Proton Dissociation Constants Research Articles

Transferrin's mechanism of interaction with receptor 1.

The kinetics and thermodynamics of the interactions of transferrin receptor 1 with holotransferrin and apotransferrin in neutral and mildly acidic media are investigated at 37 degrees C in the presence of CHAPS micelles. Receptor 1 interacts with CHAPS in a very fast kinetic step (<1 micros). This is followed in neutral media by the interaction with holotransferrin which occurs in two steps after receptor deprotonation, with a proton dissociation constant (K(1a)) of 10.0 +/- 1.5 nM. The first step is detected by the T-jump technique and is associated with a molecular interaction between the receptor and holotransferrin. It occurs with a first-order rate constant (k(-1)) of (1.6 +/- 0.2) x 10(4) s(-1), a second-order rate constant (k(1)) of (3.20 +/- 0.2) x 10(10) M(-1) s(-1), and a dissociation constant (K(1)) of 0.50 +/- 0.07 microM. This step is followed by a slow change in the conformation with a relaxation time (tau(2)) of 3400 +/- 400 s and an equilibrium constant (K(2)) of (4.6 +/- 1.0) x 10(-3) with an overall affinity of the receptor for holotransferrin [(K'1)(-1)] of (4.35 +/- 0.60) x 10(8) M(-1). Apotransferrin does not interact with receptor 1 in neutral media, between pH 4.9 and 6, it interacts with the receptor in two steps after a receptor deprotonation (K(2a) = 2.30 +/- 0.3 microM). The first step occurs in the range of 1000-3000 s. It is ascribed to a slow change in the conformation which rate-controls a fast interaction between apotransferrin and receptor 1 with an overall affinity constant [(K(3))(-1)] of (2.80 +/- 0.30) x 10(7) M(-1). These results imply that receptor 1 probably exists in at least two forms, the neutral species which interacts with holotransferrin and not with apotransferrin and the acidic species which interacts with apotransferrin. At first, the interaction of the neutral receptor with holotransferrin is extremely fast. It is followed by the slow change in conformation, which leads to an important stabilization of the thermodynamic structure. In the acidic media of the endosome, the interaction of apotransferrin with the acidic receptor is sufficiently strong and rate-controlled by a very slow change in conformation which allows recycling back to the plasma membrane.

Acid-base equilibria of various oxidation states of aqua-ruthenium complexes with 1,10-phenanthroline-5,6-dione in aqueous media.

Syntheses and pH dependent electrochemical properties of aqua-ruthenium(II) complexes, [Ru(trpy)(PDA-N,N')(OH2)](ClO4)2 ([1](ClO4)2) and [Ru(trpy)(PD-N,N')(OH2)](ClO4)2 ([2](ClO4)2) (trpy = 2,2':6',2''-terpyridine, PDA = 6-acetonyl-6-hydroxy-1,10-phenanthroline-5-one, PD = 1,10-phenanthroline-5,6-dione) are presented. Treatment of [Ru(trpy)(PD-N,N')Cl](PF6) with AgClO4 in a mixed solvent of acetone and H2O selectively produced the acetonyl-PD complex [1](ClO4)2, and the similar treatment in a mixed solvent of 2-methoxyethanol and H2O gave the PD complex [2](ClO4)2. The molecular structures of both complexes were determined by X-ray structural analysis. The proton dissociation constants of various oxidations state of [1]2+ and [2]2+ were evaluated by simulation of E(1/2) values of those redox potentials depending on pH. The simulation revealed that the acetonyl-PD complex [1]2+ underwent successive Ru(II)/Ru(III) and Ru(III)/Ru(IV) redox couples though the two redox reactions were not separated in the cyclic voltammograms. The redox behavior of [2]2+ in H2O is reasonably explained by not only the similar successive metal-centered redox reactions but also simultaneous two-electron quinone/catechol redox couple of the PD ligand including the contribution of hydration on a carbonyl carbon.

