Divalent paramagnetic counterions site binding in polyelectrolyte solutions. Analysis of the frequency dependence of the water protons magnetic relaxation and the characteristic parameters of "site binding"

Chemical shift and relaxation time measurements on the water protons in polyelectrolyte solutions containing divalent paramagnetic counterions have shown the existence of three types of counterions: - site bound with loss of water molecules and partial or complete release of the electrostriction in the first hydration sphere, - atmospherically trapped with no change in hydration, - free. The overall stoichiometry of the two former is in agreement with Manning's fraction of condensed counterions. A complete analysis of the frequency dependent contribution of site bound counterions to the water protons relaxation times leads us to interesting conclusions on the modifications of the first hydration shell and on the life time of site binding.     

Limiting-laws of polyelectrolyte solutions. Ionic distribution in mixed-valency counterions systems. I: The model

An extension of the counterion-condensation (CC) theory of linear polyelectrolytes has been developed for the case of a system containing a mixture of counterions of different valency, i and j. The main assumption in the derivation of the model is that the relative amount of the condensed counterions of the type i and j is strongly correlated and it is determined by the overall physical bounds of the system. The results predicted by the model are consistent, in the limiting cases of single species component, with those of the original CC theory. The most striking results are obtained for the cases of low charge density and excess of counterion species: in particular, an apparent positive "binding" cooperativity of divalent ions is revealed for small, increasing additions of M2+ ions to a solution containing a swamping amount of monovalent salt and a polyelectrolyte of low charge density. Apparent "competitive binding" of mono- and divalent ions derives as a bare consequence of the electrostatic interactions. Theoretical calculations of experimentally accessible quantities, namely single-(counter) ion activity coefficients, confirm the surprising predictions at low charge density, which qualitatively agree with the measured quantities.     

Thermodynamic properties of polyelectrolyte solutions containing mixtures of monovalent and trivalent counterions

The osmotic coefficients, heats of dilution, and volume changes on dilution of aqueous solutions containing mixtures of polystyrenesulfonic acid and its lanthanum salt have been determined at 25°C. The curve representing the osmotic coefficient as a function of the equivalent fraction of the acid has a maximum; the corresponding curves for the enthalpy and volume changes on dilution have a sigmoidal shape. Experimental results have been compared with predictions of the theory based on the cell model with cylindrical symmetry. A semiquantitative agreement between theory and experiment has been found.     

A new model of counterion condensation in polyelectrolyte solutions. III. Theoretical predictions of competitive condensation between counterions of different valences

Our model has been extended for theoretical estimation of competitive condensation of counterions of different valences onto polyelectrolytes in solution. The estimations are compared with those obtained from Manning theory and with experimental data on counterion activity coefficients. The agreement with the data for sodium polystyrenesulfonate/MgCl2, CaCl2 is satisfactory.     

Limiting-laws of polyelectrolyte solutions. Ionic distribution in mixed-valency counterions systems. II. A comparison of conductometric data and theoretical predictions

The competitive binding of monovalent and divalent counterions (M+ and M2+, respectively) has been studied by a conductometric procedure as described by De Jong et al. (Biophysical Chemistry 27 (1987) 173) for aqueous solutions of alkali metal polymethacrylates in the presence of Ca (NO3)2 or Mg(NO3)2. The experimentally obtained fractions of conductometrically free counterions are compared with theoretical values computed according to a new thermodynamic model recently developed by Paoletti et al. (Biophysical Chemistry, 41 (1991) 73). For the systems studied, the fractions of free monovalent and divalent counterions can be fairly well described by the theory. In fact, the results support the assumption that under the present conditions the conductometrically obtained distribution parameters (l) and (2) approximate the equilibrium fractions of free monovalent and divalent counterions. For a degree of neutralization of 0.8 and a molar concentration ratio of divalent counterions and charged groups on the polyion up to 0.25, the mean M+/M2+, exchange ratio nu has been found to be 1.39 +/- 0.03 and 1.33 +/- 0.03 for the alkali metal/Ca/PMA and alkali metal/Mg/PMA systems, respectively. These values agree well with the theoretical value, which for this particular case is 1.38.     

