The relation between activity and zinc and chloride binding of Escherichia coli alkaline phosphatase.

The relation between Zn²⁺ binding of E. coli alkaline phosphatase and enzymatic activity and anion binding (using ³⁵Cl NMR) has been investigated. The results suggest the existence of two forms of the enzyme with different zinc binding properties. The anion binding associated with the enzyme's function appears to be an amino acid residue and not the Zn²⁺ ions; furthermore, there is a rapid internal motion at the anion binding site. ³⁵Cl relaxation studies in the presence of Mg²⁺ ions point to a marked interdependence of Mg²⁺ and Zn²⁺ binding.     

127I-NMR studies of anion binding to κ-carrageenan

A preliminary ¹²⁷I-NMR relaxation study of the binding of the I^? ion to κ-carrageenan is presented. Signal loss caused by motional correlation times out of extreme narrowing makes it difficult to analyze line widths. Longitudinal relaxation times are more suitable for a comprehensive relaxation study. From the observation of nonextreme motional narrowing, the lower limit of the residence time at the binding site is estimated to be on the order of 10 ns. © 1993 John Wiley & Sons, Inc.     

The pulse radiolysis pH-jump in aqueous solutions: applications to lanthanide(III)-methyl red systems

Use of the pulse radiolytic technique for studying acid-base and complex formation reactions in aqueous solutions is described. The method is based on the conversion of the hydrated electron e⁻_aq to the anion of a strong acid, thereby obtaining a fast pH-jump. Rate constants are calculated from relaxation times measured with either spectrophotometric or conductometric detection systems. The technique has been validated with previously studied chemical systems. Rate constants for the complex formation between La³⁺ and Gd³⁺ with Methyl Red indicator have also been determined.     

Notice

¹⁷O NMR studies of ¹⁷O enriched water solutions containing superoxide dismutase have been performed between pH 7.5 and 11.7. Whereas T₁ measurements do not reveal any interaction between ¹⁷O and the paramagnetic copper center, the linewidth results appreciably increased with increasing pH with an apparent pK_a of 11.3. Comparison with ¹H NMR relaxation studies allows to interpret the present data as due to binding to the copper ion of an OH^? anion at high pH. The binding position should be of “equatorial” type, not involving the binding position of the coordinated water.     

Kinetics of monensin complexation with sodium ions by 23Na NMR spectroscopy

The kinetics of the sodium binding to the ionophore monensin (Mon) in methanol has been studied by ²³Na NMR spectroscopy. Fast quadrupole relaxation of the bound sodium affected the relaxation rate of the free sodium through an exchange process between these two species. The exchange was found to be dominated by the reaction: Na⁺ + Mon^? ? MonNa. The dissociation rate constant at 25°C is 63 s^?1, with an activation enthalpy of 10.3 $\text{kcal}\text{mol}$ and activation entropy of ?15.8 $\text{cal}\text{mol}$ deg. These results indicate that the specificity of the binding of sodium ions to monensin is reflected in the relatively slow dissociation process. The entropy changes indicate that the activated monensin-sodium complex undergoes a conformational change, but the existence of a conformational change in monensin anion prior to complexation is excluded.     

Conformational and kinetic features of the morphine-Mn(II) interaction as probed by nuclear paramagnetic resonance investigations

The nature of binding between manganese ions and morphine was studied using Fourier transform proton nuclear magnetic resonance techniques. Proton relaxation times in the presence of Mn(II) ions were determined together with their temperature dependence. Slow exchange conditions were observed for the NCH₃ group, while fast exchange conditions applied for all the other protons. The rotational correlation time of the complex was approximated by that of the free morphine molecule, as measured by selective and nonselective proton relaxation rate measurements. The distances between the metal ion and proton nuclei of morphine were evaluated on the basis of an association constant, measured from water proton spin-lattice relaxation rate binding studies. The results indicate that the metal binds directly to the two oxydryls with K_ass = 9.7 × 10^?3.The rate constant for the interaction of Mn(II) with the opiate is 2.25 × 10⁴ sec^?1 at 27°C, as determined from the temperature dependence of longitudinal relaxation rate of the NCH₃ group.     

DNA replication of SV40-infected cells. VII. Formation of SV40 catenated and circular dimers

The binding of methyl isonitrile (CH₃Nandz.tbnd;C) to hemoglobin β chains has been studied by measuring the ¹H nuclear magnetic resonance transverse relaxation times for methyl isonitrile as a function of protein concentration, temperature and ¹⁴N decoupling. Binding of methyl isonitrile both at the heme iron and at a non-specific site (or sites) has an effect upon the measured nuclear spin relaxation times. The results yield a value of 57 ± 12 seconds^?1 (20 °C) for the “off” rate constant K_?1 for specific binding and an Arrhenius activation energy for k_?1 of 14 ± 3 kcal mol^?1.     