Effect of pH on measurement of strong copper binding ligands in lakes

We investigated the effect of pH on Cu2+ binding by natural organic ligands in two New Zealand lakes on the South Island, New Zealand, using competitive ligand equilibration with salicylaldoxime and detection by cathodic stripping voltammetry. When the pH of a lakeߚwater sample was adjusted to different values in the range 6.3ߚ8.0, the conditional Cu2+ binding constant K" was found to increase, with a slope log K" versus pH of nearly 12, as would be expected for functional groups having a proton dissociation constant pKa &gt; 9. In support of this, a comparison of log K" values measured on samples taken from surface and subsurface waters of two New Zealand alpine lakes over a 2ߚyr period showed a very similar log K"ߚpH dependence. These results imply that the functional group chemistry of strong Cu2+ߚbinding ligands in such lakes is relatively uniform and may involve phenolic OH groups. In Lake Hayes, the Cuߚbinding ligand concentration [L]T exceeded that of total dissolved Cu, [Cu]T at almost all times of the year and all depths. However, in Lake Manapouri, little evidence of Cuߚbinding ligand was observed during late summer in the mixed layer, which suggests a seasonal cycle in Cuߚbinding ligands that is perhaps driven by enhanced ultraviolet irradiation in summer or by seasonal changes in phytoplankton community structure. This may have important consequences for the toxicity of Cu2+ to organisms in these lakes during summer.

새로운 N2O계 세 자리 리간드의 합성과 전이금속 착물 안정도상수

피리딘과 페놀을 포함하는 N2O계 세 자리 리간드 2-[(2-pyridine-2- ethylamino)-methyl]-phenol(H-PEMP)를 합성하였다. 또한 H-PEMP 페놀의 5-위치에 치환기로 브롬, 염소 및 메톡시를 가지는 Br-PEMP, Cl-PEMP 및 <TEX>$CH_3O-PEMP$</TEX>를, 그리고 2-hydroxy-1-naphthalene기를 가진 Naph-PEMP을 합성하였다. 합성된 각 리간드들을 수용액에서 전위차 적정 결과 양성자 해리는 3 단계로 일어났으며, 계산된 각 리간드의 총괄 양성자 해리상수(log<TEX>${\beta}$</TEX>) 값은 <TEX>$CH_3O-PEMP$</TEX>〉Naph-PEMP〉H-PEMP〉Br-PEMP〉Cl-PEMP의 순서로 Hammett식의 치환기 상수(<TEX>${\sigma}_p$</TEX>) 값 크기 순서와 일치하였다. 전이금속 이온들의 착물 안정도상수(logML 및 log<TEX>$ML_2$</TEX>) 값의 크기는 금속이온에 따라 Co(II)< Ni(II)< Cu(II)>Zn(II)의 순서이었고, 치환기에 따른 각 리간드의 착물 안정도상수 값은 리간드의 총괄 양성자 해리상수 값의 크기순서와 일치하였다. Ligands, Br-PEMP, Cl-PEMP and <TEX>$CH_3O-PEMP$</TEX> having Br, Cl and <TEX>$CH_3O$</TEX> substituents at 5-position of the <TEX>$N_2O$</TEX> tridentate ligand, 2-[(2-pyridine-2-ethylamio)-methyl]-phenol (H-PEMP) containing pyridine and phenol were synthesized. Another ligand, Naph-PEMP having pyridine and 2-hydroxy-1-naphthalene was also synthesized. The ligands were characterized using elemental analysis, UV-visible, IR, <TEX>$^1H\;NMR\;and\;^{13}C$</TEX> NMR spectroscopy and mass analysis. The potentiometric titration study in aqueous solution revealed that the proton dissociation of the ligands occurred in three steps and the order of overall proton dissociation constants (log<TEX>${\beta}$</TEX>) was <TEX>$CH_3O-PEMP$</TEX> > Naph-PEMP > H-PEMP > Cl-PEMP > Br-PEMP. The order of stability constants (logML and log<TEX>$ML_2$</TEX>) of their transition metal complexes was Co(II) < Ni(II) < Cu(II) > Zn(II). The order in their stability constants values of each transition metal complex agreed well with that in overall proton dissociation constant value of the ligands.

Aluminum Exchange between Citrate and Human Serum Transferrin and Interaction with Transferrin Receptor 1

