Application of HPLC to the Study of the Chloroplast Atpase Mg2+ Dependent Mechanism

Abstract

The determination by HPLC of released ADP from ATP by chloroplast ATPase (CF₁) is described.

The enzymatic rate measured by this method is well defined, over several minutes.

In the case of ? subunit-depleted CF₁ or activated CF₁, the rate is proportional to the enzyme concentration, the steady state theory is followed and the K_m and V_m constants have been calculated.

The enzymatic activity of CF₁ is inhibited by endogenous ? subunit and the inhibition constant has been measured.

The influences of ionic strength, pH, magnesium ion, phosphate and ADP concentrations have been studied and the results obtained by this method have been compared to previously reported data based on rate determination of released phosphate.     

Synthesis,photophysical and NMR evaluations of thiourea-based anion receptors possessing an acetamide moiety

The synthesis and photophysical evaluation of three diaryl thiourea-based anion receptors (4–6) for comparison with their urea counterparts (1–3) is outlined. These anion receptors posses an acetamide functionality on one of the aryl groups and an electron-withdrawing CF₃ group on the other. By varying the position of the acetamide group, in the o-, m- and p-positions of 4–6, respectively, the anion binding ability was both tuneable and found to be, in some cases, significantly different from that seen for the urea analogues 1–3. The binding affinities of the receptors 4–6, as well as the binding stoichiometries, were evaluated using UV–vis absorption spectroscopy in MeCN. However, these receptors were not sufficiently emissive to quantify the anion recognition using fluorescence. The results confirmed strong binding of these receptors to anions such as fluoride, acetate, phosphate, pyrophosphate and chloride. Nevertheless, the overall results obtained did not conform to the anticipated trends seen for 1–3, which is most likely due to the enhanced binding affinity of the thiourea analogues 4–6. The binding interactions were also investigated by using ¹H NMR which confirmed that these receptors interacted with the anions in a stepwise manner, where the primary anion binding interaction occurred at the thiourea side, which led to an activation of the acetamide moiety towards the second anion binding interaction, an example of an allosteric activation mode.     

ESR study of free radicals formed upon UV irradiation of nitropentafluoroacetone in various solvents

The formation of CF₃C(O)CF₂N(O^.)O₂CF₂C(O)CF₃ free radicals upon the UV irradiation of nitropentafluoroacetone (1) in toluene and mesitylene is established by ESR. The most likely cause of their formation is the one-electron oxidation of the solvents by photoexcited1 followed by decay of the radical anion formed from1 with the expulsion of an NO₂ anion and attachment of the radical to a molecule of original1. The irradiation of 1 in triethylsilane results in the elimination of a fluoride ion and fixation of a CF₃COCFN(O^–)O' radical. UV irradiation of ketone1 in pentane results in the abstraction of a hydrogen atom from the solvent and the formation of a CF₃COCF₂N(OH)O ' radical.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 512–514, March, 1993.     

Nucleotide/Protein Interaction: Energetic and Structural Features of Na,K-ATPase

Microcalorimetric titrations allow to recognize and investigate high-affinity ligand binding to Na,K-ATPase. Titrations with the cardiac glycoside Ouabain, which acts as a specific inhibitor of the enzyme, have provided not only the thermodynamic parameters of high-affinity binding with a stoichiometric coefficient of about 0.6 but also evidence for low-affinity binding to the lipid. The marked enthalpic contribution of -95 kJ mol^-1 at 298.2 K is partially compensated by a large negative entropy change, attributed to an increased interaction between water and the protein. The calorimetric ADP and ATP titrations at 298.2 K are indicative of high-affinity nucleotide binding either in 3 mM NaCl, 3 mM MgCl₂ or at high ionic strength such as 120 mM choline chloride. However, no binding is detected in the buffer solution alone at low ionic strength. The affinities for ADP and ATP are similar, around 10⁶ M^-1 and the stoichiometric coefficients are close to that of Ouabain binding. The exothermic binding of ADP is characterized by a ΔH and ΔS value of -65 kJ mol^-1 and -100 J mol^-1 K^-1, respectively. TheΔH value for ATP binding is larger than for ADP and is compensated by a larger, unfavorable ΔS value. This leads to an enthalpy/entropy compensation, which could express that H-bond formation represents the major type of interaction. As for Ouabain, the negative ΔS values that are also characteristic of nucleotide binding can indicate an increase of solvate interaction with the protein due to a conformational transition occurring subsequent to the binding process. The resulting binding constants are discussed with regard to the results of other studies employing different techniques. A molecular interaction model for nucleotide binding is suggested. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enantioselective Syntheses of the Methylene- and Fluoromethylene-Phosphonate Analogues of 3-Phospho-D-Glyceric Acid