Pulsed nuclear magnetic resonance studies on 23 Na, 7 Li and 35 Cl binding to human oxy- and carbon monoxyhaemoglobin

Pulsed nuclear magnetic resonance studies of the longitudinal (T₁) and transverse (T₂) quadrupolar relaxation times of ⁷Li, ²³Na, ³⁵Cl ions in the absence and presence of human oxy- and carbon monoxyhaemoglobin have been used to investigate the interaction of the ions and the macromolecule.The relaxation data show that Cl^? interacts strongly with the haemoglobin but provide no evidence for binding of Na⁺ up to concentrations of 0.5 m. In the case of Li⁺, evidence for interaction is obtained at concentrations of about 0.1 m.The dependence of relaxation rate on frequency was followed over a limited frequency range in an attempt to separate the effects of correlation times and exchange rates of the bonded ions on the relaxation. In the case of Cl^?, an upper limit for the mean lifetime divided by the number of sites can be set at about 1 × 10^?6 second, and a lower limit at about 1 × 10^?8 second.     

The effect of ionic strength on the kinetics of fluoride binding to cytochrome c peroxidase.

The kinetics of the reaction between cytochrome c peroxidase and fluoride was investigated as a function of ionic strength over the pH range 4 to 8. The ionic strength was varied between 0.01 and 0.10 m. At 0.01 m ionic strength, the reaction rates were determined between pH 2.7 and 9.2. A consideration of the ionic strength and pH dependence of the association rate constant for the fluoride-cytochrome c peroxidase reaction leads to the conclusion that hydrofluoric acid is the only significant reactive form of the ligand between pH 2.5 and 8. Above pH 8, binding of fluoride anion contributes to the apparent association rate even though the pH-independent rate constant for fluoride anion binding is more than 3 × 10⁵ times smaller than the rate constant for hydrofluoric acid binding.     

Dielectric relaxation studies of ionic processes in lysolecithin-packaged gramicidin channels

Summary Dielectric permittivities have been determined for suspensions of lysolecithin packaged malonyl gramicidin channels over the frequency range of 5kHz to 900 MHz and under conditions of approximately equimolar concentrations (10mM) of channels and salts. The salts were lithium chloride, sodium chloride and thallium acetate. A relaxation process unique to the thallium acetate-channel system was observed which on analysis gave rise to a relaxation time at 25⁰ of 120 nsec. The permittivity data, as well as a comparison of binding constants, indicate that the relaxation process results from Tl⁺ being bound within the channel and more specifically from an intrachannel ion translocation with a rate constant of approximately 4×10⁶ sec^–1 and with an energy of activation of less than 6.7 kcal/mole. These data compare favorably with data from conductance studies on planar bilayers and with ion and carbon-13 nuclear magnetic studies on the lysolecithin packaged malonyl gramicidin channels which combine to indicate that the relaxation process is due to the jump of the thallium ion across a central barrier.     

Relaxation Kinetics of Lipid Membranes and Its Relation to the Heat Capacity

We investigated the relaxation behavior of lipid membranes close to the chain-melting transition using pressure jump calorimetry with a temperature accuracy of ∼10^-3 K. We found relaxation times in the range from seconds up to about a minute, depending on vesicular state. The relaxation times are within error proportional to the heat capacity. We provide a statistical thermodynamics theory that rationalizes the close relation between heat capacity and relaxation times. It is based on our recent finding that enthalpy and volume changes close to the melting transition are proportional functions.     

Interactions of DNA with divalent metal ions. II. Proton relaxation enhancement studies

The extent and modes of binding of the divalent metal ions Mn²⁺ and Co²⁺ to DNA and the effects of salt on the binding have been studied by measurements of the effects of these paramagnetic metal ions on the longitudinal and transverse relaxation rates of the protons of the solvent water molecules, a technique that is sensitive to overall binding. The number of water molecules coordinated to the DNA–bound Mn²⁺ and Co²⁺ is found to be between five and six, and the electron spin relaxation times and the electron-nuclear hyperfine constants associated with Mn²⁺ and Co²⁺ are little or not affected by the binding. These observations indicate little disturbance of the hydration sphere of Mn²⁺ and Co²⁺ upon binding to DNA. An average 2–3-fold reduction in the exchange rate of the water of hydration of the bound metal ions and an order-of-magnitude increase in their rotational correlation time are attributed to hydrogen-bond formation with the DNA. The binding constants of Mn²⁺ to DNA, at metal concentrations approaching zero, are found to be inversely proportional to the second power of the salt concentration, in agreement with the predictions of Manning's polyelectrolyte theory. A remarkable quantitative agreement with the polyelectrolyte theory is also obtained for the anticooperativity in the binding of Mn²⁺ to DNA, although the experimental results can be well accounted for by another simple electrostatic model. The various modes of binding of divalent metal ions to DNA are discussed.     