The kinetics and thermodynamics of Al(III) exchange between aluminum citrate (AlL) and human serum transferrin were investigated in the 7.2-8.9 pH range. The C-site of human serum apotransferrin in interaction with bicarbonate removes Al(III) from Al citrate with an exchange equilibrium constant K1 = (2.0 +/- 0.6) x 10(-2); a direct second-order rate constant k1 = 45 +/- 3 M(-1) x s(-1); and a reverse second-order rate constant k(-1) = (2.3 +/- 0.5) x 10(3) M(-1) x s(-1). The newly formed aluminum-protein complex loses a single proton with proton dissociation constant K1a = (15 +/- 3) nM to yield a first kinetic intermediate. This intermediate then undergoes a modification in its conformation followed by two proton losses; first-order rate constant k2 = (4.20 +/- 0.02) x 10(-2) s(-1) to produce a second kinetic intermediate, which in turn undergoes a last slow modification in the conformation to yield the aluminum-loaded transferrin in its final state. This last process rate-controls Al(III) uptake by the N-site of the protein and is independent of the experimental parameters with a constant reciprocal relaxation time tau3(-1) = (6 +/- 1) x 10(-5) x s(-1). The affinities involved in aluminum uptake by serum transferrins are about 10 orders of magnitude lower than those involved in the uptake of iron. The interactions of iron-loaded transferrins with transferrin receptor 1 occur with average dissociation constants of 3 +/- 1 and 5 +/- 1 nM for the only C-site iron-loaded and of 6.0 +/- 0.6 and 7 +/- 0.5 nM for the iron-saturated ST in the absence or presence of CHAPS, respectively. No interaction is detected between receptor 1 and aluminum-saturated or mixed C-site iron-loaded/N-site aluminum-loaded transferrin under the same conditions. The fact that aluminum can be solubilized by serum transferrin in biological fluids does not necessarily imply that its transfer from the blood stream to cytoplasm follows the receptor-mediated pathway of iron transport by transferrins.

Effects of an Axial Ligand on the Reduction Potential, Proton Dissociation, and Fluorescence Quantum Yield of Hydroxo(porphyrinato)antimony(V) Complexes

Abstract The substituent effects on the reduction potentials (E1/2red), the proton dissociation constants of a hydroxo ligand (Ka), and the fluorescence quantum yields (Φ) in aryloxo(hydroxo)tetraarylporphyrinatoantimony(V) complexes were investigated. The E1/2red and Ka values were affected by substituents in the axial aryloxo ligand but, little affected by substituents in the porphyrin ring. Since E1/2red of tetraarylporphyrinatoantimony(V) complexes were higher than those of other metal-porphyrin complexes, it was suggested that the metal orbital greatly contributed to the LUMO level of the complex which can be related to the E1/2red and Ka values. Therefore, the substituent effects of the axial aryloxo ligand on the E1/2red and Ka values were attributed to the electron density of the antimony ion, which affected the LUMO level of the complexes. Moreover, the Φ of the porphyrin moiety depended on both the oxidation potential of the axial aryloxo ligands and the polarity of the solvent used. The fluorescences of the porphyrin moiety were quenched by the axial aryloxo ligands at rate constants of 108 – 1011 s-1.

Solution equilibria of deferoxamine amides

The physico‐chemical solution properties of deferoxamine were modified by acylating the terminal amino group with short‐chain aliphatic, succinic, and methylsulphonic moieties. The analog iron(III)‐binding constants and stabilities under physiological conditions were determined to confirm that the iron binding ability of the parent molecule was retained following modification. The proton dissociation constants of the lipophilic deferoxamine analogs were determined by potentiometric titration and nonlinear least‐squares analysis. However, because the iron(III) binding complex is fully formed below pH 2, the metal–ligand equilibria could not be studied using potentiometric methods. The iron binding constants of the deferoxamine analogs were determined by spectrophotometrically following the proton‐dependent exchange of iron with EDTA in the pH range of 4.0 to 6.5 and solving mass balance equations. The proton‐dissociation constants and the iron binding constants of the lipophilic deferoxamine analogs were comparable to those of deferoxamine. However, at physiological conditions, the iron‐binding complex of the most lipophilic butylamide derivative was slightly less stable and the succinamide derivative complex was slightly more stable. Like deferoxamine, the hydroxamate groups of the analogs were unhindered and free to form a 1:1 coordination complex with iron(III). Consequently, changes in aqueous solvation, conformation, and steric interference, imparted by the modifications at the terminal amino group of deferoxamine, may have affected the stabilities of the iron(III) complex and the efficiency of iron binding. © 2002 Wiley‐Liss Inc. and the American Pharmaceutical Association J Pharm Sci 91:1733–1741, 2002

Manganese(iii) meso-tetrakis(ortho-N-alkylpyridyl)porphyrins. Synthesis, characterization, and catalysis of O2˙− dismutation

