Spectroscopic studies of water-soluble superstructured iron(III) porphyrin. Interaction with the bovine serum albumin protein

Abstract

Acid-base equilibrium of the “one-face”-hindered sulfonated porphyrin, α5,15-[2,2′(dodecamethyleneoxy),(5-sulfonato)diphenyl]-10,20-bis(2-hydroxy,5-sulfonatophenyl)porphyrinato iron(III), has been studied by paramagnetic ¹H NMR. The isotropically shifted signals change in a fast exchange regime on the NMR time-scale. ¹H longitudinal relaxation times and temperature dependence of the chemical shifts were measured and analyzed. The electronic structure of hydroxo specie is characteristic of a six- or five-coordinate high-spin iron(III) porphyrin with an S = 5/2 ground state. The ¹H NMR titration allowed determination of the acidity constant, pK_a 6.2 (0.1 M KNO₃, 25 °C). In addition, we also report the interaction between the monohydroxo iron(III) porphyrin and the bovine serum albumin protein. From a ¹H NMR titration, we have determined the affinity apparent constant, log K_ap 3.2 (pH 7, KNO₃ 0.1 M, 25 °C). The formation of superstructured iron porphyrin-albumin protein adduct was confirmed by electronic absorption spectroscopy and electron paramagnetic resonance.     

Serum Albumin Targeted,pH‐Dependent Magnetic Resonance Relaxation Agents

The objective of this work was the synthesis of serum albumin targeted, Gd^III‐based magnetic resonance imaging (MRI) contrast agents exhibiting a strong pH‐dependent relaxivity. Two new complexes ( Gd‐glu and Gd‐bbu ) were synthesized based on the DO3A macrocycle modified with three carboxyalkyl substituents α to the three ring nitrogen atoms, and a biphenylsulfonamide arm. The sulfonamide nitrogen coordinates the Gd in a pH‐dependent fashion, resulting in a decrease in the hydration state, q, as pH is increased and a resultant decrease in relaxivity (r₁). In the absence of human serum albumin (HSA), r₁ increases from 2.0 to 6.0 mM ^?1 s^?1 for Gd‐glu and from 2.4 to 9.0 mM ^?1 s^?1 for Gd‐bbu from pH 5 to 8.5 at 37 °C, 0.47 T, respectively. These complexes (0.2 mM ) are bound (>98.9 %) to HSA (0.69 mM ) over the pH range 5–8.5. Binding to albumin increases the rotational correlation time and results in higher relaxivity. The r₁ increased 120 % (pH 5) and 550 % (pH 8.5) for Gd‐glu and 42 % (pH 5) and 260 % (pH 8.5) for Gd‐bbu . The increases in r₁ at pH 5 were unexpectedly low for a putative slow tumbling q=2 complex. The Gd‐bbu system was investigated further. At pH 5, it binds in a stepwise fashion to HSA with dissociation constants K_d1=0.65, K_d2=18, K_d3=1360 μM . The relaxivity at each binding site was constant. Luminescence lifetime titration experiments with the Eu^III analogue revealed that the inner‐sphere water ligands are displaced when the complex binds to HSA resulting in lower than expected r₁ at pH 5. Variable pH and temperature nuclear magnetic relaxation dispersion (NMRD) studies showed that the increased r₁ of the albumin‐bound q=0 complexes is due to the presence of a nearby water molecule with a long residency time (1–2 ns). The distance between this water molecule and the Gd ion changes with pH resulting in albumin‐bound pH‐dependent relaxivity.     

Solvent effect on the equilibrium constant of the chain reversible reaction of <Emphasis Type="Italic">N,N</Emphasis>′-diphenyl-1,4-benzoquinonediimine with 2,5-dichlorohydroquinone

The temperature dependences of the equilibrium constant K of the reversible chain reaction of N,N′-diphenyl-1,4-benzoquinonediimine with 2,5-dichlorohydroquinone in benzene, chlorobenzene, anisole, benzonitrile, and CCl₄ were studied. The enthalpies and entropies of the reaction in these solvents were determined, and a linear dependence between them in aromatic solvents was found. The equilibrium constant depends on the solvent nature: the replacement of CCl₄ by benzene at T = 298 K increases K from 13.6 to 140. The solvation effects are caused by several types of intermolecular interactions of participants of equilibrium with the medium. The decrease in K in the benzene-anisole-benzonitrile series is related, to a great extent, to complex formation with hydrogen bonding between 2,5-dichlorohydroquinone and the solvents. In anisole a charge-transfer complex is formed between the solvent and reaction product (2,5-dichloroquinone). The constant and enthalpy of the complexation were estimated. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2296–2302, December, 2007.     