Relaxation and electrophoretic effects in polyelectrolyte solutions. I. Salt-free solutions

Relations between relaxation and electrophoretic effects in salt-free polyelectrolyte solutions are studied in terms of nonequilibrium thermodynamics and of binary friction coefficients. The significance and the method of determining these coefficients are explained. Consideration of experimental results and of long- and short-range interactions suggests that the concept of “ion condensation” is needed to provide a consistent desciption of transport processes for linear flexible polyelectrolytes. Empirical relations previously used are shown to have only limited validity.     

Electron paramagnetic resonance characterization of membrane bound iron-sulfur clusters and aconitase in plant mitochondria

Electron paramagnetic resonance (EPR) characteristics of the iron-sulfur clusters of potato tuber mitochondria have been examined in various subfractions of the mitochondria. We confirm that EPR signals comparable to those of the iron-sulfur proteins of mammalian mitochondria respiratory complexes are also present in plant mitochondria. Two distinct iron-sulfur centers paramagnetic in the oxidized state exhibit signals which differ in their detailed line shape and field position. One of these which is present in the inner membrane corresponds to center S.3. The EPR spectrum of the soluble fraction revealed the presence of another center with a low field maximum at g = 2.03 and is associated with aconitase. The EPR signal observed in the mitochondrial matrix from potato tuber and characteristic of 3Fe cluster is significantly changed in shape after addition of citrate and differs clearly from the spectrum of pig heart mitochondrial aconitase. The aconitase in plant mitochondria differs from that of mammalian mitochondria by several features.     

Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison between internal microviscosity of low-density erythrocytes and the microviscosity of hemoglobin solutions: an electron paramagnetic resonance study.

The hypothesis that the internal viscosity of erythrocytes is governed by the intracellular hemoglobin (Hb) concentration is examined. Here viscosity is determined by labeling of the cytoplasmic reduced glutathione with the spin label maleimido-Tempo. Erythrocyte populations with different Hb concentrations in isosmotic conditions were obtained through incomplete lysis, followed by cell resealing, and discontinuous density gradient separation. This procedure maintains normal cell shape and volume. Microviscosity of membrane-free Hb solutions was measured by addition of spin labeled glutathione. It was found that microviscosity values are similar for the erythrocyte cytoplasm and for Hb solutions of equivalent concentrations, showing that the erythrocyte membrane does not have any influence on internal microviscosity. The dependence of the microviscosity on the concentration of Hb solutions was compared with results of macroscopic viscosity obtained by other authors. It is concluded that microviscosity is sensitive to individual properties of the Hb molecule (intrinsic viscosity), but that it is not sensitive to intermolecular interactions. As the microviscosity behavior as a function of Hb concentration is the same in Hb solutions as in the erythrocyte cytoplasm, the inferences regarding macroscopic viscosity in Hb solutions could be translated to the rheological properties of the erythrocyte cytoplasm. Thus, these properties could be predicted from the values of the mean corpuscular Hb concentration.     

Magnetic resonance studies of the binding site interactions between phosphorylcholine and specific mouse myeloma immunoglobulin

The interaction of phosphorycholine-binding mouse myeloma protein M603 and the isotopically substituted hapten phosphoryl[methyl-13C] choline has been investigated using 13C and 31P nuclear magnetic resonance (NMR) spectroscopy. Upon binding to antibody, upfield shifts of 0.7 and 1.5 ppm are observed for the hapten 13C and 31P resonances, respectively, and both spectra are in the "slow" exchange limit. Linewidth analysis indicates some immobilization of the phosphate group but essentially unrestricted methyl group rotation for the bound hapten. Hapten-antibody dissociation rate constants of 10 and 38 s-1 are calculated from 13C and 31P NMR spectra, respectively, suggesting the possibility of differential dissociation rates for the two opposing ends of the phosphorylcholine molecule. The NMR data are entirely consistent with the known x-ray structure of the M603 Fab'-phosporylcholine complex (Segal,D.M., Padlan, E.A., Cohen G.H., Rudikoff S., Potter,M., and Davies, D.R. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 4298).     