Abstract

The racemic methylene analogue of 3-phospho-D-glyceric acid^1–4 has been shown to be a viable substrate for the combined action of 3-phosphoglycerate kinase, PGK, and glyceraldehyde 3-phosphate dehydrogenase, GPD. We have shown that replacement of CH₂ by CHF or CF₂ in a variety of nucleotide analogues^4,5 can lead to improved Performance as enzyme substrates or inhibitors. We have therefore undertaken enantiospecific syntheses of the methylene- and fluoro-methylene-analogues of 3-phospho-D-glyceric acid to investigate their interaction with PGK and GPD and explore whether the fluorine atom in the latter can mimic an oxygen lone-pair in binding to enzymes.     

Supramolecular interactions in a family of dinuclear helicates in the solid-state

ABSTRACT

Metallo-helicates are a commonly encountered assembly within supramolecular chemistry. Interest in these architectures stems from their inherent helical chirality which positions them for a diverse range of applications such as catalysis and non-linear optics (NLO). The current study uses Co(II) dinuclear double helicates as versatile supramolecular synthons. The ditopic ligand, L, features two tridentate quinolinyl-hydrazone binding sites imparting it with hydrogen bond donors and π-faces for secondary supramolecular interactions. Incorporation of L into [Co₂(L)₂]⁴⁺ helical assemblies results in a helical cationic supramolecular synthon with moieties predisposed to forming π-π stacking and hydrogen bond interactions. The single-crystal X-ray structures of [Co₂(L)₂]X₄ (X = ClO₄ ^?, BF₄ ^? and CF₃SO₃ ^?) revealed a variety of anion dependent hydrogen bond networks arising through the interaction of the hydrazide hydrogen with the anion. These interactions in turn strongly influence the nature of the π-π stacking interactions of the quinoline moieties which can be analysed via the Hirshfield surface.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate.

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.     

Mono Halogen Substituted Calix[4]pyrroles: Fine-tuning the Anion Binding Properties of Calix[4]pyrrole

Abstract

Single halogen atom (i. e. I, Br, Cl and F) substituted calix[4]pyrroles, compounds 2, 3, 4 and 5, were synthesized. Studies of these systems reveal that replacement of a single β-pyrrolic hydrogen atom can increase the anion binding ability of calix[4]pyrroles for a variety of anions (e. g. Cl^?, Br^?, H₂PO₄ ^? and HSO^? ₄) relative to normal non-halogen substituted calix[4]pyrrole 1. In the case of chloride anion, the expected relative affinity sequence of 5 > 4 > 3 > 2 was observed. This was not found to be true for Br^?, H²PO^? ₄, and HSO^? ₄. Here, the chlorine substituted calix[4]pyrrole 4 was found to display a slightly higher affinity in the case of each anion than the fluorine-bearing derivative 5. This was rationalized in terms of intermolecular NH … F hydrogen bonding interactions being present in CD₂Cl₂ solutions of 5. Support for this latter conclusion came from concentration and temperature-dependent NMR spectroscopic studies.

A matched set of mono halogen substituted calix[4]pyrroles was used to study in detail, the extent to which halogen substituents may be used to fine-tune the anion binding properties of calix[4]pyrroles.     

