Relaxation Rate Of Solvent Protons Research Articles

Gd3+ Tethered Gold Nanorods for Combined Magnetic Resonance Imaging and Photo-Thermal Therapy

Near infrared (NIR) mediated photothermal therapy and magnetic resonance imaging (MRI) are promising treatment and imaging modalities in the field of cancer theranostics. Gold nanorods are the first choice of materials for NIR-mediated photothermal therapy due to their strong localized surface plasmon resonance (LSPR) at NIR region. Similarly, gadolinium based MRI contrast agents have an ability to increase the ionic and molecular relaxivity, thereby enhancing the solvent proton relaxation rate resulting in contrast enhancement. Herein, the effort has been made to engineer a dual front theranostic agent with combined photothermal and magnetic resonance imaging capacity using gadolinium tethered gold nanorods (Gd3+-AuNR). NIR-responsive gold nanorods were surface fabricated by means of Au-thiol interaction using a thiolated macrocyclic chelator that chelates Gd3+ ions, and further stabilized by thiolated polyethylene glycol (PEG-SH). The magnetic properties of the Gd3+-AuNR displayed an enhanced r 1 relaxivity of 12.1 mM–1s–1, with higher biological stability, and contrast enhancement in both solution state and in cell pellets. In-vitro (cell-free) and ex-vivo (on pig skin) analysis of the Gd3+-AuNR shows enhanced photothermal properties as equivalent to that of the raw AuNR. Furthermore, Gd3+-AuNR showed competent cellular entry and intracellular distribution as revealed by hyperspectral microscopy. In addition, Gd3+-AuNR also exhibits significant thermal ablation of B16–F10 cells in the presence of NIR. Thus, Gd3+-AuNR features a significant theranostic potential with combined photothermal and imaging modality, suggesting a great potential in anticancer therapy.

Magnetic, optical and relaxometric properties of organically coated gold–magnetite (Au–Fe3O4) hybrid nanoparticles for potential use in biomedical applications

We present the magnetic, optical and relaxometric properties of multifunctional Au–Fe3O4 hybrid nanoparticles (HNPs), as possible novel contrast agents (CAs) for magnetic resonance imaging (MRI). The HNPs have been synthesized by wet chemical methods in heterodimer and core–shell geometries and capped with oleylamine. Structural characterization of the samples have been made by X-ray diffraction and transmission electron microscopy, while magnetic properties have been investigated by means of Superconducting Quantum Interference Device-SQUID magnetometry experiments. As required for MRI applications using negative CAs, the samples resulted superparamagnetic at room temperature and well above their blocking temperatures. Optical properties have been investigated by analyzing the optical absorbtion spectra collected in UV–visible region. Relaxometric measurements have been performed on organic suspensions of HNPs and Nuclear Magnetic Resonance (NMR) dispersion curves have been obtained by measuring the longitudinal 1/T1 and transverse 1/T2 relaxation rates of solvent protons in the range 10kHz/300MHz at room temperature. NMR relaxivities r1 and r2 have been compared with ENDOREM®, one of the commercial superparamagnetic iron oxide based MRI contrast agents. MRI contrast enhancement efficiencies have been investigated also by examining T2-weighted MR images of suspensions. The experimental results suggest that the nanoparticles' suspensions are good candidates as negative CAs.

Thermodynamic Stability and Physicochemical Characterization of Ligand (4S)-4-Benzyl-3,6,10-tris(carboxymethyl)-3,6,10-triazadodecanedioic Acid (H5[(S)-4-Bz-ttda]) and Its Complexes Formed with Lanthanides, Calcium(II), Zinc(II), and Copper(II) Ions

