Influence of Mn(II) on NMR relaxation times of biological fluids

The extent that various concentrations of the paramagnetic metal ion manganese [Mn(II)] affect nuclear magnetic resonance (NMR) relaxation times was studied in vitro. Serial dilutions of Mn(II) were prepared in distilled water, 4% human serum albumin, dog plasma, dog gallbladder bile, and dog hepatic bile. T1 and T2 of each were measured at 10.7 M Hz using magnetization recovery and spin-echo radiofrequency sequences, respectively. The results show that relaxation rates (1/T1 and 1/T2) increase in a linear manner with increasing concentration of Mn(II) in all of the solutions tested. Mn(II) dissolved in dog gallbladder and hepatic bile, dog plasma, and 4% human serum albumin reduced relaxation times to a greater extent than Mn(II) in water. T1 times were reduced to a greater extent than T2 values. Thus, in T1 weighted magnetic resonance images, the NMR signal used to produce images would be more sensitive to the presence of Mn(II) in these biological fluids than in water. Furthermore, the magnitude of this in vivo effect of Mn(II) on NMR relaxation parameters depends not only on the concentration of this paramagnetic ion, but also on the constituents comprising the biological fluids (intra- and extracellular water, bile, plasma) and the nature of the chemical molecular interactions between these constituents and Mn(II).     

Water proton relaxation rate enhancements and association constants for Mn(II) to serum proteins determined by NMR T1 measurements

The water proton relaxation rate enhancement of Mn(II) bound to bovine serum albumin (BSA) and the association constant for manganese to BSA have already been determined, but such determinations have not been done for human serum albumin (HSA) and other human serum proteins and also for human serum. In this work, NMR T1 values in aqueous solutions of serum proteins and serum were measured versus increasing concentration of Mn(II). Proton relaxation rate enhancements (epsilon*) caused by different manganese concentrations were determined for each solution and 1/epsilon* was fitted against concentrations of Mn(II). Proton relaxation rate enhancements (epsilonb) of Mn(II) bound to albumin, gamma-globulin, (alpha+beta)-globulins and serum were found to be 13.69, 3.09, 8.62, and 10.87, respectively. Free and bound manganese fractions, resulted from each addition of Mn(II) to the sample, were determined by using corresponding (epsilon*) and the epsilonb values. Association constants for Mn(II) to HSA and gamma-globulin were calculated as 1.84 x 10(4) M(-1) and 2.35 x 10(4) M(-1), respectively. Present data suggest that the proton relaxation rate enhancement of Mn(II) in serum is caused by Mn(II) bound to various serum constituents. Data also suggest that association constants for Mn(II) to gamma-globulin are nearly the same as that to HSA.     

Relaxometric evaluation of novel manganese(II) complexes for application as contrast agents in magnetic resonance imaging

Three novel Mn(II) complexes bearing benzyloxymethyl functionalities are reported and their ability to enhance water (1H and 17O) relaxation times is investigated in detail. Two of them contain one coordinated water molecule and display relaxivity values only slightly smaller than those shown by the most clinically used contrast agents (e.g. [Gd(DTPA)(H2O)]2-). Moreover, in these Mn(II) chelates the exchange rate of the coordinated water is ca. one order of magnitude higher if compared to the exchange rates previously reported for Gd(III) complexes with octadentate ligands. The occurrence of such fast exchange rates of the coordinated water is exploited in the formation of macromolecular adducts with human serum albumin to attain systems displaying relaxivity values in the upper range of those so far reported for analogous Gd(III) systems. These results strongly support the view that Mn(II) complexes, in spite of the lower effective magnetic moment, can be considered as viable alternatives to the currently used Gd(III) complexes as contrast agents for MRI applications.     

Magnetic resonance studies of Mn(II)-, Mn(III)-, and Fe(III)-conalbumin complexes.

Apoconalbumin binds Mn(II) at two sites with association constants of K1 = 7 (+/- 1) X 10(4) and K2 = 0.4 (+/- 0.25) X 10(4) M-1. The binding is tighter in the presence of excess bicarbonate resulting in K1 = 1.8 (+/- 0.2) X 10(5) and K2 = 3 (+/- 2) X 10(4) M-1. The electron paramagnetic resonance spectrum (at both 9 and 35 GHz) of Mn(II) bound at the tight site reveals a rhombic distortion (lambda = E/D approximately equal to 0.25-0.31) in the protein ligand environment of the mental ion. An evaluation of the 1/pT1p, paramagnetic contribution to the longitudinal relaxation rate of solvent protons with Mn(II)-, Mn(III)-, and Fe(III)-derivatives of conalbumin revealed that the mental ion in each site of conalbumin is accessible to one water molecule. For Mn(II)-conalbumin and Mn(III)-conalbumin species, inner coordination sphere protons are rapidly exchanging with the bulk solvent, while slow exchange conditions prevail for Fe(III)-conalbumin.     