N-(二茂铁酰胺基)硫脲类阴离子受体

我们将N-(苯甲酰胺基)硫脲类阴离子识别受体研究拓展至N-(二茂铁酰胺基)硫脲, 设计合成了N-(二茂铁甲酰胺基)-N'-(取代苯基)硫脲(3a～3e, 取代基X＝p-OCH₃, p-CH₃, H, m-Br, m-CF₃), 其中二茂铁基系芳香性的电化学活性基团、“—NH—”为连接臂．研究的目的是进一步理解 “—NH—”连接臂的特性和3a～3e作为阴离子的电化学响应受体的可行性. 我们发现, 3a～3e分子中的N'-苯环取代基不影响其半波电位(0.31V vs. Ag/AgNO₃); 二茂铁环芳香质子和酰胺基—NH质子的核磁化学位移不受取代基的影响, 而N'-苯基芳香质子和硫脲—NH质子的化学位移显著地受制于取代基; 说明受体3分子中酰肼基N—N单键高度扭曲, 阻碍了N'-苯环取代基电子效应传递至二茂铁基. 我们观察到乙腈中3的氧化电位因阴离子如CH₃CO₂^－和F^－的结合负移200 mV, 二茂铁基芳香质子的化学位移向高场移动, 意味着阴离子结合诱导了受体3分子中酰肼基N—N单键构型变化, 使阴离子结合信息得以传递至二茂铁环. 吸收光谱滴定实验表明, 乙腈中3a～3e与阴离子结合时在约315 nm处出现新的吸收峰, 光谱红移达5820 cm^－1, 阴离子如CH₃CO₂^－和F^－的结合常数在10⁶ mol^－1•L, 均远高于传统的二苯基硫脲类受体(1); 我们还发现, 尽管1和3a～3e分子中硫脲—NH质子酸性对取代基的依赖性相近, 3a～3e之阴离子结合常数的取代基效应强于1. 我们认为, 这是由于N—N键的变构作用导致3a～3e的吸收光谱红移、阴离子结合常数和阴离子结合常数的取代基效应远高于传统的N,N'-二苯基硫脲类受体, 因而体现了“—NH—”连接臂的独特性质.     

Anion binding properties of indolylmethanes

The anion binding properties of the indolylmethanes (1) were investigated by ¹H-NMR spectroscopy in CDCl₃. Tris(3-methylindol-2-yl)methane (1a) selectively bound a chloride anion the over other tested anions (Br^?, I^?, HSO ₄ ^? , and NO ₃ ^? ). In contrast, analogous compounds, phenyl bis(3-methylindol-2-yl)methane (1b), 2-hydroxyphenyl bis(3-methylindol-2-yl)methane (1c), tri(indol-3-yl)methane (1d), and phenyl di(indol-2-yl)methane (1e), showed a low anion binding ability and selectivity. These results indicate that the number and a position of the binding sites (indole NH protons) of the indolylmethanes are important factors for the formation of the complex with an anion. The high binding ability and selectivity of 1a toward a chloride anion is attributed to the proper size of the binding pocket for a chloride anion and the formation of multiple hydrogen bonds between the three indole NH protons and a chloride anion. The anion affinity of 1a was significantly affected by the cation component of quaternary ammonium salts, indicating that it is ion pair binding and not solely anion binding.     

Study on Synthesis and Binding Ability of a New Anion Receptor Containing NH Binding Sites

A new colorimetric recognition receptor 1 based on the dual capability containing NH binding sites of selectively sensing anionic guest species has been synthesized. Compared with other halide anions, its UV/Vis absorption spectrum in dimethyl sulfoxide showed the response toward the presence of fluoride anion with high selectivity, and also displayed dramatic color changes from colorless to yellow in the presence of TBAF （5 × 10^-5 mol/L）. The similar UV/Vis absorption spectrum change also occurred when 1 was treated with AcO^- while a little change with H2PO^-4 and OH^-. Receptor 1 has almost not affinity abilities to Cl^-, Br^- and I^-. The binding ability of receptor 1 to fluoride with high selectivity over other halides contributes to the anion size and the ability of forming hydrogen bonding. While the different ability of binding with geometrically triangular （AcO^-）, tetrahedral （H2PO^-4 ） and linear （OH^-） anions maybe result from their geometry configuration.     

Liquid Chromatographic Determination of the Myocardial Isozyme of Creatine Kinase

Abstract

Creatine kinase serum enzymes were separated by low pressure liquid chromatography with a Glycophase DEAE column. The separated MB creatine kinase isozyme was assayed by measurinq the amount of ATP formed from ADP and creatine phosphate in the presence of the enzyme. High performance 1iquid chromatography was used to determine ATP.     

