Solomon-Bloembergen Equations Research Articles

Allosteres to regulate neurotransmitter sulfonation

Catecholamine neurotransmitter levels in the synapses of the brain shape human disposition-cognitive flexibility, aggression, depression, and reward seeking-and manipulating these levels is a major objective of the pharmaceutical industry. Certain neurotransmitters are extensively sulfonated and inactivated by human sulfotransferase 1A3 (SULT1A3). To our knowledge, sulfonation as a therapeutic means of regulating transmitter activity has not been explored. Here, we describe the discovery of a SULT1A3 allosteric site that can be used to inhibit the enzyme. The structure of the new site is determined using spin-label-triangulation NMR. The site forms a cleft at the edge of a conserved ∼30-residue active-site cap that must open and close during the catalytic cycle. Allosteres anchor into the site via π-stacking interactions with two residues that sandwich the planar core of the allostere and inhibit the enzyme through cap-stabilizing interactions with substituents attached to the core. Changes in cap free energy were calculated ab initio as a function of core substituents and used to design and synthesize a series of inhibitors intended to progressively stabilize the cap and slow turnover. The inhibitors bound tightly (34 nm to 7.4 μm) and exhibited progressive inhibition. The cap-stabilizing effects of the inhibitors were experimentally determined and agreed remarkably well with the theoretical predictions. These studies establish a reliable heuristic for the design of SULT1A3 allosteric inhibitors and demonstrate that the free-energy changes of a small, dynamic loop that is critical for SULT substrate selection and turnover can be calculated accurately.

Immersion Depths of Lipid Carbons in Bicelles Measured by Paramagnetic Relaxation Enhancement.

Myriads of biological processes occur in or at cellular lipid membranes. Knowledge about the localization of proteins, lipids, and other molecules within biological membranes is thus crucial for the understanding of such processes. Here, we present a method to determine the immersion depths of lipid carbon atoms in membranes by paramagnetic relaxation enhancement (PRE) caused by the presence of doxylated lipids. As membrane mimetics, we employ small isotropic bicelles made of synthetic lipids and of natural Escherichia coli phospholipid extract. Bicelles are particularly suitable for solution state NMR since they maintain a lipid bilayer while they are at the same time amenable to solution state NMR experiments. PREs were measured in the presence of different doxylated lipids with the nitroxide radical located in the headgroup and at various positions in the acyl chain. Theoretical PREs were calculated assuming a simple bicelle model using the Solomon-Bloembergen equations. Immersion depths of the lipid carbon atoms were obtained by a least-squares fit of the theoretical to the experimental PREs. The carbon immersion depths correspond well to results obtained by other methods and differences do not exceed 3-5 Å. This means that the method presented here provides sufficient resolution to distinguish the localization of carbons in different regions of the lipid bilayer, despite considerable simplifications of the underlying theory. These simplifications include a simple form of the spectral density function, which we find is sufficient to reliably determine immersion depths. A more complicated spectral density function that includes bicelle, lipid, and local motions may only improve the results if its parametrization is good enough. The approach presented here may be extended to the determination of protein localization in membranes employing realistic membrane mimetics like the bicelles made of E. coli phospholipid extract used here.

NMR Spectra Transformed by Electron-Nuclear Coupling as Indicator of Structural Peculiarities of Magnetically Active Molecular Systems.

The peculiarities of nuclear spin relaxation in the paramagnetic systems have been analyzed taking into account the exchange processes. The analysis is based on the modified Solomon-Bloembergen equations. In this line, the conditions of detecting of the NMR signals of samples are discussed depending on resonance frequency of the NMR spectrometer and characteristic relaxation time. On this basis, (1)H NMR spectra of cobalt semiquinolate complex have been analyzed. It has been shown that the satellite signals observed in the spectrum are caused by hyperfine coupling of the tert-butyl group protons with α and β states (localized on pz orbital of the aromatic carbon) of unpaired electron spin. The relaxation process of the resonance protons is controlled by paramagnetic dipole-dipole coupling. The contact hyperfine coupling does not contribute to the paramagnetic broadening. A mechanism involving paramagnetic molecular structures, which are responsible for intramolecular exchange processes in the cobalt semiquinolate complex, is given.