Binding of thiocyanate and cyanide to manganese(III)-reconstituted horseradish peroxidase: a 15N nuclear magnetic resonance study

Binding of thiocyanate and cyanide ions to Mn(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by relaxation rate measurements (at 50.68 MHz) of 15N resonance of SC15N- and C15N-. At pH = 4.0 the apparent dissociation constant (KD) for thiocyanate and cyanide binding to Mn(III)HRP was deduced to be 156 and 42 mM, respectively. The pH dependence of the 15N line width as well as apparent dissociation constant for thiocyanate and cyanide binding were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate and cyanide in deprotonated form bind to the enzyme in a protonated form. The binding of thiocyanate and cyanide to Mn(III)HRP was found to be facilitated by protonation of an ionizable group on the enzyme [Mn(III)HRP] with a pKa = 4.0. From competitive binding studies it was shown that iodide, thiocyanate and cyanide bind to Mn(III)HRP at the same site; however, the binding site for resorcinol is different. The apparent dissociation constant for iodide binding deduced from competitive binding studies was found to be 117 mM, which agrees very well with the iodide binding to ferric HRP. The binding of thiocyanate and cyanide was shown to be away from the metal center and the distance of the 15N of thiocyanate and cyanide from the paramagnetic manganese ion in Mn(III)HRP was found to be 6.9 and 6.6 A, respectively. Except for cyanide binding, these observations parallel with the iodide and thiocyanate ion binding to native Fe(III)HRP. Water proton relaxivity measurements showed the presence of a coordinated water molecule to Mn(III)HRP with the distance of Mn-H2O being calculated to be 2.6 A. The slow reactivity of H2O2 towards Mn(III)HRP could be attributed to the presence of water at the sixth coordination position of the manganese ion.     

Studies on human lactoferrin by electron paramagnetic resonance, fluorescence, and resonance Raman spectroscopy

Investigations of metal-substituted human lactoferrins by fluorescence, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy confirm the close similarity between lactoferrin and serum transferrin. As in the case of Fe(III)- and Cu(II)-transferrin, a significant quenching of apolactoferrin's intrinsic fluorescence is caused by the interaction of Fe(III), Cu(II), Cr(III), Mn(III), and Co(III) with specific metal binding sites. Laser excitation of these same metal-lactoferrins produces resonance Raman spectral features at ca. 1605, 1505, 1275, and 1175 cm-1. These bands are characteristic of tyrosinate coordination to the metal ions as has been observed previously for serum transferins and permit the principal absorption band (lambda max between 400 and 465 nm) in each of the metal-lactoferrins to be assigned to charge transfer between the metal ion and tyrosinate ligands. Furthermore, as in serum transferrin the two metal binding sites in lactoferrin can be distinguished by EPR spectroscopy, particularly with the Cr(III)-substituted protein. Only one of the two sites in lactoferrin allows displacement of Cr(III) by Fe(III). Lactoferrin is known to differ from serum transferrin in its enhanced affinity for iron. This is supported by kinetic studies which show that the rate of uptake of Fe(III) from Fe(III)--citrate is 10 times faster for apolactoferrin than for apotransferrin. Furthermore, the more pronounced conformational change which occurs upon metal binding to lactoferrin is corroborated by the production of additional EPR-detectable Cu(II) binding sites in Mn(III)-lactoferrin. The lower pH required for iron removal from lactoferrin causes some permanent change in the protein as judged by altered rates of Fe(III) uptake and altered EPR spectra in the presence of Cu(II). Thus, the common method of producing apolactoferrin by extensive dialysis against citric acid (pH 2) appears to have an adverse effect on the protein.     

Effect of manganese on the nuclear magnetic relaxivity of water protons in chloroplast suspensions

Field dispersion profiles of the proton spin-lattice relaxation rate, T1^?1, in chloroplast suspensions show a local maximum near 20 MHz, probably due to bound Mn(II); EDTA extraction eliminates, and MnCl₂ addition restores, the paramagnetic relaxivity. Since neither treatment affects water oxidation, the Mn(II) site monitored appears to lie outside the water-splitting enzyme. Intense illumination almost totally suppresses the paramagnetic relaxivity through an electron-transport-dependent mechanism. Previous reports that chloroplast nuclear magnetic relaxivity varies cyclically in flash experiments require reevaluation in terms of the probable role of Mn(II) that is nonfunctional in water oxidation.     