A series of ortho isomers of meso-tetrakis(N-alkylpyridyl)porphyrins (alkyl being methyl, ethyl, n-propyl, n-butyl, n-hexyl, and n-octyl) and their Mn(III) complexes were synthesized and characterized by elemental analysis, uv/vis spectroscopy, electrospray ionization mass spectrometry and electrochemistry. An increase in the number of carbon atoms in the alkyl chains from 1 to 8 is accompanied by an increase in: (a) lipophilicity, as measured by the chromatographic retention factor, Rf; (b) metal-centered redox potential, E1/2 from +220 to +367 mV vs. NHE, and (c) proton dissociation constant, pKa2 from 10.9 to 13.2. A linear correlation was found between E1/2 and Rf of the Mn(III) porphyrins and between the pKa2 and Rf of the metal-free compounds. As the porphyrins become increasingly more lipophilic, the decrease in hydration disfavors the creation of charge, while enhancing the electron-withdrawing effect of the positively charged pyridyl nitrogen atoms. Consequently, E1/2 increases linearly with the increase in pKa2, a trend in porphyrin basicity opposite from the one we previously reported for other water-soluble Mn(III) porphyrins. All of these Mn(III) porphyrins are potent catalysts for superoxide dismutation (disproportionation). Despite the favorable increase of E1/2 with the increase in chain length, the catalytic rate constant decreases from methyl (log kcat = 7.79) to n-butyl, and then increases such that the n-octyl is as potent a SOD mimic as are the methyl and ethyl compounds. The observed behavior originates from an interplay of hydration and steric effects that modulate electronic effects.

Interaction of partially ionized poly(acrylic acid) with a surfactant counterion at constant pH values

Binding of a cationic surfactant ion, dodecylpyridinium ion, to poly(acrylic acids) of low charge densities was examined by potentiometry using surfactant-selective electrodes in the solutions, where the pH was kept constant by employing a pH buffering system. The binding of the surfactant counterions was thus able to be studied at a constant pH during the binding process. The binding took place in two steps, the first cooperative binding step and the second gradual binding step. The critical association concentration decreased as the pH increased, indicating the predominant role of the electric interaction in the binding. The binding isotherms obtained at different but constant pH values were analyzed by the matrix method, taking into account the nearest-neighbor interactions among three different kinds of sites on the polymer: ionized, protonated, and surfactant-bound. The theoretical analysis could describe only the first step but could not explain the second step. A relatively large cooperativity parameter, u, was found for the first step and it can be between 3 × 103 and 1 × 104. When the ionic strength was decreased tenfold, the cooperativity of the binding decreased (u∼1 × 103). The binding constants of the isolated site were 5.5–6.0 × 104 kg mol−1 and slightly increased to 6.5 × 104 kg mol−1 as the ionic strength decreased. The deviation of the second step from the theoretical analysis was supposed to arise from a change of proton dissociation constant in the nonpolar space formed by the bound surfactants.

Kinetics and Mechanism of Water Substitution at Half-Sandwich Iridium(III) Aqua Cations Cp*Ir(A−B)(H2O)2+/+ in Aqueous Solution (Cp* = η5-Pentamethylcyclopentadienyl Anion; A−B = Bidentate N,N or N,O Ligand)

The perchlorate complexes of a series of half-sandwich monoaqua cations Cp*Ir(A−B)(H2O)2+/+ with A−B = prol (D/L-proline anion), picac (picolinic acid anion), R,R-dach [(−)-(1R,2R)-1,2-diaminocyclohexane], R,R-dpen [(+)-(1R,2R)-1,2-diphenylethylenediamine], phen (o-phenanthroline), and bpy (2,2′-bipyridine) (Cp* = η5-pentamethylcyclopentadienyl anion) have been prepared and characterized. An X-ray structure analysis of Cp*Ir(R,R-dach)(H2O)(ClO4)2·H2O has revealed that the cation Cp*Ir(R,R-dach)(H2O)2+ has a distorted pseudo-octahedral coordination geometry. In the case of A−B = prol, crystallization from water led to the trinuclear complex [Cp*Ir(D-prol)]3(ClO4)3, which has also been characterized by X-ray structure analysis. The experimental data suggest that in aqueous solution the trinuclear proline complex dissociates to form the cation Cp*Ir(D-prol)(H2O)+. The proton dissociation constants of the coordinated water in Cp*Ir(A−B)(H2O)2+/+ have been determined as pKa = 7.5 (A−B = bpy) and pKa = 7.1 (A−B = R,R-dach and picac). Substitution of the water in Cp*Ir(A−B)(H2O)2+/+ by the monodentate ligands L = py (pyridine), DMS (dimethyl sulfide), TU (thiourea), and monodentate anions according to the Equation Cp*Ir(A−B)(H2O)2+/+ + L Cp*Ir(A−B)L2+/+ + H2O has been studied by multi-wavelength stopped-flow spectrophotometry in aqueous solution at I = 0.2 M. This kinetic investigation, carried out at different concentrations, temperatures, and pressures, showed that the process obeys second-order kinetics, where rate = kL[Cp*Ir(A−B)H2O2+/+][L]. The magnitude of the second-order rate constant kL depends on the nature of both A−B and L. The data for kL have been found to range from 6.4 × 104M−1s−1 (A−B = D-prol; L = TU) to 10.5 M−1s−1 (A−B = bpy; L = py) at 298 K. The activation parameters for water substitution at Cp*Ir(A−B)(H2O)2+/+ (A−B = bpy, R,R-dach, and picac) by L = TU have been evaluated. The activation volumes of ΔV≠ = +2.3, +7.4, and +7.3 cm3 mol−1, respectively, are supportive of an Id mechanism. The results regarding the kinetic lability of the coordinated water in the monoaqua cations Cp*Ir(A−B)(H2O)2+/+ are compared to those obtained for the triaqua cation Cp*Ir(H2O)32+.