A derivative of H5ttda (=3,6,10-tris(carboxymethyl)-3,6,10-triazadodecanedioic acid=N-{2-[bis(carboxymethyl)amino]ethyl}-N-{3-[bis(carboxymethyl)amino]propyl}glycine), H5[(S)-4-Bz-ttda] (=(4S)-4-benzyl-3,6,10-tris(carboxymethyl)-3,6,10-triazadodecanedioic acid=N-{(2S)-2-[bis(carboxymethyl)amino]-3-phenylpropyl}-N-{3-[bis(carboxymethyl)amino]propyl}glycine; 1) carrying a benzyl group was synthesized and characterized. The stability constants of the complexes formed with Ca2+, Zn2+, Cu2+, and Gd3+ were determined by potentiometric methods at 25.0±0.1° and 0.1M ionic strength in Me4NNO3. The observed water proton relaxivity value of [Gd{(S)-4-Bz-ttda}]2− was constant with respect to pH changes over the range pH 4.5–12.0. From the 17O-NMR chemical shift of H2O induced by [Dy{(S)-4-Bz-ttda}]2− at pH 6.80, the presence of 0.9 inner-sphere water molecules was deduced. The water proton spin-lattice relaxation rate for [Gd{(S)-4-Bz-ttda}]2− at 37.0±0.1° and 20 MHz was 4.90±0.05 mM−1 s−1. The EPR transverse electronic relaxation rate and 17O-NMR transverse-relaxation time for the exchange lifetime of the coordinated H2O molecule (τM), and 2H-NMR longitudinal-relaxation rate of the deuterated diamagnetic lanthanum complex for the rotational correlation time (τR) were thoroughly investigated, and the results were compared with those previously reported for the other lanthanide(III) complexes. The exchange lifetime (τM) for [Gd{(S)-4-Bz-ttda}]2− (2.3±1.3 ns) was significantly shorter than that of the [Gd(dtpa)(H2O)]2− complex (dtpa=diethylenetriaminepentaacetic acid). The rotational correlation time τR for [Gd{(S)-4-Bz-ttda}]2− (70±6 ps) was slightly longer than that of the [Gd(dtpa)(H2O)]2− complex. The marked increase of relaxivity of [Gd{(S)-4-Bz-ttda}]2− mainly resulted from its longer rotational time rather than from its fast water-exchange rate. The noncovalent interaction between human serum albumin (HSA) and the [Gd{(S)-4-Bz-ttda}]2− complex containing the hydrophobic substituent was investigated by measuring the solvent proton relaxation rate of the aqueous solutions. The association constant (KA) was less than 100 M−1, indicating a weaker interaction of [Gd{(S)-4-Bz-ttda}]2− with HSA.

Water-Proton Relaxation by a Noncovalent Albumin-Binding Gadolinium Chelate: An NMRD Study of a Potential Blood Pool Agent

Magnetic dipolar interactions between solvent water protons and paramagnetic solutes, such as chelated Gd3+ ions, provide an efficient means for increasing both the longitudinal (1/T1) and transverse (1/T2) relaxation rates of solvent protons. For small paramagnetic chelate complexes, interactions by direct water coordination to the metal ion (inner sphere) and diffusion in the outer sphere environment of the metal complexes are the two main contributors to relaxation enhancement. At magnetic resonance imaging (MRI) fields and physiologic conditions, these contributions are additive and generally comparable in magnitude, whereas for macromolecular complexes, inner sphere contributions tend to dominate relaxation. The inner sphere contribution to 1/T1 is given by: 1/T1pis =q[Gd3+]/55.5(T1M + τM). Here, q is the number of coordinated inner sphere water molecules, T1M is the longitudinal relaxation time of the coordinated water protons, and τM is their lifetime on the metal ion. For coordinated Gd3+ ions with q = 1, both T1M and τM are of the order of microseconds. 1/T1 is a complicated function of the strength of the interaction, the magnitude of the applied MRI field B0, and an overall correlation time, τC. τC is given by τC−1 = τR−1 + τS−1 + τM−1, the inverse of the sums of the reciprocals of the orientational relaxation time of the complex τR, the electronic relaxation time of the paramagnetic metal ion τS, and τM, respectively. For Gd3+ ions, τS can also be a function of B0, often characterizable by another correlation time τV. In any event, the enhancement is maximized for kinetically labile complexes, when exchange of the inner sphere water is rapid (but not too rapid), such that τC << τM << T1M ( 1 Outhred RK George EP A nuclear magnetic resonance study of hydrated systems using the frequency dependence of the relaxation processes. Biophys J. 1973; 13: 83-96 Abstract Full Text PDF PubMed Scopus (30) Google Scholar , 2 Koenig SH Brown III, RD Relaxation of solvent protons by paramagnetic ions: dependence on magnetic field and chemical environment—implications for NMR imaging. Magn Reson Med. 1984; 1: 478-495 Crossref PubMed Scopus (187) Google Scholar , 3 Bertini L Luchinat C NMR of paramagnetic molecules in biological systems. Benjamin/Cummins, Menlo Park, Calif1986 Google Scholar ).

