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Nuclear magnetic relaxation dispersion (NMRD) profiles and ESR linewidths have been measured for a series of neutral Gd(3+)-triazamacrocyclic complexes and compared with previous data for the simplest member of the macrocyclic triaza series, Gd(NOTA), and for the widely studied linear triaza complex, Gd(DTPA)2-. Water proton relaxivities and their temperature dependence were found to vary widely with the size of the triaza macrocycle and the identity of the side-chain chelating groups. The number of rapidly exchanging water molecules directly coordinated to the Gd3+ ion (q) was found to vary from 2 to 4 for eight of the ten complexes examined and a linear relationship between the 50-MHz relaxivity value and integral values of q is presented for this series of complexes. tau s values derived from ESR linewidths for some of the complexes are in reasonable agreement with those derived from their NMRD profiles; however, those complexes which either tended to form aggregates in solution or gave evidence for multiple averaged solution structures showed broad, near Lorentzian linewidths which were clearly not dominated by the electron spin relaxation time (tau s).

By using near-UV circular dichroism (CD) and solvent proton nuclear magnetic relaxation dispersion measurements, three different conformational states have been detected in Ca(2+)-Mn(2+)-concanavalin A upon binding a variety of asparagine-linked carbohydrates. Two of these transitions have been described previously, one for the binding of monosaccharides such as methyl alpha-D-mannopyranoside and oligosaccharides with terminal alpha-Glc or alpha-Man residues, and the second for the binding of oligomannose and complex type carbohydrates (Brewer, C. F., and Bhattacharyya, L. (1986) J. Biol. Chem. 261, 7306-7310). The third transition occurs upon binding a bisected biantennary complex type carbohydrate with terminal GlcNAc residues. Temperature-dependent nuclear magnetic relaxation dispersion and CD measurements have identified regions of the protein near the two metal ion binding sites that are associated with the conformation changes, and Tyr-12, which is part of the monosaccharide binding site, as responsible for the CD changes. The results support our previous conclusions that the rotamer conformation of the (alpha 1,6) arm of bisected complex type oligosaccharides binds to concanavalin A with dihedral angle omega = -60 degrees whereas nonbisected complex type oligosaccharides bind with omega = 180 degrees (Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1294-1299). The present findings also explain the effects of increasing chain length of bisected complex type carbohydrates on their interactions with the lectin.

A preparation of monocrystalline iron oxide nanoparticles with an oxidized starch coating, currently in clinical trials (NC100150 Injection; CLARISCAN), was characterized by magnetization measurements, relaxometry, and photon correlation spectroscopy. By combining the results with a measure of iron content, one can obtain the size and magnetic attributes of the iron cores, including the relevant correlation times for outer sphere relaxation (tau(SO) and tau(D)), and information about the interaction of the organic coating with both core and solvent. The results are 6.43 nm for the iron oxide core diameter, a magnetic moment of 4.38x10(-17) erg/G, and a water-penetrable coating region of oxidized oligomeric starch fragments and entrained water molecules. The latter extends the hydrodynamic diameter to 11.9 nm and lowers the average diffusivity of solvent about 64% (which increases tau(D) accordingly). The nanoparticles show little size-polydispersity, evidenced by the lowest value of r(2)/r(1) at 20 MHz reported to date, an asset for magnetic resonance angiography.