Reaction of β-Alkannin (Shikonin) with Reactive Oxygen Species: Detection of β-Alkannin Free Radicals

β-Alkannin (shikonin), a compound isolated from the root of Lithospermum erythrorhizon Siebold Zucc., has been used as a purple dye in ancient Japan and is known to exert an anti-inflammatory activity. This study aimed to understand the biological activity in terms of physico-chemical characteristics of β-alkannin. Several physico-chemical properties including proton dissociation constants, half-wave potentials and molecular orbital energy of β-alkannin were elucidated. This compound shows highly efficient antioxidative activities against several types of reactive oxygen species (ROS), such as singlet oxygen (1O2), superoxide anion radical (O2), hydroxyl radical (OH) and tert-butyl peroxyl radical (BuOO) as well as iron-dependent microsomal lipid peroxidation. During the reactions of β-alkannin with 1O2, O2 and BuOO, intermediate organic radicals due to β-alkannin were detectable by ESR spectrometry. Compared with the radicals due to naphthazarin, the structural skeleton of β-alkannin, the β-alkannin radical observed as an intermediate in the reactions with 1O2 and O2 was concluded to be a semiquinone radical. On the other hand, during the reactions of β-alkannin and naphthazarin with BuOO, ESR spectra different from the semiquinone radical were observed, and proposed to result from the abstraction of hydrogen atoms from phenolic hydroxyl groups of β-alkannin by BuOO. Based on the ROS-scavenging abilities of β-alkannin, the compound was concluded to react directly with ROS and exhibits antioxidative activity, which in turn exerts anti-inflammatory activity.

Histidine residues underlie Congo red binding to Aβ analogs

The binding mechanism of Congo red (CR) to Alzheimer's disease (AD) amyloid fibrils (Aβ) in terms of binding affinity and number of sites was quantitated from absorption spectroscopy (at 200-700 nm) by measuring the concentration of CR bound (CR-B) to AD Aβ assemblies as a function of CR concentration and pH in 80% ethanol. The rationale for the use of this high concentration of ethanol derives from its use in histological screens for amyloid in tissue sections. Moreover, free CR can be separated from bound CR by filtration in ethanolic but not aqueous medium. The Aβ analogs studied here included: (1) peptides having different lengths: Aβl-40, Aβ11-28, Aβ13-28, Aβ19-28, Aβ11-25; (2) wildtype, control sequences ofAβ1-40 and sequences having different natural amino acid substitutions: primate Prl-40, rodent Rol-40, hereditary cerebral haemorrhage with amyloidosis, Dutch type (HCHWA-D) Dul-40, primate reverse sequence Pr40-1; and (3) A β11-25 sequences having different substitutions: H13D, H14D, and D23K. Negative-staining showed that A βl-40 fibrils in buffer were indistinguishable from those in buffered ethanolic medium. For all amyloid analogs except A βl 9-28, which has no histidine residues and showed no CR binding over the entire pH range 4.0-9.5, CR-B decreased as a function of increasing pH. The decrease was steepest at about pH 5 and became zero above pH 7. For analogs having the same number of histidines, CR-B fell on the same binding curve, indicating that histidine residues are the likely binding sites for CR in this medium. The pH titration of the binding was parameterized by the stoichi-ometry of dye to the sites, the number ofhistidines per molecule, the binding dissociation constant Kd, and the apparent proton dissociation constant pK of the histidine; and the calculated pH-titration curves were found to fit the observed ones. For the peptides having 1-3 histidines the average pK was 5. 0-5. 5, which was similar to the expected pK of histidine in low dielectric medium (80% ethanol), and the Kd s were 2.8-5.9 μM. That histidine residues underlie CR binding in Aβ amyloid is consistent with previous findings that Aβ peptides sediment as fibrillar assemblies atpH∼3–7 and bind Congo red over the same pH range in aqueous medium. Further, the conformation near the binding motif His l3-Hisl4-Glnl 5-Lysl6 in Aβ assemblies is not greatly altered in 80% ethanol.