35-Cl nuclear magnetic resonance studies of anion-binding sites in proteins: lactate dehydrogenase.

³⁵Cl nmr relaxation rate measurements have been used to study anion-binding sites in pig heart lactate dehydrogenase. These studies reveal two types of sites, one is intimately associated with the active site, the other is not. The nonactive site has been ascribed to a subunit site in analogy with crystallographic results from the dogfish M₄ enzyme. The binding of either the reduced or the oxidized form of NAD results in an increase in the ³⁵Cl nmr relaxation rate by a factor of 1.8–2. The enhanced nmr relaxation rate of the binary lactate dehydrogenase-NAD complex is reduced on binding of the substrate inhibitor molecules oxamate or oxalate to a value less than that exhibited by lactate dehydrogenase alone. The enhancement of the nmr relaxation rate is attributed to a decrease in the dissociation constant of Cl for the enzyme. The K_p values for Cl binding to the active center site of lactate dehydrogenase is 0.85 m and for lactate dehydrogenase-NADH is 0.25 m. The ratio of these constants, 3.4, agrees well with the measured enhancement value 3.7. The effect of coenzyme analogs on the ³⁵Cl nmr relaxation rate has been examined. 3-Acetylpyridine NAD produces an enhancement of 4.3, thionicotinamide NAD of 2.3, whereas 3-pyridinealdehyde, adenosinediphosphoribose, and adenosine diphosphate do not affect the nmr relaxation state of Cl bound to lactate dehydrogenase.     

1H NMR Relaxometry in Natural Humous Soil Samples: Insights in Microbial Effects on Relaxation Time Distributions

¹H NMR relaxometry is applied for the investigation of pore size distributions in geological substrates. The transfer to humous soil samples requires the knowledge of the interplay between soil organic matter, microorganisms and proton relaxation. The goal of this contribution is to give first insights in microbial effects in the ¹H NMR relaxation time distribution in the course of hydration of humous soil samples. We observed the development of the transverse relaxation time distribution of the water protons after addition of water to air dried soil samples. Selected samples were treated with cellobiose to enhance microbial activity. Besides the relaxation time distribution, the respiratory activity and the total cell counts were determined as function of hydration time. Microbial respiratory activities were 2–15 times higher in the treated samples and total cell counts increased in all samples from 1×10⁹ to 5×10⁹ cells g⁻¹ during hydration. The results of ¹H NMR relaxometry showed tri-, bi- and mono-modal relaxation time distributions and shifts of peak relaxation times towards lower relaxation times of all investigated soil samples during hydration. Furthermore, we found lower relaxation times and merging of peaks in soil samples with higher microbial activity. Dissolution and hydration of cellobiose had no detectable effect on the relaxation time distributions during hydration. We attribute the observed shifts in relaxation time distributions to changes in pore size distribution and changes in spin relaxation mechanisms due to dissolution of organic and inorganic substances (e.g. Fe³⁺, Mn²⁺), swelling of soil organic matter (SOM), production and release of extracellular polymeric substances (EPS) and bacterial association within biofilms.     

15N relaxation study of the cold shock protein CspB at various solvent viscosities

For a detailed NMR study of the dynamics of the cold shock protein CspB from Bacillus subtilis, we determined ¹⁵N transverse and longitudinal relaxation rates and heteronuclear nuclear Overhauser effects at different solvent viscosities. Up to a relative viscosity of 2, which is equivalent to 27% ethylene glycol (EG), the overall correlation time follows the linear Stokes-Einstein equation. At a relative viscosity of 6 (70% EG) the correlation time deviates from linearity by 30%, indicating that CspB tumbles at a higher rate as expected from the solvent viscosity probably due to a preferential binding of water molecules at the protein surface. The corresponding hydrodynamic radii, determined by NMR diffusion experiments, show no variation with viscosity. The amplitudes of intramolecular motions on a sub-nanosecond time scale revealed by an extended Lipari–Szabo analysis were mainly independent of the solvent viscosity. The lower limit of the NMR `observation window' for the internal correlation time shifts above 0.5 ns at 70% EG, which is directly reflected in the experimentally derived internal correlation times. Chemical exchange contributions to the transverse relaxation rates derived from the Lipari-Szabo approach coincide with the experimentally determined values from the transverse ¹H-¹⁵N dipolar/¹⁵N chemical shift anisotropy relaxation interference. These contributions originate from fast protein folding reactions on a millisecond timescale, which get retarded at increased solvent viscosities.     