Uranyl ion complexation of 2,2′-dihydroxyazobenzene enhanced on a backbone of poly(ethylenimine)

The formation constant (K_f) for the uranyl complex of 2,2′-dihydroxyazobenzene (DHAB) was measured with DHAB attached to poly(ethylenimine) (DHAB-PEI) at pH 7.7 to 9.4. The value of K_f was estimated from the equilibrium constant for extraction of uranyl ion from the uranyl complex of DHAB-PEI (UO₂DHAB-PEI) with carbonate ion, which in turn was measured from the absorbance change observed on addition of bicarbonate ion to the solution of UO₂DHAB-PEI. At pH 8.0, the uranyl-binding ability of DHAB was enhanced by about 10⁴ times on attachment of DHAB to PEI. The major origin of the increased ability of uranyl ion complexation is the basic local microenvironment of PEI, which encourages ionization of the phenol groups of DHAB. Various other possible origins are discussed also. The log K_f for DHAB-PEI at pH 8.0 indicates that DHAB moieties of DHAB-PEI are mostly occupied, whereas DHAB unattached to PEI is mostly unoccupied by uranyl ion under conditions of seawater when only the pH and concentrations of bicarbonate and uranyl ions of seawater are considered. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 3936–3942, 1999     

A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases

Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance (¹HNMR) techniques.On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy.Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants K_a were determined for binary (i.e. PBZ–SA and ASA–SA) and ternary complexes (i.e. PBZ–[ASA]–SA and ASA–[PBZ]–SA).PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants K_aI of PBZ–SA complex. Similarly, PBZ influences K_aI of ASA–SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of K_aII values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of ¹HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.     

On the Stability of the Schiff Bases of Pyridoxal 5′-Phosphate with Polypeptides Containing L-Lysine

We determined the apparent equilibrium constant of formation, K_pH, of the Schiff bases of pyridoxal 5′-phosphate (PLP) and poly- and copolymers containing L -lysine, as a function of pH at 25° and a constant ionic strength of 0.1 M . The K_pH values obtained at acidic and neutral pH were larger that those reported for Schiff bases of PLP and hexylamine. We determined calorimetrically ΔH of formation of Schiff bases of PLP and poly(L -lysine) (?4.5′kcal/mol), and PLP and hexylamine (?3.4 kcal/mol) at pH 7.00. Semi-empirical theoretical calculations (INDO and AMI methods) of a model compound of Schiff base of PLP and polypeptide containing L -lysine show the capability of specific interactions between groups of PLP and the peptide skeleton.     

Factors Contributing to Solubility Synergism of Some Basic Drugs with β-Cyclodextrin in Ternary Molecular Complexes

Ternary complexes exploiting solubility synergism (SSn) between basic drugs and β-cyclodextrin (β-CD) in the presence of an organic hydoxy acid have been reported to provide the pharmaceutical technology with highly soluble ternary complexes, even with the least soluble β-CD. In this work, phase solubility techniques were used to study factors affecting SSn in aqueous solution, which may help in understanding the mechanism involved in ternary complex formation in solution, under equilibrium conditions. The equilibrium solubility of both β-CD and each of 8 structurally unrelated drugs were measured in tandem in the presence of different acid types at low and high pHs, and at different time intervals over a period of 1–40 days. The results indicate that SSn is evident regardless of acid type (organic and inorganic) at low pH, but the extent of SSn is acid type dependant and is limited by the drug salt solubility product constant (pK _sp). Among different drugs, no apparent trend exists between drug salt solubility and the extent of SSn, but lowering drug salt solubility by increasing pH depresses SSn. The results also reveal no apparent trend between the magnitude of the complex formation constant (K _ij) and SSn. For example, drugs of low K _ij values such as astemizole, cisapride and sildenafil do not show any SSn, yet ketotifen and pizotifen, which also have low K _ij values, exhibit substantial SSn. However, the solublizing power of β-CD represented by the slope of phase solubility diagram can be used as a marker for SSn (slopes exceeding 0.4 induce SSn).     