Calculation of aqueous proton dissociation constants of quinoline and hydroxyquinolines: A comparison of solvation models

Absolute pKas of the hydroxyquinolines in aqueous solution are calculated through application of high level molecular orbital gas-phase Gibbs energies and solvation Gibbs energies using a variety of different methods. The gas-phase energies were obtained at the G3(MP2) level, while solvation energies were calculated using six different methods; the Langevin dipole (LD) and Poisson–Boltzmann (PB) methods, two semi-empirical quantum mechanical methods SM2 and SM5.42R/A, and the polarizable continuum models PCM and IEF-PCM. The pKas obtained using the SM2, SM5.42R/A and LD solvation energies showed significantly larger deviations from experiment (unsigned average deviations of 1.09–5.33) than the PB, PCM or IEF-PCM methods (unsigned average deviations of 0.46–1.60) for the calculation for amphoteric molecules. None of the methods studied appear to be able to accurately predict solvation energies of the zwitterionic species. For the dissociation constants involving these species, large deviations from experiment were observed with all models. The LD method performed least well, showing consistently large deviations from experiment.

Mechanistic Investigation on the Water Substitution in the η5-Organometallic Complexes Cp*Ir(H2O)32+and Cp*Rh(H2O)32+

The substitution of water in the half-sandwich complexes Cp*Rh(H2O)32+ and Cp*Ir(H2O)32+ (Cp* = η5-pentamethylcyclopentadienyl anion) by Cl-, Br-, I-, SCN-, py-CN (4-cyanopyridine), py-nia (nicotinamide), py (pyridine), TU (thiourea), and DMS (dimethylsulfide) was studied by stopped-flow spectroscopy at variable concentration, temperature, and pressure. The proton dissociation constants of the triaqua complexes, pKa = 6.47 (for rhodium) and pKa = 3.86 (for iridium), as well as the equilibrium constants for the formation of the dinuclear species (Cp*M)2(μ-OH)3+ were obtained by spectrophotometric titrations. The equilibrium constants K1 for the formation of the monosubstituted complexes Cp*M(H2O)2L+/2+, as determined for anionic and neutral ligands L, lie in the range 102−105 M-1 and follow the sequences K(Cl-) < K(Br-) < K(I-) and K(py-CN) < K(py-nia) < K(py) < K(TU,DMS). Assuming the Eigen−Wilkins mechanism for the formation of the monosubstituted complexes, second-order rate constants kf,1 were correcte...

Relationship among Redox Potentials, Proton Dissociation Constants of Pyrrolic Nitrogens, and in Vivo and in Vitro Superoxide Dismutating Activities of Manganese(III) and Iron(III) Water-Soluble Porphyrins

The log kcat values for the dismutation of O2•- by a series of monohydroxoiron(III) and aquamanganese(III) porphyrins, including ortho, meta, and para isomers of 5,10,15,20-tetrakis(N-alkylpyridiniumyl)porphyrins, were found to vary linearly with the metal-centered redox potentials (E1/2) for the M(III)/M(II) couple. Each 120 mV increase in E1/2 imparted a 10-fold increase in kcat. The observed behavior is in accord with the Marcus equation for outer-sphere electron-transfer reactions, suggesting that the same mechanism is operative for iron and manganese porphyrins. The Marcus plot enabled us to estimate the self-exchange rate constants of monohydroxoiron porphyrins to be ∼1 order of magnitude higher than those of aquamanganese porphyrins. Furthermore, E1/2 values for all of the metalloporhyrins investigated were linearly related to the acid dissociation constants (pKa3) of the pyrrolic nitrogen of the metal-free porphyrins, indicating that either E1/2, or the more readily measured pKa3, may be useful in predicting SOD activity in vitro. The most potent compounds investigated, with respect to SOD activity, are those of the ortho N-alkylpyridiniumyl series. Ortho N-alkylpyridiniumyl groups are more electron withdrawing than are the meta or para groups, thus imparting a more positive redox potential and a correspondingly higher SOD activity. Sufficiently positive potentials, or sufficiently low pKa3 values, are necessary for useful SOD activity, but so is the absence of toxicity. Despite their favorable redox potentials and SOD activities, all Fe(III) porphyrins investigated were toxic to Escherichia coli under both aerobic and anaerobic conditions and to both SOD-deficient and SOD-proficient strains. Only the ortho and meta manganese isomers of the N-alkylpyridiniumyl series (MnIIITE-2-PyP5+, MnIIITM-2-PyP5+ and MnIIITM-3-PyP5+) significantly protected SOD-deficient E. coli and allowed growth in an aerobic minimal medium. In previous work, we established that the lower toxicity of these compounds is due to diminished ability to bind to nucleic acids. The Mn(III) complexes are preferable to the Fe(III) complexes for SOD mimics possibly due to a lower tendency for axial ligation. We propose E1/2 ≥ +0.05 V vs NHE and/or pKa3 ≤ 2.0 as necessary requirements for Mn porphyrins to be considered useful SOD mimics.