Non-ionic bulky Gd(III) DTPA-bisamide complexes as potential contrast agents for magnetic resonance imaging.

A series of new diethylene triamine pentaacetic acid (DTPA)-bisamide chelates containing bulky alkyl and aryl side chains have been prepared and characterized. Nuclear magnetic relaxation dispersion profiles were measured for the neutral gadolinium [Gd(III)] DTPA-bisamide complexes in water solution, and their chemical exchange times (tau(m)) were found to be in the range of 1.4 to 4.9 micros. Significant enhancements of solvent proton relaxation rates were observed between 10 and 50 MHz for one of the complexes of the series [Gd(III)-DTPA-bis-2-ethylhexylamide] in human serum albumin (HSA) solution, indicating the formation of a paramagnetic macromolecular adduct. The binding association constant K(A) of the complex and the albumin 5.7 x 10(3) M(-1) was obtained, and the relaxivity of the fully bound adduct was determined to be 13.8 mM(-1) s(-1) at 20 MHz and 25 degrees C. The high value of K(A) makes the above derivative a good potential blood pool contrast agent at the physiological HSA concentration.

Gd(III) complexes as contrast agents for magnetic resonance imaging: a proton relaxation enhancement study of the interaction with human serum albumin

The non-covalent interaction between human serum albumin (HSA) and DOTA-like Gd(III) complexes containing hydrophobic benzyloxymethyl (BOM) substituents has been thoroughly investigated by measuring the solvent proton relaxation rates of their aqueous solutions. The binding association constants (K A) to HSA are directly related to the number of hydrophobic substituents present on the surface of the complexes. Furthermore, an estimation of ΔH° and ΔS° has been obtained by the temperature dependence of K A. Assays performed with the competitor probes warfarin and ibuprofen established that the complexes interact with HSA through two nearly equivalent binding sites located in the subdomains IIA and IIIA of the protein. Strong relaxation enhancements, promoted by the formation of slowly tumbling paramagnetic adducts, have been measured at 20 MHz for complexes containing two and three hydrophobic substituents. The macromolecular adduct with the latter species has a relaxivity of 53.2±0.7 mM–1 s–1, which represents the highest value so far reported for a Gd(III) complex. The temperature dependence of the relaxivity for the paramagnetic adducts with HSA indicates long exchange lifetimes for the water molecules dipolarly interacting with the paramagnetic centre. This is likely to be related to the formation, upon hydrophobic interaction of the complexes with HSA, of a clathrate-like, second-coordination-sphere arrangement of water molecules. Besides affecting the dissociative pathway of the coordinated water molecule, this water arrangement may itself significantly contribute to enhancement of the bulk solvent relaxation rate.

Inclusion complexes between β‐cyclodextrin and β‐benzyloxy‐α‐propionic derivatives of paramagnetic DOTA‐ and DPTA‐Gd(III) complexes

AbstractThe addition of β‐cyclodextrin to a solution containing β‐benzyloxy‐α‐propionic‐substituted DOTA‐ and DTPA‐Gd(III) complexes leads to the formation of inclusion compounds whose equilibrium constants have been determined by NMR titrations. The main effect associated with the formation of this ‘host‐uest’ interaction is a marked elongation of the molecular reorientational time, τr, which in turn results in increased solvent proton relaxation rates.