Influence of the pH on the Formation of the Catanionic n-Tetradecylammonium Cholate Surfactant Salt

The catanionic n-tetradecylammonium cholates, with the 1:1 and 1:2 cationic:anionic surfactant molar ratio, have been synthesized by reaction of tetradecylammonium chloride and sodium cholate. The stoichiometry of the compounds formed is strongly dependent on pH of the aqueous reaction solution. In the neutral and slightly acidic medium the isolated surfactant was detected as 1:1, at acidic medium (pH ≈ 3) as 1:2, while the cholic acid alone precipitated from the strong acidic solution (pH ≈ 2). The catanionic surfactants have been characterized by elemental and spectroscopic (IR, ¹H, and ¹³C NMR) analyses, and microscopic observations. The mole fractions of individual components in precipitate present in the heterogeneous aqueous system have been determined as a function of pH. The data interpretation in the pH regions where two different kinds of precipitates coexist pointed to the complex equilibrium in the examined systems. The evaluation of the relevant equilibrium constants was solved by the nonlinear regression analysis. In the acidic region where cholic acid and 1:2 precipitate coexisted the equilibrium constants of dissolution were K ₁ = 8.4 × 10^?10 and K ₄ = 3.7 × 10^?27, respectively. In the region where 1:2 and 1:1 precipitate coexisted the equilibrium constant of dissolution of 1:1 tetradecylammonium cholate was K ₂ = 4.2 × 10^?20.     

Rationalizing nuclear overhauser effect data for compounds adopting multiple-solution conformations

An algorithm is described for refining the populations of a set of multiple-solution conformers using experimental nuclear Overhauser effects (nOes). The method is based upon representing the effective relaxation matrix for the set of interconverting proposed conformers as a linear combination of relaxation matrices (LCORMs) due to each conformer. The conformer population derivative of the nOe is derived from a Taylor series expression for the calculated nOe. This derivative may then be used in a standard nonlinear least-squares refinement procedure. The LCORM nOe procedure is tested using a monosaccharide system, 1-O-methyl-α-L -iduronate, that is known to exhibit conformational variability. The measured nOes for this system are used to refine the populations of a set of three static conformers, namely, the ¹C₄, ⁴C₁, and ²S₀ ring conformers. The populations thus derived are compared to those previously obtained using nuclear magnetic resonance proton-proton coupling constant information. Two possible extensions to the method are discussed: The first uses combined nOe and coupling constant data while the second removes the restrictions that the conformers used for fitting be rigid entities. © 1994 by John Wiley & Sons, Inc.     

Fluorescence resonance energy transfer from tryptophan in human serum albumin to a bioactive indoloquinolizine system

The interaction between a bioactive molecule, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), with human serum albumin (HSA) has been studied using steady-state absorption and fluorescence techniques. A 1:1 complex formation has been established and the binding constant (K) and free energy change for the process have been reported. The AODIQ-HSA complex results in fluorescence resonance energy transfer (FRET) from the tryptophan moiety of HSA to the probe. The critical energy-transfer distance (R ₀) for FRET and the Stern-Volmer constant (K _sv) for the fluorescence quenching of the donor in the presence of the acceptor have been determined. Importantly, K _SV has been shown to be equal to the binding constant itself, implying that the fluorescence quenching arises only from the FRET process. The study suggests that the donor and the acceptor are bound to the same protein at different locations but within the quenching distance.     

Sorption of tungsten on alumina in dynamic conditions

Summary The sorption of tungstate anions on alumina from aqueous solutions of sodium chloride was investigated in dynamic conditions. The breakthrough curves as the function of W and NaCl concentrations, pH and the flow rates were determined. The breakthrough capacities, the capacities at C/C₀=0.5, the total column sorption capacities and the utilization degrees or column efficiencies, were determined. The obtained tungstate anion sorption data fit with a Langmuir-type isotherm. The values of the breakthrough capacity, Q_0,9^max,the Langmuir equilibrium constant or affinity parameter, K_L, and the free energy change, ΔG, of tungsten sorption were also determined.     

Effects of Alkyl Chain Length and Hydrogen Bonds on the Cooperative Self‐Assembly of 2‐Thienyl‐Type Diarylethenes at a Liquid/Highly Oriented Pyrolytic Graphite (HOPG) Interface