The pH-dependent changes of intramolecular electron transfer on copper-containing nitrite reductase.

Electron transfer over 12.6 A from the type 1 copper (T1Cu) to the type 2 copper (T2Cu) was investigated in the copper-containing nitrite reductases from two denitrifying bacteria (Alcaligenes xylosoxidans GIFU 1051 and Achromobacter cycloclastes IAN 1013), following pulse radiolytical reduction of T1Cu. In the presence of nitrite, the rate constant for the intramolecular electron transfer of the enzyme from A. xylosoxidans decreased 1/2 fold to 9 x 10(2) s-1 (20 degrees C, pH 7.0) as compared to that for the same process in the absence of nitrite. However, the rate constant increased with decreasing pH to become the same (2 x 10(3) s-1) as that in the absence of nitrite at pH 6.0. A similar result was obtained for the enzyme from A. cycloclastes. The pH profiles of the two enzymes in the presence of nitrite are almost the same as that of the enzyme activity of nitrite reduction. This suggests that the intramolecular electron transfer process is closely linked to the following process of catalytic reduction of nitrite. The difference in redox potential (DeltaE) of T2Cu minus T1Cu was calculated from equilibrium data for the electron transfer. The pH-dependence of DeltaE was in accord with the equation: DeltaE = DeltaE(0)+0.058 log (Kr[H+]+[H+]2)/(K(0)+[H+]), where K(r) and K(0) are the proton dissociation constants for the oxidized and reduced states of T2Cu, respectively. These results raise the possibility that amino acid residues linked by the redox of T2Cu play important roles in the enzyme reaction, being located near T2Cu.

Novel Pentacyano Complexes of Tri- and Tetravalent Platinum.

New pentacyano complexes of tri- and tetravalent platinum were obtained in aqueous solution and characterized by multinuclear NMR ((195)Pt, (13)C) supported by Raman spectroscopy. The complexes form as products of redox decomposition of metal-metal bonded platinum-thallium compounds. The trimetallic [(NC)(5)Pt-Tl-Pt(CN)(5)](3)(-) yields a new dimeric compound of Pt(III), [(NC)(5)Pt-Pt(CN)(5)](4)(-). The latter is a rare representative of unbridged dimeric complexes of trivalent platinum; it was obtained through an oxidation of monomeric square-planar platinum(II) species by a metal complex. From the bimetallic compounds [(NC)(5)Pt-Tl(CN)(n)()](n)()(-) (n = 0-2) tetravalent platinum complexes are formed. Depending on the Pt-Tl species, electron transfer is initiated either by heat or by exposition to light; it results in [Pt(CN)(6)](2)(-) or in the hitherto unknown complexes [Pt(CN)(5)(OH)](2)(-) and [Pt(CN)(5)(H(2)O)](-), with the (195)Pt NMR chemical shift values 1638.7 (+/-0.6) and 1766.7 (+/-0.6), respectively. Proton dissociation constant of [Pt(CN)(5)(H(2)O)](-) has been determined, pK(a) = 2.51 (+/-0.01). In both Pt(III) and Pt(IV) pentacyano complexes platinum is hexacoordinated forming a pseudo-octahedron with two types of cyano ligands: four equivalent equatorial cyanides and one apical. Related platinum(IV) species, [Pt(CN)(5)X](2)(-) (X = Cl, Br, I), have also been studied. In all the pentacyano complexes a pronounced trans influence is reflected in a substantial difference between the (195)Pt-(13)C spin-spin coupling constant for the apical (trans) and the equatorial (cis) carbon sites. In this respect, the studied X ligands can be ordered in a series of decreasing (195)Pt-(13)C(trans) coupling constant: H(2)O > Cl(-) > Br(-) > I(-) > OH(-) > CN(-).