The accessibility of iron at the active site of recombinant human phenylalanine hydroxylase to water as studied by 1H NMR paramagnetic relaxation. Effect of L-Phe and comparison with the rat enzyme

The high-spin (S = 5/2) Fe(III) ion at the active site of recombinant human phenylalanine hydroxylase (PAH) has a paramagnetic effect on the longitudinal relaxation rate of water protons. This effect is proportional to the concentration of enzyme, with a paramagnetic molar-relaxivity value at 400 MHz and 25 degrees C of 1. 3 (+/- 0.03) x 10(3) s-1 M-1. The value of the Arrhenius activation energy (Ea) for the relaxation rate was -14.4 +/- 1.1 kJ/mol for the resting enzyme, indicating a fast exchange of water protons in the paramagnetic environment. The frequency dependence of the relaxation rate also supported this hypothesis. Thus, the recombinant human PAH appears to have a more solvent-accessible catalytic iron than the rat enzyme, in which the water coordinated to the metal is slowly exchanging with the solvent. These findings may be related to the level of basal activity before activation for these enzymes, which is higher for human than for rat PAH. In the presence of saturating (5 mM) concentrations of the substrate L-Phe, the paramagnetic molar relaxivity for human PAH decreased to 0.72 (+/- 0.05) x 10(3) s-1 M-1 with no significant change in the Ea. Effective correlation times (tauC) of 1.8 (+/- 0.3) x 10(-10) and 1.25 (+/- 0.2) x 10(-10) s-1 were calculated for the enzyme and the enzyme-substrate complex, respectively, and most likely represent the electron spin relaxation rate (tauS) for Fe(III) in each case. Together with the paramagnetic molar-relaxivity values, the tauC values were used to estimate Fe(III)-water distances. It seems that at least one of the three water molecules coordinated to the iron in the resting rat and human enzymes is displaced from coordination on the binding of L-Phe at the active site.     

Field-dispersion profiles of the proton spin-lattice relaxation rate in chloroplast suspensions. Effect of manganese extraction by EDTA,Tris, and hydroxylamine

Proton spin-lattice relaxation rates (R₁) have been measured in a variety of dark-adapted chloroplast suspensions over a range of field strengths between 1 and 15 kG (4–5 MHz). When the effects of EDTA or Tris washing on chloroplast relaxivities are compared, the pool of Mn associated with oxygen evolution is seen not to contribute significantly to relaxivity. Instead, nearly all of the observed relaxivity, which is characterized by a paramagnetic maximum near 20.7 MHz in the field dispersion profile of R₁, appears to arise from contaminating non-functional Mn(II) that can be removed by EDTA during the isolation procedure. These observations, which contradict previous reports ascribing chloroplast relaxivity to the water-oxidizing system, require a reevaluation of proposed models, derived from NMR studies, of the state of Mn in the water-splitting reaction.Chloroplasts from which loosely bound non-functional Mn has been removed by EDTA washing do show an enhancement of relaxivity when exposed to NH₂OH at concentrations known to inactivate water oxidation. This NH₂OH-induced relaxivity is comprised of Mn(II) in two distinct paramagnetic sites. One site is chelatable by EDTA, whereas the other site is not. This finding suggests that some Mn(II) tightly bound to thylakoid membranes can contribute to relaxivity after inactivation of the oxygen-evolving reaction.     

A proton-relaxation enhancement study of the interaction of manganous ions with phospholipids in aqueous dispersions.

An interaction of dipalmitoylphosphatidylcholine (PC) and phosphatidylserine (PS) with manganous ions has been investigated by measuring the effect of bound manganese upon the longitudinal relaxation rate, 1/T1, of the solvent water protons and evaluating the enhancement factor epsilon b. The observed enhancement values were used to determine the number of interacting sites per polar head group, n, and the values of association constants, KA, of manganese to PC and PS. Changes in epsilon b correlate with structural changes at the interacting site. By increasing the temperature one can see an abrupt decrease in epsilon b within the temperature interval from 40 to 50 degrees C indicating the thermal phase transition of PC as established by calorimetry, fluorescence and high-resolution NMR measurements. That an enhancement of 1/T1 of the solvent-water protons occurs at all is explained by assuming a restricted rotation of the Mn2+-aquo complex in the bound state. In addition we suppose that the rotation of the Mn2+-aquo complex is the mechanism which dominates the relaxation of the water protons in teh bulk solvent when phospholipids are present.     