An appropriate understanding of the process of self‐assembly is of critical importance to tailor nanostructured order on 2D surfaces with functional molecules. Photochromic compounds are promising candidates for building blocks of advanced photoresponsive surfaces. To investigate the relationship between molecular structure and the mechanism of ordering formation, 2‐thienyl‐type diarylethenes with various lengths of alkyl side chains linked through an amide or ester group were synthesized. Their self‐assemblies at a liquid/solid interface were investigated by scanning tunneling microscopy (STM). The concentration dependence of the surface coverage was analyzed by using a cooperative model for a 2D surface based on two characteristic parameters: the nucleation equilibrium constant (K_n) and the elongation equilibrium constant (K_e). The following conclusions can be drawn. 1) The concentration at which a stable 2D molecular ordering is observed by STM exponentially decreases with increasing length of the alkyl chain. 2) Compounds bearing amide groups have higher degrees of cooperativity in self‐assembly on 2D surfaces (i.e., σ, which is defined as K_n/K_e) than compounds with ester groups. 3) The self‐assembly process of the open‐ring isomer of an ester derivative is close to isodesmic, whereas that of the closed‐ring isomer is cooperative because of the difference in equilibrium constants for the nucleation step (i.e., K_n) between the two isomers.     

Solvent Extraction–Photometric Determination of Palladium Using Complexation with 1-(2-Pyridylazo)-2-Naphthol

Palladium(II) complexation with 1-(2-pyridylazo)-2-naphthol (PAN) in aqueous solutions followed by extraction with chloroform and photometric detection was studied. The best conditions were found for the formation of the complex in an aqueous solution and for its extraction with chloroform that provided a sufficient degree of binding palladium ions. The stability constant of the complex cation PdX⁺, which is extracted by chloroform as an ion pair [PdX]⁺[An^–] (An^–– CH₃COO^–), was calculated using the methods proposed by Rossotti (log K _stab= 18.73) and Komar' (log K _stab= 18.82). The equilibrium constant of the complexation reaction was also calculated (5.45 × 10⁴). It was shown that components of nonferrous alloys affect the determination of palladium by photometry as its complex or ion pair with PAN in chloroform.     

荧光法研究3-氨基苯硼酸与牛血清白蛋白间的相互作用

为了了解分子印迹反应的机理和最适宜的反应条件, 应用荧光猝灭法研究了3-氨基苯硼酸(APBA)与牛血清白蛋白(BSA)的相互作用, 二者的反应受到体系pH值、离子强度等关键因素的影响. 实验结果表明: 适宜的离子强度和pH值为6.25时, APBA与BSA的色氨酸残基的荧光猝灭反应的物质的量比为2∶1, 表观结合常数K_A＝1.0×10¹¹ L²• mol^－2, 说明二者间形成了较强的化学键. 通过上述研究, 明晰了3-氨基苯硼酸与牛血清白蛋白之间的作用机理, 有助于分离或富集蛋白质中BSA组分, 从而能够改进印迹和洗脱的效率.     

Equilibrium constant for coordinative polymerization of an o,o′-dihydroxyazobenzene derivative with Ni(II) ion in water

Equilibrium constant (K_CP) for coordinative polymerization is measured for the first time. Constant K_CP is defined as [L]_cp/[M][L], where [L]_cp represents the concentration of the ligand present in the coordination polymer. Plot of absorbance changes measured for 3, a water-soluble derivative of o,o′-dihydroxyazobenzene, against the concentration of Ni(II) ion indicates formation of a 1 : 1-type complex in water at pH 7.74 and 25°C when Ni (II) is added in excess of 3. The 1 : 1-type complex can be either Ni 3, the monomeric complex, or (Ni 3 )_n, the coordination polymer. The equilibrium constant for formation of the 1 : 1-type complex is estimated as 10^13.10 by using UO₂²⁺ ion as the competing metal ion. For the Ni(II) complex of an o,o′-dihydroxyazobenzene derivative attached to poly(ethylenimine), the formation constant is estimated as 10^5.36. Due to the structure of the polymer, possibility of coordinative polymerization is excluded for the polymer-based ligand. The much greater equilibrium constant for formation of the Ni(II) complex of 3, therefore, indicates formation of (Ni 3 )_n instead of Ni 3. The value of K_CP for (Ni 3 )_n shows that only 10⁻⁷% of the initially added 3 is left unpolymerized when Ni(II) is added in excess of 3 by 10⁻⁴ M. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35 : 1825–1830, 1997     

Tautomerization of Phenols,Part III,The Anthrone−Anthrol Equilibrium in Aqueous Solution

The equilibrium between 10H‐anthr‐9‐one and 9‐anthrol favors the ketone, which ionizes as a carbon acid in aqueous base. Rates of equilibration were measured over the pH range 1 – 13 in aqueous solution (25°, ionic strength I=0. M ). Five independent thermodynamic and kinetic parameters were determined by analysis of the pH‐rate profile: the equilibrium constant of enolization, pK_E=2.17, the ionization quotient of anthrol, pQ=7.84, and the rate constants of enolization catalyzed by acid, k=2.2⋅10⁻⁴ M ⁻¹ s⁻¹, base, k=51.0 M ⁻¹ s⁻¹, and water, k=1.21⋅10⁻⁵ s⁻¹. Structure‐reactivity relationships strongly support the view that pH‐independent enolization of anthrone in water proceeds by rate‐determining ionization of the C‐acid.     