Nature of Polyoxometalate Intramolecular Coordination to Quaternary Ammonium Salts from Paramagnetic Relaxation Enhancement

Polyoxometalates (POMs) exhibit catalytic activity toward a variety of harmful chemicals such as chemical warfare agents, qualifying them as promising candidates as additives to create self-decontaminating surfaces and materials. However, POMs exhibit poor solubility and dispersion behavior in organic matrices, including polymeric coatings. In an effort to improve compatibility with polymer coatings and impart surface segregating behavior, we describe the encapsulation of a Ni(II)-containing POM, α2-K8P2W17O61(Ni2+·OH2)·17H2O (Ni-POM), with a series of amphiphilic alkyl ethoxy dimethyl quaternary ammonium salts (QASs) and elucidate their structural coordination. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA) were utilized to confirm that QASs are coordinated to the Ni-POM and that the average number of QAS coordinated to each Ni-POM increases with increasing alkyl moiety length. The 1H NMR spectra of the QAS(Ni-POM) complexes show marked site-specific broadening and reduced spin–lattice relaxation times T1 compared to either a nonparamagnetic QAS(POM) complex or the neat QAS ligand. These paramagnetic relaxation enhancement (PRE) effects were used to obtain structural and dynamical information about the binding of QASs to the Ni-POM. The single-exponential saturation recovery behavior observed in all cases indicated that all bound QAS molecules are rapidly moving about the entire (Ni-POM) surface on a time scale less than tens of milliseconds. Motionally averaged distances of the QAS protons to the paramagnetic Ni2+ center were estimated using a modified Solomon–Bloembergen equation. Comparisons of relative distances for protons at different sites on the QAS molecule provide key insights into the structural nature of the bonding. Surprisingly, the ethylene oxide moiety of the amphiphilic QAS was found to coordinate more closely with the surface of the Ni-POM than the quaternary ammonium nitrogen cation, and the alkyl moieties extended outward from the Ni-POM center. These results suggest that QAS(Ni-POM) complexes should behave effectively as a hydrophobic nonpolar complexes in their desired roles as catalytic centers in coatings.

Interaction of H2@C60 and Nitroxide through Conformationally Constrained Peptide Bridges

We synthesized two molecular systems, in which an endofullerene C60 , incarcerating one hydrogen molecule (H2 @C60 ) and a nitroxide radical are connected by a folded 310 -helical peptide. The difference between the two molecules is the direction of the peptide orientation. The nuclear spin relaxation rates and the para → ortho conversion rate of the incarcerated hydrogen molecule were determined by (1) H NMR spectroscopy. The experimental results were analyzed using DFT-optimized molecular models. The relaxation rates and the conversion rates of the two peptides fall in the expected distance range. One of the two peptides is particularly rigid and thus ideal to keep the H2 @C60 /nitroxide separation, r, as large and controlled as possible, which results in particularly low relaxation and conversion rates. Despite the very similar optimized distance, however, the rates measured with the other peptide are considerably higher and thus are compatible with a shorter effective distance. The results strengthen the outcome of previous investigations that while the para → ortho conversion rates satisfactorily obey the Wigner's theory, the nuclear spin relaxation rates are in excellent agreement with the Solomon-Bloembergen equation predicting a 1/r(6) dependence.