Rationale and objectives Gd-DTPA-BMEA, a nonionic bis(methoxyethylamide) derivative of Gd-DTPA, is the active ingredient of OptiMARK, now awaiting FDA approval. In this study, we compare the relaxivities of Gd-DTPA-BMEA (OptiMARK) with those of the commercially available DTPA-based agents Gd-DTPA2- (Magnevist) and Gd-DTPA-BMA (Omniscan) at different field strengths (1/T1 nuclear magnetic relaxation dispersion (NMRD) profiles). In addition, we study how changes in structural attributes of small paramagnetic chelate complexes of Gd3+ ions influence 1/T1 NMRD profiles. Methods 1/T1 NMRD profiles of Gd-DTPA-BMEA (OptiMARK) were measured at 5 degrees and 35 degrees C and a set of values for the parameters that describe relaxation by Gd(3+)-proton magnetic dipolar interactions was obtained. The rotational (tau R) and the diffusional (tau D) correlation times for Gd-DTPA-BMA were adjusted for the 15% greater molecular weight of Gd-DTPA-BMEA. tau M (the resident lifetime of Gd(3+)-bound water) was obtained from available 17O NMR relaxation data. For tau S0 and tau V (the low-field relaxation time of the Gd3+ moment and its correlation time), Gd-DTPA-BMA values were taken as initial values and tau S0 refined as needed. Results Although, at 35 degrees C, tau M is comparable for the two neutral agents and an order of magnitude longer than that for Gd-DTPA2-, the 1/T1 NMRD profiles of Gd-DTPA-BMEA are indistinguishable from those of Gd-DTPA2- and Gd-DTPA-BMA. A 40% increase in the value of tau S0 from Gd-DTPA2- is required for agreement of data and theory for Gd-DTPA-BMEA. Conclusions Based on their 1/T1 NMRD profiles, the efficacy of the three agents should be identical in typical clinical MRI applications. The data can be fit reliably to theory, and differences in the fit parameters (and structure) have no effect on the three profiles at 35 degrees C. The relatively long values of tau M for the two neutral agents would only be of importance at low temperatures.

The parameters that govern water proton magnetic relaxation (e.g. water exchange rates, and rotational and electronic correlation times) of representatives of two classes of Gd(III) complexes have been estimated, using two different approaches and the results compared with those derived for known analogs. The complexes studied are: (i) the non-ionic GdDTPA-bis(-methoxyethyl-amide) [Gd(DTPA-BMEA)], a typical small-molecule extracellular MR agent, and (ii) the ionic Gd(III) complex of 4-pentylbicyclo[2.2.2]octane-1-carboxyl-di-L-aspartyl-lysine-deriv ed-DTPA [GdL]4-, a prototype MR blood pool agent, which binds to serum albumin in vivo through non-covalent hydrophobic interactions. An 17O-NMR study of [Gd(DTPA-BMEA)] gives a water exchange rate constant of k(ex)298 = (0.39 +/- 0.02) x 10(6) s(-1), identical to that for the bismethylamide analog [Gd(DTPA-BMA)]. Both approaches yield longer rotational correlation times for [Gd(DTPA-BMEA)], consistent with its higher molecular weight. An 17O-NMR study of [GdL]4- gives a water exchange rate constant of k(ex)298 = (4.2 +/- 0.1) x 10(6) s(-1), identical to that for [Gd(DTPA)]2-. The water exchange rate on [GdL]4- did not decrease considerably when bound to albumin, the lowest limit is k(ex,GdL-BSA) = k(ex,GdL)/2. Both approaches yield identical rotational correlation times for [GdL]4-, however, it was difficult to derive a consistent rotational constant for the albumin-bound [GdL]4- using the different approaches (values ranged between 1.0 and 23.0 ns).

Water content and water-proton relaxation rates are reported for fresh, histologically characterized, surgical specimens of calcified human intracranial meningiomas and compared with results for noncalcified meningiomas from an earlier study and with calcium hydroxyapatite (CaHA) suspensions to elucidate the influence of calcification on magnetic resonance imaging (MRI) signal intensity of calcified meningiomas.The magnetic field dependence of 1/T1 of water protons (nuclear magnetic relaxation dispersion profile) and dry weights are reported for 38 calcified nonhemorrhagic and 3 hemorrhagic specimens of known histologic subtype, a subset of the 67 specimens measured earlier. Calcification was considered mild or heavy when the dry weight was within or above the range for noncalcified meningiomas. Preliminary 1/T1 profiles for pure CaHA and a single high-field 1/T2 value also are reported.The ranges of dry weights and of low-field 1/T1 values were twice as large for calcified as for noncalcified meningiomas. No correlation was found between low-field 1/T1 and either histologic subtype or dry weight. Mild calcification produced the highest low-field 1/T1 values; the most heavily calcified tumor had slightly increased low-field 1/T1. Calcium hydroxyapatite increases low-field 1/T1 significantly but not high-field 1/T1; high-field 1/T2 is large. For calcified hemorrhagic meningiomas, increases in both low-field and high-field 1/T1 were seen.For mild calcification, MRI signal voids result from an increased high-field 1/T2; for heavier calcification, reduced proton density (from excluded water) becomes of increasing importance. Cellular CaHA appears to brighten the signal in T1-weighted MRI in the presence of hemorrhage.