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).     

Design Principles and Theory of Paramagnetic Fluorine‐Labelled Lanthanide Complexes as Probes for 19F Magnetic Resonance: A Proof‐of‐Concept Study

The synthesis and spectroscopic properties of a series of CF₃‐labelled lanthanide(III) complexes (Ln=Gd, Tb, Dy, Ho, Er, Tm) with amide‐substituted ligands based on 1,4,7,10‐tetraazacyclododecane are described. The theoretical contributions of the ¹⁹F magnetic relaxation processes in these systems are critically assessed and selected volumetric plots are presented. These plots allow an accurate estimation of the increase in the rates of longitudinal and transverse relaxation as a function of the distance between the Ln^III ion and the fluorine nucleus, the applied magnetic field, and the re‐rotational correlation time of the complex, for a given Ln^III ion. Selected complexes exhibit pH‐dependent chemical shift behaviour, and a pK_a of 7.0 was determined in one example based on the holmium complex of an ortho‐cyano DO3A‐monoamide ligand, which allowed the pH to be assessed by measuring the difference in chemical shift (varying by over 14 ppm) between two ¹⁹F resonances. Relaxation analyses of variable‐temperature and variable‐field ¹⁹F, ¹⁷O and ¹H NMR spectroscopy experiments are reported, aided by identification of salient low‐energy conformers by using density functional theory. The study of fluorine relaxation rates, over a field range of 4.7 to 16.5 T allowed precise computation of the distance between the Ln^III ion and the CF₃ reporter group by using global fitting methods. The sensitivity benefits of using such paramagnetic fluorinated probes in ¹⁹F NMR spectroscopic studies are quantified in preliminary spectroscopic and imaging experiments with respect to a diamagnetic yttrium(III) analogue.     

Laser light scattering studies of poly(L-lysine HBr) in aqueous solutions

A simple theory for the relaxation of concentration fluctuations in polyelectrolyte solutions is presented, and particular results for the high-salt and no-salt limiting cases are discussed. Autocorrelation functions for the fluctuating intensity of scattered light from dilute aqueous solutions of poly(L -lysine HBr) (PLL-HBr) with and without added salt have been observed over a wide range of pH. The observed autocorrelation functions are in general very satisfactorily represented by single exponentials except at high pH (>10.5), where considerable aggregation is manifested. Solutions of PLL HBr without added salt exhibit extraordinary behavior, evident at low pH, involving a species with a very slowly decaying autocorrelation function. Though this species is readily annealed to a more ordinary individual free-molecule form by cycling the pH to 9.5 or higher and back, the resulting molecules are found to require unusually long times to reach internal configuration equilibrium under low pH conditions. Solutions of PLL-HBr in 0.2M NaBr and 0.1M NaCl are apparently free of similar extraordinary effects and show the normal isothermal helix–coil transition accompanied by a 15–30% rise in the diffusion coefficient to a maximum at pH 10.5, which is interpreted in terms of a change in molecular dimension of an interrupted helix. The predicted K² dependence of the reciprocal relaxation time and enhancement of the apparent diffusion coefficient of the polyelectrolyte in the absence of salt is confirmed.     

Interaction of magnolol with bovine serum albumin: a fluorescence-quenching study

The interaction of magnolol with bovine serum albumin(BSA) was studied using fluorescence spectroscopy under physiological conditions. The binding constants, K, and the ratio of quantum yields of protein fluorescence for complex and free protein, f, at 298 K, 304 K, and 310 K were obtained; the values were 6.799×10⁵ L mol^–1, 5.541×10⁵ L mol^–1, and 4.344×10⁵ L mol^–1 and 0.17, 0.30, and 0.34, respectively. The standard enthalpy change (H°) and the standard entropy change (S°) were calculated to be –28.53 kJ mol^–1 and 15.88 J mol^–1 K^–1, which indicated that hydrophobic forces played major role in the interaction of magnolol and BSA. The binding average distance between magnolol and BSA (4.32 nm) was obtained on the basis of the theory of Förster energy transfer.