Trends in NMR studies of paramagnetic Gd(III) complexes as potential contrast agents in MRI

The paramagnetic Gd(III) complexes with polyaminocarboxylate ligands are intensively studied as possible Contrast Agents for Magnetic Resonance Imaging (M.R.I.). Their ability to enhance the solvent proton relaxation rate is mainly determined by the molecular reorientational time (τ R). In order to increase τ R we studied the formation of non-covalent interactions between functionalized Gd(III)-complexes and micelles. The N.M.R.D. (Nuclear Magnetic Relaxation Dispersion) profiles of aqueous solutions of these paramagnetic complexes with hexadecyltrimethylammonium bromide (CTABr) were measured and the results could be accounted for by difference in negative charge and number of hydrophobic aromatic residues among the different complexes.

Relaxometry of ferritin solutions and the influence of the Fe3+ core ions

The magnetic field dependence of 1/T1 over the range 0.01 to 50 MHz proton Larmor frequency (NMRD profile) is reported for water protons in solutions of horse spleen apoferritin, and of ferritin reconstituted at both low and high iron levels. The apoferritin results are in every way typical of diamagnetic spherical proteins of their size (K. Hallenga and S. H. Koenig, Biochemistry 15, 4255 (1976)). Titration of up to 24 ferrous ions per protein molecule, with subsequent oxidation to ferric, shows a nonlinear saturating contribution to the NMRD profile which is interpreted as arising from a small number of ferric ions (six to eight) bound close to the outside of each ferritin molecule, and a comparable number of interior sites. The latter become multiply occupied as the core grows and do not contribute measurably to 1/T1 in this state. The former sites are never more than singly occupied, and their contribution to the solvent proton relaxation rates is independent of the loading of the core. Measurements of 1/T2 at 20 MHz are quite in accord with theoretical expectations for apoferritin and ferritin with up to 24 ferric ions per molecule. However, a marked increase in 1/T2 is observed at higher iron loadings that we are unable to account for within the framework of the theory of outer sphere relaxation, even when the effects arising from inhomogeneities in the local magnetic field are included. A sample of human spleen hemosiderin was found to have the same 1/T1 NMRD profile as a comparable sample of ferritin.

Flash-induced enhancements in the proton NMR relaxation rate of Photosystem II particles

Flash-induced enhancements in the NMR spin-lattice relaxation rate of solvent protons have been detected in suspensions of Photosystem II particles. The relaxation enhancements are small (less than 1% of background) and have been detected using signal-averaging techniques. The enhancements correlate with the known properties of the S states with respect to (1) decay kinetics, (2) extractants of manganese, (3) sensitivity to atrazine-type inhibition of electron transport, (4) reagents which accelerate S-state decay (ADRY reagents), and (5) the two-flash retardation in the S-state advancement that is produced by low concentrations of NH 2OH. The transient proton relaxation enhancement observed after a single flash arises from a strongly relaxing paramagnetic species that is produced by the S 1 → S 2 transition of the water-oxidizing center. The appearance of such a species on an oxidative transition is suggestive of an Mn(III) → Mn(IV) oxidation.

Magnetic field dependence of solvent proton relaxation rates induced by Gd3+ and Mn2+ complexes of various polyaza macrocyclic ligands: Implications for NMR imaging

The magnetic field dependence of the solvent water proton longitudinal relaxation rate 1/T1 (the NMRD profile) has been measured for solutions of chelates of Gd3+ and Mn2+ ions with two different polyaza macrocyclic ligands: 1,4,7-triazacyclononane-N,N',N",-triacetic acid (NOTA) and 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA). Studies were carried out mainly near physiological pH, but the pH dependence was also examined in some cases. The results are compared with published data for complexes of Gd3+ and Mn2+ ions with ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). Competition experiments for the NOTA and DOTA chelates with EDTA and DTPA were also performed. It is found that, over the field range in which NMR imaging is currently being done, different symmetries of otherwise similar chemical ligands can alter 1/T1 of solvent protons by factors of up to three. The ligand environment can influence the relaxation times of the electronic spin moments of the ions, as well as their coordination number, thereby changing both the inner and outer sphere contributions to the relaxivities of the complexes. The relevance of these results to questions of efficiency and toxicity of these chelates as agents for enhancement of contrast in NMR images is discussed.

Electron spin resonance and magnetic relaxation studies of gadolinium(III) complexes with human transferrin.