Relaxometric characterization of human hemalbumin

Hemalbumin [i.e., Fe(III)-protoporphyrin IX-human serum albumin; Fe(III)heme-HSA] is an important intermediate in the recovery of heme iron following hemolysis. Relaxometric data are consistent with the occurrence of a hexacoordinated high-spin Fe(III) center with no water in the inner coordination sphere. The relatively high relaxation enhancement observed for an aqueous solution of Fe(III)heme-HSA (r1p=4.8 mM(-1)s(-1) at 20 MHz, pH 7, and 25 C) is ascribed to the occurrence of a strong contribution from water molecules in the second coordination sphere. Structural analysis of the putative binding region has been performed by a Monte Carlo simulated annealing procedure, which allowed us to identify His105 and Tyr148 as axial ligands. The role of a tyrosinate as the sixth Fe(III)heme ligand is supported by the pH-dependent analysis. Interestingly, when Fe(III) is replaced by Mn(III), the occurrence of a fast exchanging water molecule at pH values close to neutrality is detected. As the pH is increased, the Mn(III) containing system behaves analogously to Fe(III)heme-HSA. At higher pH, the phenolate ligand is eventually displaced by OH- from both Fe(III) and Mn(III) centers. Support for the proposed bonding scheme has been gained also from competitive binding assays for the sixth coordination site by fluoride, azide, and imidazole ligands.     

Effects of manganese (Mn++) and iron (Fe+++) on magnetic resonance imaging (MRI) characteristics of human placenta and amniotic fluid

The contrast and intensity of a magnetic resonance image (MRI) is affected in part by the spin-lattice relaxation time (T1) and spin-spin relaxation time (T2). Certain paramagnetic metal ions can alter these parameters suggesting that they may be useful contrast agents in MRI. In this study, Mn++ and Fe+++ were examined for their effects on T1 and T2 in human placenta and amniotic fluid (AF) at concentrations between 0.002 and 2.0 mM. Both Mn++ and Fe+++ produced a dose-dependent decrease in placental and AF T1. The effects of Fe+++ were not pronounced, decreasing T1 only at the highest concentrations, and not to the same degree as Mn++. Placental T2 was also significantly decreased by Mn++, whereas Fe+++ had no effect. These differences may be due to molecular binding, uptake by the placenta, or the paramagnetic characteristics of the metals. The results suggest that Mn++ will alter human placental MRI for T1 and to a lesser extent T2-dependent imaging processes. Fe+++ should have little or no effect on human placental MRI, except at very high concentrations.     

Towards MRI contrast agents of improved efficacy. NMR relaxometric investigations of the binding interaction to HSA of a novel heptadentate macrocyclic triphosphonate Gd(III)-complex

 A novel heptacoordinating ligand consisting of a thirteen-membered tetraazamacrocycle containing the pyridine ring and bearing three methylenephosphonate groups (PCTP-[13]) has been synthesized. Its Gd(III) complex displays a remarkably high longitudinal water proton relaxivity (7.7 mM^–1 s^–1 at 25  °C, 20 MHz and pH 7.5) which has been accounted for in terms of contributions arising from (1) one water molecule bound to the metal ion, (2) hydrogen-bonded water molecules in the second coordination sphere, or (3) water molecules diffusing near the paramagnetic chelate. Variable-temperature ¹⁷O-NMR transverse relaxation data indicate that the residence lifetime of the metal-bound water molecule is very short (8.0 ns at 25  °C) with respect to the Gd(III) complexes currently considered as contrast agents for magnetic resonance imaging. Furthermore, GdPCTP-[13] interacts with human serum albumin (HSA), likely through electrostatic forces. By comparing water proton relaxivity data for the GdPCTP-[13]-HSA adduct, measured as a function of temperature and magnetic field strength, with those for the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephosphonate complex lacking a metal-bound water molecule), it has been possible to propose a general picture accounting for the main determinants of the relaxation enhancement observed when a paramagnetic Gd(III) complex is bound to HSA. Basically, the relaxation enhancement in these systems arises from (1) water molecules in the hydration shell of the macromolecule and protein exchangeable protons which lie close to the interaction site of the paramagnetic complex and (2) the metal bound water molecule(s). As far as the latter contribution is concerned, the interaction with the protein causes an elongation of the residence lifetime of the metal-bound water molecule, which limits, to some extent, the potential relaxivity enhancement expected upon the binding of the paramagnetic complex to HSA. Received: 27 January 1997 / Accepted: 12 May 1997     