Enzymatic Synthesis of an RNA Fragment Corresponding to the Anti-Codon Loop and Stem of tRNAPhe from Yeast

Abstract

A hexadecamer corresponding to the anticodon loop and stem of tRNA^Phe yeast has been prepared using T₄ RNA ligase and isolated by high performance liquid chromatography. The two oligonucleotides used in the ligation were isolated from a ribonuclease T₁ digest of the tRNA which was resolved by HPLC on an anion exchange column. To prepare the “acceptor” oligonucleotide for the RNA ligase reaction a 3′ terminal phosphate was removed. To prepare the “donor” oligomer a 5′ terminal phosphate was added. Analysis of the product hexadecamer was by nucleoside and nucleotide-3′-mono-phosphate composition.     

Formation and Stability of Difluoromethylene Phospho-Raiies,R3P=CF2

Abstract

Carbonyl compounds react with CBr₂F₂ in the presence of phosphanes, RP (R = Ph, NR;), and metals (M = Zn, Cd, Pb) forming geminal difluoroolefins (eq. 1)¹.

R′CHO + CBr₂F₂ + R₃P + M → R′CH=CF₂ + MBr₂ + R₃PO (1)

Without any doubt this reaction has to occur via the intermediate formation of difluoromethylene phosphoranes, which then undergo the Wittig reaction with carbonyl compounds (eq. 2). R₃P=CF₂ + R′CHO → R₃PO + R′CH=CF₂ (2).     

N-H…O,C-H… O hydrogen-bonded supramolecular frameworks in 4-fluoroanilinium and dicyclohexylaminium picrate salts

The asymmetric unit of compound (I), 4-fluoroanilinium picrate, C₆H₇NF⁺.C₆H₂N₃O₇^? contain one 4-fluoroanilinium cation and one picrate anion whereas in compound (II), dicyclohexylaminium picrate, C₁₂H₂₂N⁺.C₆H₂N₃O₇^? the asymmetric unit contains two sets of dicyclohexylaminium cation and picrate anion due to conformational difference between the molecules. In (I), all three nitro groups of the picrate anion are positionally disordered over two sites refined to major and minor components. The molecular ions of (I), interlinked through N–H???O and C–H???O hydrogen bonds forming two-dimensional supramolecular sheet along (-1 0 1) plane. Whereas in (II), the symmetry-independent molecules labeled as A and B molecule form independent one-dimensional supramolecular tape extending along (1 1 0) and (1 0 0) direction. The supramolecular tapes are interlinked through C–H???O interaction to form three-dimensional network in the crystalline solid in (II).

     

Study of Binding of Benzyl-Thiouracil to Human Serum Albumin by Gel Filtration Chromatography

Abstract

The binding of benzyl-thiouracil to human serum albumin was studied at 37°C and pH 7.4 by Sephadex filtration chromatography based upon Hummel and Dreyer's method. As the benzyl-thiouracil (ligand) was adsorbed on to the gel matrix, the free ligand concentrations had to be corrected through the ligand distribution between the stationary and mobile phases.

A good agreement was found between binding parameters—calculated by this method and by the classical method (equilibrium dialysis). Binding is characterized by one binding site with a moderate association constant (K₁ = 5.7 × 10⁴ M^-1) and two binding sites with a low association constant (K₂ = 7.8 × 10³ M^-1).     

Coordination Centres of Purine Nucleotides: Adenosine‐5′‐diphosphate and Adenosine‐5′‐triphosphate in their Reactions with Nickel(II), Cobalt(II) and Tetramines

Interactions between the nucleotides: adenosine‐5′‐diphosphate (ADP) and adenosine‐5′‐triphosphate (ATP) with Ni^II and Co^II ions, as well as with spermine (Spm) and 1,11‐diamine‐4,8‐diazaundecane (3,3,3‐tet) are the subject of this study. Composition and stability constants of mixed complexes thus formed have been determined on the basis of the potentiometric measurements, whereas interaction centres in ligands have been identified by VIS and NMR spectral parameter analysis. Mixed tetraprotonated complexes with Ni^II, i.e. Ni(ADP)H₄(Spm), Ni(ATP)H₄(Spm), Ni(ADP)H₄(3,3,3‐tet) and Ni(ATP)H₄(333‐tet), are identified as ML·······L′ type adducts, in which the main coordination centre is the nucleotide nitrogen N(1) or N(7) donor atom, and the fully protonated polyamine is engaged in noncovalent interactions with nucleotide phosphate group oxygen atoms. Ni(ADP)H₂(Spm), Ni(ATP)H₂(Spm), Ni(ADP)H₂(3,3,3‐tet) and Ni(ATP)H₂(3,3,3‐tet) complexes represent the {N3} coordination type In diprotonated mixed complexes of Ni^II with spermine are weak noncovalent interligand interactions, providing an additional stabilising effect. Formation of ML·······L′ type molecular complexes has been observed in systems with Co^II: Co(ADP)H₄(Spm), Co(ATP)H₄(Spm), Co(ADP)H₄(3,3,3‐tet) and Co(ATP)H₄(3,3,3‐tet), in which the N(7) atom and oxygen atoms of the phosphate group are involved in coordination and the fully protonated polyamine is engaged in noncovalent interactions with the nucleotide N(1).     