A human serum transferrin complex was prepared in which Gd(III) was substituted for Fe(III) at the two metal-binding sites. Characteristic changes upon metal binding in both the UV absorption of ligated tyrosines and the solvent proton longitudinal magnetic relaxation rates demonstrated 2/1 metal stoichiometry and pH-dependent binding constants. Binding studies were complicated both by binding of Gd(III) to nonspecific sites on transferrin at pH less than or equal to 7 and by complexation of the Gd(III) by the requisite bicarbonate anion at pH greater than or equal to 6.0. A unique Gd(III) electron spin resonance spectrum, with a prominent signal at g = 4.96, was observed for the specific Gd(III)-transferrin complex. The major features of this spectrum were fit successfully by a model Hamiltonian which utilized crystal field parameters similar to those determined for Fe(III) in transferrin [Aasa, R. (1970) J. Chem. Phys. 52, 3919-3924]. The magnetic field dependence of the solvent proton relaxation rate was measured as a function of both pH and metal ion concentration. An observed biphasic dependence of the relaxation rate on metal concentration is attributed to either sequential metal binding to the two iron-binding sites with different relaxation properties or random binding to two sites that are similar but show conformationally induced changes in relaxation properties as the second metal is bound. The increase in the solvent proton relaxation rate with pH is consistent with a model in which a proton of a second coordination sphere water molecule is hydrogen bonded to a metal ligand which becomes deprotonated at pH 8.5.

The electron-nucleus dipolar coupling in slow rotating systems. 4. The effect of zero-field splitting and hyperfine coupling when [formula omitted] and [formula omitted

Solvent proton relaxation rates as a function of magnetic field have been calculated for slow rotating high-spin manganese(II)-containing systems. The static zero-field splitting of the S = 5 2 manifold has been included in the spin Hamiltonian and found to be quite meaningful with respect to understanding the experimental data. The effect of the hyperfine coupling between the manganese nuclear spin and the electron spin has been evaluated and found to be minor. The available experimental data on manganese(II) concanavalin A have been used to test the influence of the above spin-Hamiltonian terms on the parameters describing the electron-nucleus coupling.

Magnetic relaxation of solvent protons by Cu2+- and VO2+-substituted transferrin: theoretical analysis and biochemical implications.

Measurements of the magnetic field dependence of the longitudinal nuclear magnetic relaxation rates of solvent protons (NMRD profiles) in solutions of paramagnetic proteins have contributed significantly to the elucidation of the physical biochemistry of a number of metalloprotein systems. In many cases, NMRD profiles were used as indicators of chemical state, both static and dynamic [cf. Brewer, C. F., Brown, R. D., III, & Koenig, S. H. (1983) J. Biomol. Struct. Dyn. 1, 961-997], in part because a proper theoretical description of the data, with realistic assumptions for a model system, was computationally intractable. This has been particularly true for Cu2+-protein complexes, attributable in part to the S = 1/2 ground-state configuration of the Cu2+ ions; significant progress in interpreting such data has been made only recently [Bertini, I., Briganti, F., Luchinat, C., Mancini, M., & Spina, G. (1985) J. Magn. Reson. 63, 41-55]. We report NMRD profiles for solutions of Cu2+ - and VO2+-substituted human transferrin, both S = 1/2 ions, as well as computations that include the effects of the anisotropic hyperfine interactions of the paramagnetic ions with their respective nuclei. The description of the data that results from these computations is quite good, sufficiently so that one can say with confidence that the protons that contribute to the relaxation are rather distant (approximately 3.5 A) from the ions and in rapid exchange (approximately 10(8) s-1) with solvent. A possible view, consistent with what is known of the biochemistry of these substituted transferrins, is that relaxation occurs in the second coordination sphere: the exchanging entity is a water molecule hydrogen bonded to a donor atom of the metal ion complex.

Magnetic field dependence of solvent proton relaxation in aqueous solutions of Fe3+ complexes.