1H NMR techniques in studies of transport of paramagnetic ions in multicellular systems

Two different pulse sequences used in 1H NMR spectroscopy termed free induction decay amplitude recovery (FIDAR) and spin-echo recovery (SER) were applied to studies of transport of paramagnetic ions in multicellular systems. The molar relaxivity of several paramagnetic species (Fe3+, Co2+, Ni2+, Cu2+, Mn2+, MnEDTA2-, dextran-magnetite) in water solutions was measured at 32 MHz resonance frequency. Ionic transport was studied using Mn2+ and MnEDTA2- as models for cations and anions, respectively, and plant root tissue as a model of a multicellular system.     

NMR studies of primary and secondary sites of parvalbumins using the two paramagnetic probes Gd (III) and Mn (II)

The binding of cations by parvalbumins was studied by the proton relaxation enhancement (PRE) method using the paramagnetic probes Gd(III) and Mn(II). Gd(III) appears as a specific probe of the primary sites CD and EF with the following binding parameters: n = 2, KdGd = 0.5 x 10(-11) M and epsilon b = 2.3. The low value of epsilon b is the result of a nearly complete dehydration of the protein bound ions. Competition experiments between Gd(III) and various diamagnetic cations show the following order of affinity for the EF and CD sites: Mg2+ less than Zn2+ less than Sr2+ less than Ca2+ less than Cd2+ less than La3+ less than or equal to Gd3+. Mn 2+ is a specific probe of a secondary site with the following binding parameters: n = 1, KdMn = 0.6 x 10(-3) M and epsilon b = 17. The high value of epsilon b suggests that the protein bound Mn(II) has retained most of its hydration shell. Competition experiments between (Mn(II) and different cations show similar affinities for this site: Ca2+ less than or equal to Mg2+ less than or equal to Cd2+ less than or equal to Mn2+. This secondary site is located near the EF primary site.     

Metal ion binding properties of hen ovalbumin and S-ovalbumin: characterization of the metal ion binding site by 31P NMR and water proton relaxation rate enhancements

In this study, water proton relaxation rate (PRR) enhancements have been used to characterize the binding of metal ions to native ovalbumin, ovalbumin in which phosphate has been enzymatically cleaved from one or both of the two protein phosphoserines, and a heat-stabilized form of the protein (S-ovalbumin). With Scatchard plots constructed from water PRR enhancements, it was found that native ovalbumin and S-ovalbumin had one strong binding site for Mn2+ ion (KD approximately equal to 6.0 X 10(-4) M). Alkaline phosphatase treated ovalbumin, a protein having a single phosphoserine, had one Mn2+ binding site of slightly weaker affinity (KD approximately equal to 8.3 X 10(-4) M), while acid phosphatase treated ovalbumin, a dephosphorylated protein, had two much weaker Mn2+ ion binding sites (KD approximately equal to 1.3 X 10(-3) M). Competitive binding studies on the native protein suggested that Zn2+ ion competes with Mn2+ for the single strong-affinity site (KD approximately equal to 6.1 X 10(-3) M) while Mg2+ and Ca2+ do not. In a second set of experiments, the paramagnetic contribution to the 31P spin-lattice (T1P) and spin-spin (T2P) relaxation times at three separate magnetic field strengths was measured. Correlation times tau c characterizing Mn2+-31P dipolar relaxation were estimated from the ratios of T1P/T2P at a single field and from the ratios of spin-lattice relaxation rates at three different field strengths. The correlation times so obtained, ranging from about 0.7 to 7.7 ns at the three field strengths, were used in calculating distances from the bound Mn2+ ion to the phosphoserines of native ovalbumin, S-ovalbumin, and alkaline phosphatase treated ovalbumins. It was determined that the phosphate of phosphoserine-68 was 5.95 +/- 0.26 and 6.29 +/- 0.18 A from the Mn2+ in the native and alkaline phosphatase treated protein, respectively, and 6.99 +/- 0.30 A away from the Mn2+ in S-ovalbumin. The phosphate of phosphoserine-344 was determined to be 5.31 +/- 0.20 and 5.75 +/- 0.10 A from the Mn2+ ion in native ovalbumin and S-ovalbumin, respectively. The 13C nucleus of [1-13C]galactose enzymatically transferred to the nonreducing end of the ovalbumin oligosaccharide chain was not found to be significantly relaxed by Mn2+ bound to the protein, even at 1:1 stoichiometric ratio of metal:protein. Using this, we estimate the nonreducing terminal of the ovalbumin oligosaccharide to be at least 39 A from the metal ion binding site on the protein.     