Determination of the Interaction of Arsenic and Human Serum Albumin by Online Microdialysis Coupled to LC with Hydride Generation Atomic Fluorescence Spectroscopy

Study on the stoichiometry and affinity of the arsenicals bound to HSA is an important step toward a better understanding of arsenic toxic effects. After incubation of As^III or As^V with HSA at the physiological conditions (pH 7.43 and 37 °C), the free arsenicals and arsenic-HSA complexes were separated and detected by the combined techniques of microdialysis and liquid chromatography with hydride generation atomic fluorescence spectroscopy (MD–LC–HGAFS). The decrease of As^III peak response rather than As^V indicated that HSA reacted with As^III but not As^V. The binding plots indicated that the binding between HSA and As^III was in Scatchard pattern when the concentration ratios of As^III to HSA were ≤1:1. The strong binding sites (n ₁) were 1.6 and the stability constant (K ₁) was 1.54 × 10⁶ M⁻¹. When the concentration ratios of As^III to HSA were >1:1, the binding was in Plasvento pattern with the stability constant K ₂ ≅ 0 and no specific binding of As^III with HSA. On the contrary, As^V did not show binding with HSA. The results showed that As^III reacted with HSA more readily than As^V, which provides a chemical basis for arsenic toxicity.

     

基于分子内电荷转移的阴离子比率荧光分子探针

设计合成了一种基于4-甲基-1-羟基二苯甲酮对硝基苯腙的比色和比率荧光阴离子受体1。此类受体以羟基和腙单元为识别位点,以硝基苯基为信号报告基团。向受体1的DMSO溶液中加入AcO-、H2PO4-、F-后,溶液颜色由黄色变为紫红色,而加入所研究的其它阴离子则无变化,从而实现对AcO-、H2PO4-、F-这三种离子的裸眼识别。利用紫外-可见吸收光谱、荧光光谱考察了其与AcO-,H2PO4-,F-,Cl-,Br-,I-等阴离子的识别作用。1H NMR滴定为受体分子与阴离子之间氢键作用本质提供了有力证据。     

Elucidation of spermidine interaction with nucleotide ATP by multiple NMR techniques

Interaction of polyamines with nucleotides plays a key role in many biological processes. Here we use multiple NMR techniques to characterize interaction of spermidine with adenosine 5′‐triphosphate (ATP). Two‐dimensional ¹H‐¹⁵N spectra obtained from gs‐HMBC experiments at varied pH show significant shift of N‐1 peak around pH 2.0–7.0 range, suggesting that spermidine binds to N‐1 site of ATP base. The binding facilitates N‐1 deprotonation, shifting its pK_a from 4.3 to 3.4. By correlating ¹⁵N and ³¹P chemical shift data, it is clear that spermidine is capable of concurrently binding to ATP base and phosphate sites around pH 4.0–7.0. The self‐diffusion constants derived from ¹H PFG‐diffusion measurements provide evidence that binding of spermidine to ATP is in 1:1 ratio, and pH variations do not induce significant nucleotide self‐association in our samples. ³¹P spectral analysis suggests that at neutral pH, Mg²⁺ ion competes with spermidine and shows stronger binding to ATP phosphates. From ³¹P kinetic measurements of myosin‐catalyzed ATP hydrolysis, it is found that binding of spermidine affects the stability and reactivity of ATP. These NMR results are important for advancing the studies on nucleotide–polyamine interaction and its impact on nucleotide structures and activities under varied conditions. Copyright © 2009 John Wiley & Sons, Ltd.