It might appear that the Fe3+ ion would be particularly useful as an agent for enhancing contrast in NMR images since it has a relatively large magnetic moment and occurs in vivo in a variety of forms. Moreover, the concentration of Fe3+ changes locally in certain disease states (e.g., beta-thalassemia) and in trauma (formation of methemoglobin), and can be altered in the gastrointestinal tract by the ingestion of readily available dietary supplements. However, the Fe3+ ion is insoluble above pH approximately 4, and soluble chelate and protein complexes of Fe3+ tend to sequester the ions from solvent; hence, the efficacy of Fe3+ ions for relaxing water protons ought to be low under typical physiological conditions. We report the magnetic field dependence of the relaxation rate of solvent protons (NMRD profiles) for solutions of a variety of Fe3+ complexes to demonstrate the phenomenology relevant to NMR imaging. From these data we make some estimates to show that, despite the low relaxation rates of solvent protons in solutions of Fe3+ complexes, certain observed changes in image contrast are consistent, quantitatively, with inferences that can be drawn from solution data.

RELAXATION OF SOLVENT PROTONS BY PARAMAGNETIC IONS AND ITS DEPENDENCE ON MAGNETIC FIELD AND CHEMICAL ENVIRONMENT

Paramagnetic ions have been used to alter the magnetic relaxation rates 1/T1 and 1/T2 of solvent water protons since the first observations of a proton resonance signal almost four decades ago. The earliest theories of relaxation indicated that the influence of solute paramagnetic ions on relaxation rates of solvent protons should depend both on the chemical environment of the solute ions and on the magnetic field strength. Much knowledge, both experimental and theoretical, has since been amassed regarding relaxation effects in solutions of hydrated ions (aquoions) and of complexes of these ions with macromolecules, mainly proteins. The phenomena are well understood at this point, though the understanding is more retrospective than predictive for ion-protein complexes. Nonetheless, from what is now known about homogeneous solutions, and from current reports on the introduction of paramagnetic ions into tissue to alter contrast in NMR images by affecting relaxation rates, it is clear that the solution results are particularly germane, and transferable, to tissue investigations. The main features of relaxation of solvent protons in the presence of solute paramagnetic ions, as hydrated aquoions and complexed with protein, are presented here, with attention to those ions most relevant to in vivo studies, and with emphasis on the influence of the magnetic field and the chemical environment of these ions on solvent proton relaxation rates.

Relaxation of solvent protons and deuterons by protein-bound Mn 2+ ions. Theory and experiment for Mn 2+-concanavalin A

Despite the demonstrated utility of measurements of the magnetic field dependence of the magnetic relaxation rates of solvent protons in solutions of metalloproteins as an indicator of biochemical changes, it is becoming increasingly evident that quantitative comparisons of such data with the theory of relaxation, limited by the approximations and assumptions usually made, yield results for the strength of the solvent-paramagnetic ion interaction that generally do not make chemical sense. These results, when expressed as the number of solvent-donated ligands of the ions, usually give too large a value, typically by about twofold. It has been suggested by several investigators that a comparison of proton and deuteron relaxation rates could resolve the problem. Data are presented for the longitudinal relaxation rates of solvent protons and deuterons over more than four decades of magnetic field (from 0.01 to 270 MHz proton Larmor frequency) for solutions of Mn 2+-concanavalin A, a protein for which the physical biochemistry is thoroughly documented, one that should be particularly tractable for such comparisons. The main conclusion is that, in the general case, there is no decade of magnetic field over which the mathematical criterion of best agreement of data with theory can be relied upon to yield quantitatively correct biochemical results; rather, biochemistry must still be a guide for elucidating relaxation pathways.

Determinants of proton relaxation rates in tissue.