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.     

Metal ion complexes of apoferritin. Evidence for initial binding in the hydrophilic channels

Metal binding to the iron storage protein apoferritin is the first step in the process by which iron accumulates within the protein shell. In the present study, the stoichiometry of metal binding to apoferritin in solution has been examined using the probe ions Mn(II), VO(IV), and Cd(II) in conjunction with EPR spectroscopic and cadmium ion selective electrode measurements. Binding studies were carried out with the individual ions, in competition with one another, and in competition with Fe(II), Fe(III), and Tb(III). All three probe ions show binding stoichiometries near 0.3 and 0.7 metal ion per subunit, close to the theoretically predicted values of 0.33 and 0.67 for the binding of one and two metal ions, respectively, per three subunits. These results in conjunction with other data are consistent with the binding of one, and possibly two, metal ions within each of the eight hydrophilic channels which are located on 3-fold axes leading to the interior of the protein. Pairs of cadmium binding sites have been located in these channels by x-ray crystallography (Rice, D. W., Ford, G. C., White, J. L., Smith, J. M. A., and Harrison, P. M. (1983) Adv. Inorg. Biochem. 5, 39-49). The possibility that some metal binding occurs elsewhere on the protein is not precluded by the present data, however. In competition experiments between various metal ions, approximately 0.3 metal ion per subunit is readily displaced implying common binding sites in the channels for all of them. The stoichiometry of Mn(II) displacement by Fe(II) is less clear. Oxidation of Fe(II) to Fe(III) by molecular oxygen in the presence of Mn(II) regenerates some Mn(II) binding on the protein, suggesting migration of iron(III) to other protein sites, or perhaps to core.     

Synthesis and evaluation of nanoglobular macrocyclic Mn(II) chelate conjugates as non-gadolinium(III) MRI contrast agents

Because of the recent observation of the toxic side effects of Gd(III) based MRI contrast agents in patients with impaired renal function, there is strong interest on developing alternative contrast agents for MRI. In this study, macrocyclic Mn(II) chelates were conjugated to nanoglobular carriers, lysine dendrimers with a silsesquioxane core, to synthesize non-Gd(III) based MRI contrast agents. A generation 3 nanoglobular conjugate of Mn(II)-1,4,7-triaazacyclononane-1,4,7-triacetate-GA amide (G3-NOTA-Mn) was also synthesized and evaluated. The per ion T(1) and T(2) relaxivities of G2, G3, G4 nanoglobular Mn(II)-DOTA monoamide conjugates decreased with increasing generation of the carriers. The T(1) relaxivities of G2, G3, and G4 nanoglobular Mn(II)-DOTA conjugates were 3.3, 2.8, and 2.4 mM(-1) s(-1) per Mn(II) chelate at 3 T, respectively. The T(1) relaxivity of G3-NOTA-Mn was 3.80 mM(-1) s(-1) per Mn(II) chelate at 3 T. The nanoglobular macrocyclic Mn(II) chelate conjugates showed good in vivo stability and were readily excreted via renal filtration. The conjugates resulted in much less nonspecific liver enhancement than MnCl(2) and were effective for contrast-enhanced tumor imaging in nude mice bearing MDA-MB-231 breast tumor xenografts at a dose of 0.03 mmol Mn/kg. The nanoglobular macrocyclic Mn(II) chelate conjugates are promising nongadolinium based MRI contrast agents.     

Influence of paramagnetic ions bound to human serum albumin on water 1HNMR relaxation times

The extent to which various paramagnetic ions (Cu2+, Mn2+ and Gd3+) free and bound to human serum albumin alter the water proton relaxation times at two frequencies has been investigated. NMR relaxation parameters, T1 and T2, were measured at 5 and 10 MHz using a saturation recovery (90 degrees-tau-90 degrees) and a spin-echo (90 degrees-tau-180 degrees) sequence respectively. We found that all three ions enhance their effectiveness in inducing water proton magnetic relaxation when they are bound to human serum albumin and that Gd3+ is the most effective in pure water and Mn2+ in the presence of the protein. Cu2+ has a smaller effect, but it presents an interesting behaviour correlated with the existence of two different binding sites, which is also confirmed by electronic paramagnetic resonance spectra. The results indicate the potential usefulness of large molecular paramagnetic complexes as contrast agents in NMR Imaging.