ratios of 1, 2, or 4 TFP per CaM. In all of these, CaM domains adopt the “open” conformation seen in CaM‐kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca²⁺)₄‐CaM and explore differential effects on the N‐ and C‐domains of CaM, stoichiometric TFP titrations of CaM were monitored by ¹⁵N‐HSQC NMR. Two TFP bound to apo CaM, whereas four bound to (Ca²⁺)₄‐CaM. In both cases, the preferred site was in the C‐domain. During the titrations, biphasic responses for some resonances suggested intersite interactions. TFP‐binding sites in apo CaM appeared distinct from those in (Ca²⁺)₄‐CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ‐motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N‐domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the “closed,” “semi‐open,” and “open” domains of CaM. In physiological processes, apo CaM, as well as (Ca²⁺)₄‐CaM, needs to be considered a potential target of drug action. Proteins 2010. © 2010 Wiley‐Liss, Inc.