Predicting the paramagnet-enhanced NMR relaxation of H2encapsulated in endofullerene nitroxides by density-functional theory calculations

We have investigated the structure and nuclear magnetic resonance (NMR) spectroscopic properties of some dihydrogen endofullerene nitroxides by means of density-functional theory (DFT) calculations. Quantum versus classical roto-translational dynamics of H₂ have been characterized and compared. Geometrical parameters and hyperfine couplings calculated by DFT have been input to the Solomon-Bloembergen equations to predict the enhancement of the NMR longitudinal relaxation of H₂ due to coupling with the unpaired electron. Estimating the rotational correlation time via computed molecular volumes leads to a fair agreement with experiment for the simplest derivative; the estimate is considerably improved by recourse to the calculation of the diffusion tensor. For the other more flexible congeners, the agreement is less good, which may be due to an insufficient sampling of the conformational space. In all cases, relaxation by Fermi contact and Curie mechanisms is predicted to be negligible.

Predicting the 1H and 13C NMR spectra of paramagnetic Ru(III) complexes by DFT

Nuclear shieldings, including the Fermi-contact and pseudocontact terms, have been calculated with density functional theory (DFT) (nonrelativistic and relativistic) methods in several Ru(III) complexes, thereby predicting (1)H and (13)C paramagnetic shifts. A fair agreement with experimental values is observed. Structural, magnetic and dynamic parameters have also been input to the Solomon-Bloembergen equation in order to predict signal lineshapes. It is shown that DFT-predicted paramagnetic shifts can greatly aid in obtaining and understanding NMR spectra of paramagnetic Ru(III) complexes.

Predicting the NMR Spectra of Paramagnetic Molecules by DFT: Application to Organic Free Radicals and Transition‐Metal Complexes

Nuclear shieldings, including the Fermi contact and pseudocontact terms, have been calculated with DFT methods in a variety of open-shell molecules (nitroxides, aryloxyl and various transition-metal complexes), thereby predicting (1)H and (13)C chemical shifts. In general, when experimental data are reliable a good agreement with experimental values is observed, thus demonstrating the predictive power of DFT also in this context. However, the general accuracy is lower than that for closed-shell species. A few inconsistencies in literature values are reconciled by reassigning some shifts. Structural, magnetic, and dynamic parameters have also been put into the Solomon-Bloembergen equations to predict signal line shapes, in particular those of signals that are difficult to locate or are undetectable. Guidelines are provided to predict the order of magnitude of relaxation rates. It is shown that DFT-predicted paramagnetic shifts can greatly assist in obtaining and understanding the NMR spectra of paramagnetic molecules, which generally require different experimental strategies and exhibit problems in detection and assignment.

Structure and Dynamics of Micelle-bound Human α-Synuclein

Misfolding of the protein alpha-synuclein (aS), which associates with presynaptic vesicles, has been implicated in the molecular chain of events leading to Parkinson's disease. Here, the structure and dynamics of micelle-bound aS are reported. Val3-Val37 and Lys45-Thr92 form curved alpha-helices, connected by a well ordered, extended linker in an unexpected anti-parallel arrangement, followed by another short extended region (Gly93-Lys97), overlapping the recently identified chaperone-mediated autophagy recognition motif and a highly mobile tail (Asp98-Ala140). Helix curvature is significantly less than predicted based on the native micelle shape, indicating a deformation of the micelle by aS. Structural and dynamic parameters show a reduced helical content for Ala30-Val37. A dynamic variation in interhelical distance on the microsecond timescale is complemented by enhanced sub-nanosecond timescale dynamics, particularly in the remarkably glycine-rich segments of the helices. These unusually rich dynamics may serve to mitigate the effect of aS binding on membrane fluidity. The well ordered conformation of the helix-helix connector indicates a defined interaction with lipidic surfaces, suggesting that, when bound to larger diameter synaptic vesicles, it can act as a switch between this structure and a previously proposed uninterrupted helix.

Ensemble approach for NMR structure refinement against (1)H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecule.