35Cl nuclear magnetic resonance study of zinc and phosphate binding of E. coli alkaline phosphatase

³⁵Cl^? quadrupole relaxation was measured in the presence of metal-free alkaline phosphatase and in the presence of Zn²⁺-alkaline phosphatase. The relaxation data show that for an enzyme containing the minimum amount of zinc needed for full activity—2 g atoms of zinc per mole of protein—there appears to be no binding of halide ions to the protein-bound zinc ions. In contrast, when there is a high metal-enzyme ratio, a large relaxation enhancement is observed, demonstrating coordination of halide ions to the metal ions.Addition of inorganic phosphate causes no change in the ³⁵Cl^? relaxation in the presence of metal-free enzyme. However, marked decreases in relaxation are observed upon addition of phosphate to the Zn²⁺-alkaline phosphatase. The relaxation measurements carried out in the presence of phosphate show that substrate binding does prove to be metal-ion dependent. Furthermore, experiments with inorganic phosphate suggest the tight binding of one phosphate to the alkaline phosphatase.     

Kinetics of the co-operative reaction of Helix pomatia hemocyanin with oxygen. Oxygen binding at low and intermediate oxygen saturations

The kinetics of oxygen binding of Helix pomatia α-hemocyanin has been studied at low and intermediate levels of ligand saturation, under conditions in which oxygen binding is highly co-operative. Temperature-jump relaxation spectra are heterogeneous and can be resolved into a slow and a fast phase. The latter is related to a bimolecular reaction, i.e. the binding of oxygen. At very low degrees of fractional saturation (<0.15) the reactant concentration-dependence of the faster relaxation rate allows the combination and dissociation rate constants of the low affinity or T-state to be estimated as 1.3 × 10⁶m^?1 s^?1 and 300 s^?1, respectively. A possible interpretation of the slow component in the relaxation spectrum is discussed.In stopped-flow experiments, after mixing deoxyhemocyanin with oxygen-containing buffer, most of the binding process to the T-state is lost in the dead time. The observed initial rates of oxygen binding are between 15 and 120 s^?1. depending on the oxygen concentration, and may reflect the rate of the allosteric change from a low to a high affinity state (T→R transition), which is slower than oxygen binding.Similarities and differences in the overall kinetic properties of small and giant respiratory proteins, i.e. hemoglobin and hemocyanin, are discussed.     

A novel technique for identification of sites of anion transport in intact cells and tissues using a fluorescent probe

Abstract The purported blocker of anion transport 4, 4′ di-isothiocyano-2-2′ stilbene disulfonate (DIDS) has been shown to partially inhibit ³⁶Cl^? influx, ³⁶CIO^?₃ influx and ³⁵SO^2?₄ influx into Pisum salivum L. cv. Feltham First seedlings. This inhibitory effect could be prevented by pretreatment with the respective unlabelled medium. There was no effect of DIDS on ¹⁴C methylamine influx. The results are consistent with the hypothesis that the binding of DIDS to the site of anion-carrier interaction is responsible for its observed inhibitory effects on anion fluxes. The fluorescent properties of DIDS upon binding to membrane proteins was exploited in an attempt to examine the major sites of anion pumping in whole roots. The results show clearly that in the presence of DIDS the epidermal layers became brightly fluorescent, while cortical layers did not fiuoresce. Lycopersicum esculentum cells taken from locular fluid were plasmolysed using sucrose solution, and the patterns of fluorescence in the presence of DIDS showed in an unambiguous way that the fluorescence is associated with cell membranes. The potential usefulness of this technique to probe sites of anion transport in whole plants and tissues is discussed.     

Specificity of Na+ binding to phosphatidylserine vesicles from A 23Na NMR relaxation rate study

²³Na NMR relaxation rate measurements show that Na⁺ binds specificially to phosphatidylserine vesicles and is displaced partially from the binding site by K⁺ and Ca²⁺ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na⁺ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na⁺-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na⁺) fall in the range 0.4–1.2 M^?1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na⁺ at 0.1 M Na⁺ concentration.