Transferrins--a mechanism for iron uptake by lactoferrin.

Iron uptake by bovine lactoferrin from nitrilotriacetatoFe(III) [FeN(Ac)3] in the presence of bicarbonate has been investigated at pH 7.1-8.7. Deprotonated apolactoferrin interacting with bicarbonate or carbonate extracts iron from nitrilotriacetatoFe(III); the direct second-order rate constant k1 = (4.90 +/- 0.20)x10(4) M(-1) s(-1), a reverse second-order rate constant k(-1) = (1.80+/-0.05)x10(5) M(-1) s(-1), and the iron-exchange equilibrium constant K1 = 0.25+/-0.05. The newly formed iron-protein complex loses a single proton with proton dissociation constant K3a = (17+/-0.5) nM, then undergoes a modification in its conformation followed by the loss of two or three protons; the first-order rate constant k2 = (1.0+/-0.10) s(-1). This induces a new modification in the conformation; the first-order rate constant k3 = (8.75+/-0.40)x10(-3) s(-1). This second modification in conformation controls the rate of iron uptake by the N site of the protein and is followed by a single proton loss; K5a = 8.0 nM. Finally, the holoprotein or the monoferric lactoferrin in their final equilibrated states are produced by a third modification in the conformation occurring in about 9000 s. The mechanism of iron uptake by lactoferrin is very similar to that of serum transferrin with a cooperativity between the C and N sites upon iron uptake but with lower rates, higher affinities and at least one more proton loss involved. These differences may be the result of slight discrepancies in the intimate structures of binding sites for serum transferrin and lactoferrin. In order to analyse the cooperativity between these iron-binding sites, the three-dimensional position of the chain of amino acid residues separating the N and C lobes of human apo-, holo- and dicopper-lactoferrin have been compared by the recognition of the three-dimensional shape dissimilarity program. The interlobe peptides of human hololactoferrin and apolactoferrin showed only 75.5 % tridimensional similarity, indicating that iron uptake affects the three-dimensional structure of the interlobe chain.

Proton Reactivity and Electronic Structure of Phenoxyl Radicals in Water

Application of proton reactivity in modeling the electronic structure of reactive chemical intermediates, interpreting their resonance Raman spectra in aqueous solution, and testing theoretical procedures has been demonstrated. The case of phenoxyl radicals (p-XC6H4O•), for which there has been persistent disagreement between the structural models derived from purely spectroscopic arguments and ab initio MO calculations, is discussed. It has been shown that the neutral p-aminophenoxyl (X = NH2) radical deprotonates in basic aqueous solutions (pKa = 14.5), implying a highly zwitterionic structure with formal positive charge on amine nitrogen, an amide-like CN bond (⩽1.33 Å), and near planarity of the molecular geometry. The radical protonates at oxygen, rather than nitrogen, the proton dissociation constant (pKa = 2.2) corresponding to ∼36% negative electronic charge on the O atom and a CO bond length of ∼1.267 Å. This chemical model of the radical structure contrasts with the ab initio UNO-CAS/6-31G* (pyramidal) and ROHF/[32/2] (planar) models which predict CO/CN bond lengths of 1.237/1.395 and 1.367/1.38 Å, respectively. Both ab initio structures relate to radical protonation on the nitrogen position and not the oxygen. In the proton reactivity models of para-substituted phenoxyl radicals, the CO bond length increases in the order F < H < CH3 ≪ OCH3 ∼ OH ≪ NH2 < O-, which is quite different from the UNO-CAS/6-31G* predicted order NH2 < OH ∼ Cl < H < F ≪ O-. The near equivalence of the NH2 and O- substitutions, observed in spectroscopic and electron-transfer properties of p-aminophenoxyl (X = NH2) and p-benzosemiquinone anion (X = O-) radicals in water, is readily explainable by the structural models derived from proton reactivity but not by the calculated structures. It has been established that solvation is primarily responsible for the observed substituent effects on the spectra, structure, and chemistry of phenoxyl radicals in water.

Ionization constants of water insoluble arylpropionic acids in aqueous N, N-dimethylformamide mixtures from potentiometric pH-titrations

The ionization constants of five water insoluble anti-inflammatory arylpropionic acids were evaluated in 80% V/V N, N-dimethylformamide aqueous mixture. From these values, data on proton dissociation constants in water have been computed and compared with the results previously reported by using different approaches.