Paramagnetic relaxation enhancement (PRE) measurements on (1)H nuclei have the potential to play an important role in NMR structure determination of macromolecules by providing unique long-range (10-35 A) distance information. Recent methodological advances for covalently attaching paramagnetic groups at specific sites on both proteins and nucleic acids have permitted the application of the PRE to various biological macromolecules. However, because artificially introduced paramagnetic groups are exposed to solvent and linked to the macromolecule by several freely rotatable bonds, they are intrinsically flexible. This renders conventional back-calculation of the (1)H-PRE using a single-point representation inaccurate, thereby severely limiting the utility of the (1)H-PRE as a tool for structure refinement. To circumvent these limitations, we have developed a theoretical framework and computational strategy with which to accurately back-calculate (1)H-PREs arising from flexible paramagnetic groups attached to macromolecules. In this scheme, the (1)H-PRE is calculated using a modified Solomon-Bloembergen equation incorporating a "model-free" formalism, based on a multiple-structure representation of the paramagnetic group in simulated annealing calculations. The ensemble approach for (1)H-PRE back-calculation was examined using several SRY/DNA complexes incorporating dT-EDTA-Mn(2+) at three distinct sites in the DNA, permitting a large data set comprising 435 experimental backbone and side-chain (1)H-PREs to be obtained in a straightforward manner from 2D through-bond correlation experiments. Calculations employing complete cross-validation demonstrate that the ensemble representation provides a means to accurately utilize backbone and side-chain (1)H-PRE data arising from a flexible paramagnetic group in structure refinement. The results of (1)H-PRE based refinement, in conjunction with previously obtained NMR restraints, indicate that significant gains in accuracy can be readily obtained. This is particularly significant in the case of macromolecular complexes where intermolecular translational restraints derived from nuclear Overhauser enhancement data may be limited.

The copper-topaquinone-phenylhydrazine-adduct geometry in Escherichia coli amine oxidase derivatized with phenylhydrazines substituted with 19F-NMR relaxation measurements.

The copper quinoprotein amine oxidase from Escherichia coli was derivatized with phenylhydrazine, substituted with a F3C group at the ortho, meta, or para position. The derivatization of the topaquinone cofactor was verified by ultraviolet/visible spectroscopy. The reduction (with dithionite) of Cu(II) to Cu(I), which was required to obtain reference samples, was verified by EPR spectroscopy. 19F-NMR spectroscopy was carried out on the derivatized enzyme forms, and the spectra showed the line-broadening effect due to the paramagnetic Cu(II). The distance between the Cu and the mean of the three F positions in the F3C groups was calculated by means of the Solomon-Bloembergen equation for the distance-dependent contribution of CU(II) to the transversal-relaxation time of the F resonance. Assuming that the F3C-phenylhydrazines in the enzyme are always aligned towards the Cu in the same way, four configurations can be envisaged that should be taken into account to determine the topology of the two cofactors. Based on these configurations, two spatial positions were found where the calculated distances triangulated, each of these positions having a symmetry-related counterpart above or below the topaquinone-phenylhydrazine plane. If it is assumed that the geometric positions of the phenylhydrazine and topaquinone moieties in the adduct remain the same in the derivatized enzymes, a number of minimum distances between the Cu and certain atoms in the topaquinone moiety of the adduct can be calculated (1.52 +/- 0.06 nm from the C2-O, 1.30 +/- 0.04 nm from the C4-O, and 1.26 +/- 0.04 nm from the C5-N). However, one of the configurations yields very similar distances between the Cu and the C2-O and C4-O. Therefore, no conclusions can be made with regard to which OH group is closest to the Cu. By application of the same approach to the 19F-NMR data obtained for porcine-plasma marine oxidase [Williams, T J. & Falk, M.C.(1986) J. Biol. Chem. 261, 15949- 15954] we observed substantial differences between the topologies of the cofactors in the two enzymes. Possible reasons for this are discussed.