Magnetic circular dichroism and electron paramagnetic resonance studies of iron(II)-metallothionein

The electronic and magnetic properties of the Fe(II)-thiolate centers in Fe(II)-metallothionein have been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance spectroscopies at various levels of Fe(II) incorporation. In agreement with previous results [Good, M., & Vasák, M. (1986) Biochemistry 25, 8353-8356], rabbit liver metallothionein was found to bind a maximum of seven Fe(II) ions, with cluster formation occurring when more than four Fe(II) ions are bound at pH 8.5. The results indicate that all the iron in fully loaded Fe(II)-metallothionein is accommodated in Fe(II)-thiolate clusters that have either S = 0 or S = 2 ground states as a result of antiferromagnetic coupling between high-spin Fe(II) ions. By analogy with the cluster composition and mechanism of assembly that have been established for other divalent metal ions, the clusters with S = 0 and S = 2 ground states are attributed to tetranuclear and trinuclear centers, respectively. EPR signals indicative of S = 2 species were observed for samples containing monomeric tetrathiolate-Fe(II) centers and trinuclear Fe(II)-thiolate clusters. However, the nature of the zero-field splitting of the S = 2 ground states that is indicated by the EPR signals is not consistent with that deduced from M?ssbauer and magnetic circular dichroism studies, suggesting heterogeneity in both types of center.     

Magnetic resonance investigation of ionizable residues at the active site of thermolysin.

The details of the pH dependence of the thermodynamic and magnetic interactions of the active-site region of thermolysin in which manganese has replaced the active-site zinc atom and the inhibitor N-trifluoroacetyl-D-phenylalanine have been examined. These show a number of ionizable groups in the active-site region. A cooperative displacement of manganese at the catalytic site is observed as pH is lowered. This appears to be the result of the protonation of histidine-142 and -146 which act as metal ligands. The metal is 50% displaced at pH 6.0. At higher pH values, the environment of the bound manganese changes as a result of the ionization of at least two groups of approximate pKa = 8.5 and 9.5. These values are assigned to tyrosine-157 and to the water molecule which acts as a metal ligand at the active site. The binding behavior of the inhibitor strongly suggests that two molecules of inhibitor bind to the enzyme. The weaker site is competitive with the synthetic substrate FAGLA (furylacryloylglycyl-leucinamide), while the strong site has no effect on FAGLA hydrolysis. This second site is in the vicinity of the active site with a distance of 8 A or less between the trifluoromethyl group and manganese bound at the active site.     

Magnetic circular dichroism and electron paramagnetic resonance studies of hydrogenases I and II from Clostridium pasteurianum

The two iron-only hydrogenases (I and II) from Clostridium pasteurianum have been investigated by variable temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies. Samples were studied both reduced with dithionite under an atmosphere of H2 and after oxidation with thionine. The results are consistent with four and two [4Fe-4S]1+,2+ (F)-clusters in hydrogenases I and II, respectively. All four F-clusters are reduced and paramagnetic in reduced hydrogenase I, with up to one exhibiting an S = 3/2 ground state and the remainder having conventional S = 1/2 ground states. Both F-clusters have S = 1/2 ground states in reduced hydrogenase II; however, one appears to be only partially reduced under the conditions used for reduction. MCD studies of the oxidized enzymes show no temperature-dependent features in the visible region which can be attributed to the EPR-active S = 1/2 hydrogen-activating cluster, suggesting predominantly oxygen and nitrogen coordination for the iron atoms of this center. However, temperature-dependent MCD transitions arising from a hitherto undetected S greater than 1/2 Fe-S clusters are apparent in both oxidized hydrogenases. Detailed EPR studies of oxidized hydrogenase I revealed resonances from an S = 3/2 species, however, spin quantitation reveals this to be a trace component that is unlikely to be responsible for the observed low temperature MCD spectrum. The nature and origin of these S greater than 1/2 Fe-S clusters are discussed in light of the available spectroscopic data for these and other iron-only hydrogenases.