It is well established that the longitudinal magnetic relaxation rate of solvent water protons, 1/T1, increases markedly in homogeneous protein solutions as the magnetic field is reduced well below the traditional NMR range. For a 5% solution of protein of 10(5) Da, for example, 1/T1 increases from about 50% above the pure water rate of 20 MHz to five times the water rate at 0.01 MHz. For tissue, including blood, the behavior is similar. Data for blood show that extracellular water has ready access to the hemoglobin inside red blood cells, which causes the enhanced relaxation. The extent to which cell water can sample the spatial structure of soft tissue, and how this structure influences relaxation rates, is as yet unknown. Nonetheless, the relaxation data for tissue can be accommodated within the conceptual framework developed earlier for analyzing homogeneous solutions of diamagnetic proteins. The variation of 1/T1 with field differs among tissues, and its magnitude at a given field can vary by more than a factor of three, far more than does the water content of the tissues. Solute complexes of paramagnetic ions with macromolecules, which increase the relaxation rates of solvent protons, can be introduced intravenously in tissue. They are known to accumulate in specific organs, and therefore have potential utility as contrast-enhancing agents in NMR imaging. Mn2+ and Gd3+, for example, produce characteristic dependences of 1/T1 on magnetic field that vary with the chemical state of the agent. The possibility exists, therefore, for monitoring the in vivo biochemistry of these agents.

Relaxation of solvent protons by paramagnetic ions and its dependence on magnetic field and chemical environment: implications for NMR imaging.

Paramagnetic ions have been used to alter the magnetic relaxation rates 1/T1 and 1/T2 of solvent water protons since the first observations of a proton resonance signal almost four decades ago. The earliest theories of relaxation indicated that the influence of solute paramagnetic ions on relaxation rates of solvent protons should depend both on the chemical environment of the solute ions and on the magnetic field strength. Much knowledge, both experimental and theoretical, has since been amassed regarding relaxation effects in solutions of hydrated ions (aquoions) and of complexes of these ions with macromolecules, mainly proteins. The phenomena are well understood at this point, though the understanding is more retrospective than predictive for ion-protein complexes. Nonetheless, from what is now known about homogeneous solutions, and from current reports on the introduction of paramagnetic ions into tissue to alter contrast in NMR images by affecting relaxation rates, it is clear that the solution results are particularly germane, and transferable, to tissue investigations. The main features of relaxation of solvent protons in the presence of solute paramagnetic ions, as hydrated aquoions and complexed with protein, are presented here, with attention to those ions most relevant to in vivo studies, and with emphasis on the influence of the magnetic field and the chemical environment of these ions on solvent proton relaxation rates.

Water Exchange at the Active Site of Carbonic Anhydrase: A Synthesis of the OH- and H 2O-Models

We have measured the paramagnetic contribution to the magnetic relaxation rate of solvent protons in highly purified, buffer- and salt-free solutions of Co(2+)-substituted human carbonic anhydrase B (HCAB), as a function of pH in the range 5.5-10 and as a function of magnetic field. We have also measured the optical absorption at 640 nm to characterize the enzyme. The relaxation rates vary with pH much as does the CO(2) hydration activity, increasing with increasing pH. We find that the relaxation rates at all intermediate values of pH can be described as linear combinations of the rates obtained at the extremes of pH used, indicating the existence of low- and high-pH forms of the enzyme with pH-dependent concentrations. The optical data can be similarly represented. The fraction of high-pH form present, determined from either the relaxation or optical data, has a pK(a) of approximately 7.6 when approximated by a single ionization. The data are very similar to that for HCAB in the presence of buffer, in contrast to the bovine enzyme for which the pK(a) is affected substantially by the presence of sulfate. Previous analysis of the high relaxation rates at high pH indicated rapid exchange of Co(2+)-liganded protons, possible only if these exchanging protons were conveyed by water molecules. On the other hand, the present demonstration of the existence of two forms of HCAB in highly purified solutions, coupled with other data, argues strongly for ionization of a water molecule ligand of the metal ion at the active site, with OH(-) as the solvent-donated ligand at high pH. We propose a mechanism of ligand exchange at high pH that reconciles these ostensibly conflicting requirements by invoking a pentacoordinate intermediate having both OH(-) and H(2)O as ligands. Proton exchange can be rapid between these ligands because charge transfer without net ionization can occur, so that the leaving water can carry away the initial OH(-). The low-pH form is a thermal mixture of tetra- and pentacoordinate species, the latter having low relaxation rates by analogy with inhibitor derivatives of the enzyme and model systems. The proposed associative ligand-exchange mechanism reconciles the distinctions between the OH- and H(2)O-models of carbonic anhydrase by merging them, providing the first model is consistent with the observed pH dependence of hydration activity, optical absorption, and solvent magnetic relaxation.