A Computer Program for the Calculation of Paramagnetic Enhancements of Nuclear-Relaxation Rates in Slowly Rotating Systems

A computer program has been developed for the calculation of paramagnetic enhancement of nuclear-relaxation rates as a function of magnetic field in slowly rotating systems, when the Solomon-Bloembergen equations break down. The inclusion in the static spin-Hamiltonian parameters of g anisotropy, zero-field splitting, and hyperfine coupling with the metal nucleus provides a description of the electron spin system which is practically complete and adequate for most experimental cases. All three tensorial terms may include rhombic components and they can be treated simultaneously, provided that the tensors share the same principal axes.

Molecular flexibility demonstrated by paramagnetic enhancements of nuclear relaxation. Application to alamethicin: a voltage-gated peptide channel

A nitroxide spin label attached to the C-terminus of the channel forming peptide alamethicin produces an enhancement of the nuclear spin-lattice relaxation rates of peptide protons as a result of both intermolecular and intramolecular magnetic dipole-dipole interactions. The intermolecular contribution provides evidence that alamethicin monomers collide preferentially in a C-terminal-to-N-terminal configuration in methanol. From the intramolecular paramagnetic enhancement of nuclear spin-lattice relaxation times, effective distances between the unpaired electron on the nitroxide at the C-terminus of alamethicin and protons along the peptide backbone were calculated. These distances are much shorter than distances based on the reported crystal structure of alamethicin, and cannot be accounted for by motion in the bonds that attach the nitroxide to the peptide. In addition, the differences between distances deduced from the nuclear spin relaxation and the distances seen in the crystal structure increase toward the N-terminal end of the peptide. The simplest explanation for these data is that the alamethicin backbone suffers large structural fluctuations that yield shorter effective distances between the C-terminus and positions along the backbone. This finding can be interpreted in terms of a molecular mechanism for the voltage-gating of the alamethicin channel. When the distances between a paramagnetic center and a nucleus fluctuate, paramagnetic enhancements are expected to yield distances that are weighted by r-6, and distances calculated using the Solomon-Bloembergen equations may more nearly represent a distance of closest approach than a time average distance. Therefore, the use of paramagnetic centers such as spin labels or metal ions with long electron T1 values provides a distance measurement that reflects a dynamically averaged structure where the averaging process heavily weights short distances. The results of such measurements, when combined with other structural information, may provide particularly clear evidence for the magnitude of structural fluctuations involving distances greater than 10 A.

Preferred orientations in the binding of 4'-hydroxyacetanilide (acetaminophen) to cytochrome P450 1A1 and 2B1 isoforms as determined by 13C- and 15N-NMR relaxation studies.

The widely used analgesic/antipyretic agent 4'-hydroxyacetanilide (acetaminophen, APAP) is oxidized by cytochromes P450 to a potent cytotoxin, N-acetyl-p-benzoquinone imine (NAPQI), and a nontoxic catechol, 3',4'-dihydroxyacetanilide (3-hydroxyacetaminophen, 3-OH-APAP). There are marked differences in the ratios of these two products formed from different isoforms of cytochrome P450. For example, the ratio of NAPQI to 3-OH-APAP formed by rat liver CYP1A1 was found to be approximately 3:1, whereas the ratio of the same two products formed by rat liver CYP2B1 was approximately 1:5. Investigations of the binding of APAP to CYP1A1 and CYP2B1 were carried out to assess the possibility that different preferred orientations of APAP in the active sites of these isoforms may, in part, by responsible for their different product selectivities. Although the spectral dissociation constants (Ks congruent to 0.85 mM) and UV-vis binding spectra (type I; absorption minimum congruent to 420 nm, absorption maximum congruent to 390 nm) were similar for interactions of APAP with the two P450 isoforms, NMR longitudinal relaxation times (T1) of APAP nuclei were significantly different. Two isotopically substituted analogs of APAP, [2,3',5'-13C3]-4'-hydroxyacetanilide and 4'-hydroxyacet-[15N]-anilide, were synthesized, and their binding to purified CYP1A1 and CYP2B1 was examined by NMR spectroscopy. Paramagnetic relaxation times (T1p) for each of the labeled nuclei were calculated from the T1 values obtained before (ferric) and after (ferrous-CO) treatment with Na2S2O4 and CO. The Solomon-Bloembergen equation was then used to calculate distances of the isotopically labeled nuclei from the heme iron of each P450 isoform. The results were that the amide nitrogen approaches relatively close to the heme iron in CYP1A1 (3.64 +/- 0.51 A) whereas it is significantly further away (> 4.5 A) in CYP2B1. In contrast, the aryl carbon atoms ortho to the phenolic group of APAP approach closer to the heme iron of CYP2B1 (3.19 +/- 0.12 A) than to the heme iron of CPY1A1 (3.66 +/- 0.30 A). The results are consistent with the hypothesis that CYP1A1 produces NAPQI preferentially because of closer proximity of the heme iron to the amide nitrogen, whereas CYP2B1 produces 3-OH-APAP preferentially because of closer proximity of the heme iron to the phenolic oxygen in this isoform.

NMR relaxation studies of the interaction of thiocyanate with lactoperoxidase

The interaction of lactoperoxidase, LPO, with its substrate, thiocyanate, SCN −, has been investigated by 13C and 15N NMR relaxation measurements. When 0.1 M SCN −, enriched with either 13C or 15N, was titrated with native ferric lactoperoxidase a large change in the spin-lattice relaxation time of the respective nucleus was observed. In the presence of saturating amounts of CN −, a high affinity ligand for the heme iron, a similar but much smaller change in the relaxation time for SCN − was found. Studies of the rate of carbon relaxation as a function of temperature have shown that thiocyanate is in fast exchange between a site on the enzyme and bulk solution. When LPO in either the absence or presence of CN − was titrated with SCN − a linear increase in the relaxation time was observed. Dissociation constants (K d values) have been determined from a least-squares analysis of these data. Apparent distances between the heme iron of lactoperoxidase and either the carbon or nitrogen atoms of bound thiocyanate ion have been determined through application of the Solomon-Bloembergen equation. These distances demonstrate that the observed association does not involve iron-thiocyanate coordination, suggesting the possibility of an anion binding site.

Proton-metal distance determination in cobalt(II) stellacyanin by proton nuclear magnetic resonance relaxation measurements including Curie-spin effects: a proposed structure of the metal-binding region

1H nuclear magnetic resonance (1H NMR) experiments on Co(II)-substituted stellacyanin have been performed. Large paramagnetic hyperfine shifts are observed, the whole spectrum covering a range of 190 ppm. Experiments were mainly performed at 270 MHz from which temperature and pH* dependencies of the out-shifted resonances were reported, as well as determinations of the longitudinal (T1) and transverse (T2) relaxation times. These relaxation times are among other things, dependent on the individual proton-metal distance, and the aim of this work has been to determine these distances, by use of the Solomon-Bloembergen equations modified to include the so-called "Curie spin". The application of this method to a protein has not been reported earlier. Experiments were also performed at 100, 400, and 500 MHz in order to estimate the size of the Curie spin from the field dependence of the line widths. Furthermore, determination of the values for the rotational correlation time, tau r, and the effective magnetic moment, mu eff, was necessary for the present approach. With apostellacyanin, tau r was found to be (6.0 +/- 0.4) X 10-8 s. From the paramagnetic susceptibility of Co(II) stellacyanin, the value (4.53 +/- 0.03)beta was determined for mu eff. The proposed assignments of several paramagnetically out-shifted resonances. the proton-metal distances obtained, and the known peptide sequence of stellacyanin have allowed us to build a three-dimensional model of the metal site and its surrounding structure consistent with all the experimental data. It is revealed that both histidine ligands bind the metal with their 3-nitrogens. Also we find strong indications that a second sulfur atom is actually binding the metal, this being the long-sought-after fourth ligand. The model suggests that this sulfur belongs to Cys-59, which together with Cys-93 constitutes the disulfide bridge known to be present in the structure. A potential fifth ligand, an amide oxygen from Asn-47, is also found.

19F nuclear magnetic resonance as a probe of the spatial relationship between the heme iron of cytochrome P-450 and its substrate

The distance between the heme iron of ferrous cytochrome P-450-CAM and a fluorine label attached to the 9-methyl carbon of its substrate, (1R)-(+)-camphor, has been determined using 19F NMR. This investigation uses the Solomon-Bloembergen equation to measure the distance from a paramagnetic heme iron to a fluorine probe incorporated into a substrate that is not in fast exchange. The structural identity of the substrate analogue, 9-fluorocamphor, has been established using one- and two-dimensional NMR methods and mass spectrometry. The relaxation rate of 9-fluorocamphor bound to high-spin paramagnetic ferrous P-450-CAM has been studied at 188, 282, and 376 MHz, and the correlation time has been directly determined from the frequency dependence of the relaxation rate. When the substrate analogue was bound to the low-spin diamagnetic ferrous-CO derivative of the enzyme, the relaxation rate was found to be 100 times slower and was therefore neglected in the distance calculation. The relaxation data for the paramagnetic system and the correlation time have been used to calculate a distance of 3.8 A between the heme iron and the C-9 fluoride. A fit of the distance and the chemical shift data to the pseudocontact shift equation predicts an angle of approximately 52 degrees between the heme normal and the Fe-F vector. The solution state Fe-F distance is somewhat shorter and the angle between the heme normal and the Fe-F vector slightly larger for the substrate-bound ferrous enzyme reported herein than the analogous values for the substrate-bound ferric enzyme determined in the solid state by x-ray crystallography. These differences may reflect a structural change at the substrate-binding site upon reduction of the iron.

NMR study of the conformations of free and lanthanide-complexed glutathione in aqueous solution

The /sup 1/H and /sup 13/C NMR spectra of glutathione in its free and La(III)-complexes forms have been analyzed. The vicinal coupling constants were used to gain rotamer populations by the Karplus approach. The data were compared with the results of previous X-ray crystallographic, theoretical, and NMR studies. The gadolinium(III) complex of glutathione has been studied by the measurement of Gd(III)-induced /sup 1/H and /sup 13/C spin-lattice relaxation rate enhancements and application of the Solomon-Bloembergen equation. The two carboxylate groups of the molecule are found to be competing complexation sites; the microscopic formation constants have been determined. Complexation to the lanthanide(III) ions does not appear to change the conformation equilibrium of glutathione substantially.

Binding of copper(II) and manganese(II) to 9-methylpurine studied by nuclear magnetic resonance and potentiometric titration

The binding of Cu(II) and Mn(II) to 9-methylpurine has been examined by proton NMR and potentiometric titration. The spin-lattice relaxation rates of ring protons H2, H6 and H8, used as input to the Solomon-Bloembergen equation, give a binding pattern which qualitatively agrees with crystallographic data. At neutral pH Cu(II) and Mn(II) binding occurs at both N1 and N7 with a preference for N7 (66%). At acidic pH, where N1 is the predominant protonation site, a further shift towards N7 binding is observed. These results are not in complete agreement with those based on comparison of N1/N7 basicity.

Proton spin-lattice relaxation in aqueous solution of the nickel(II) ion

Spin-lattice relaxation rates for water protons in the presence of paramagnetic Ni(II) ions at high temperatures and low pH are reported as a function of the magnetic field up to 11.7 T. The data are analyzed using the conventional modified Solomon-Bloembergen equations and a theoretical model allowing for the slow-motion situation for the electron spin. The latter model is shown to describe the experimental data with a somewhat more reasonable set of parameters.