Resected meningiomas were examined by relaxometry and light microscopy to evaluate the potential of magnetic resonance imaging (MRI) for identifying histologic subtypes and for discriminating among benign, radiation therapy-induced, and malignant meningiomas.The magnetic field dependence of 1/T1 of water protons (nuclear magnetic relaxation dispersion [NMRD] profile) and the water content (dry weight) were measured for 67 specimens, and the data were compared with histology. Only noncalcified, nonhemorrhagic meningiomas are reported.No correlations were found between NMRD profiles, dry weight, and any histologic subtype, in contrast to an analogous study of astrocytomas. Rather, meningiomas have a broader variability of dry weight and 1/T1 than related parenchyma but a much narrower range than all grades of astrocytomas. The mean value of 1/T1, at all fields, is slightly higher in meningiomas--and the mean water content about the same--as adult cortical gray matter.Meningiomas are frequently isointense with cortex, and histologic subtypes cannot be differentiated at any magnetic field strength by MRI using only T1- or proton density-weighted MRI.

By using near-UV circular dichroism (CD) and solvent proton nuclear magnetic relaxation dispersion measurements, three different conformational states have been detected in Ca(2+)-Mn(2+)-concanavalin A upon binding a variety of asparagine-linked carbohydrates. Two of these transitions have been described previously, one for the binding of monosaccharides such as methyl alpha-D-mannopyranoside and oligosaccharides with terminal alpha-Glc or alpha-Man residues, and the second for the binding of oligomannose and complex type carbohydrates (Brewer, C. F., and Bhattacharyya, L. (1986) J. Biol. Chem. 261, 7306-7310). The third transition occurs upon binding a bisected biantennary complex type carbohydrate with terminal GlcNAc residues. Temperature-dependent nuclear magnetic relaxation dispersion and CD measurements have identified regions of the protein near the two metal ion binding sites that are associated with the conformation changes, and Tyr-12, which is part of the monosaccharide binding site, as responsible for the CD changes. The results support our previous conclusions that the rotamer conformation of the (alpha 1,6) arm of bisected complex type oligosaccharides binds to concanavalin A with dihedral angle omega = -60 degrees whereas nonbisected complex type oligosaccharides bind with omega = 180 degrees (Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1294-1299). The present findings also explain the effects of increasing chain length of bisected complex type carbohydrates on their interactions with the lectin.

The range of compounds, both paramagnetic and ferromagnetic, with potential utility as tracers for enhancing contrast in magnetic resonance imaging is very large. Fortunately, the changes in 1/T1 and 1/T2 of water protons in solution--and ultimately in tissue--induced by these agents arise from variations of only a few physical parameters. In the present work, we first consider the properties of Gd-DTPA2- in solution in some detail, including its effects on both inner and outer sphere relaxation of solvent protons. Then, by varying one or another physical parameter, we derive behavior that characterizes several classes of contrast agents with markedly different properties. These include small macrocycles, such as Gd-DOTA-; larger complexes, including oligomers and conjugates of small paramagnetic complexes with protein; magnetized particles, such as magnetite, as a function of their size; and highly anisotropic porphyrin compounds. Predictions are compared with data from the literature.

It is well established that the spin-lattice magnetic relaxation rate 1/T1 of solvent protons in homogeneous protein solutions increases dramatically as the magnetic field is reduced well below the traditional NMR range. For a 5% solution of protein of 10(5) Daltons, for example, 1/T1 increases from about 50% above the pure solvent rate at 20 MHz to five times the solvent rate at 0.01 MHz. At higher fields, the effect of protein on the relaxation rate decreases progressively toward zero. 1/T1 of solvent in erythrocyte suspension behaves similarly, indicating that extracellular water has ready access to intracellular protein. We now report analogous data for samples of various mammalian tissues: we find that the data can be accommodated within the conceptual framework developed earlier for analyzing homogeneous protein solutions. It appears that tissue water probes the macromolecular composition and structure in a tissue-specific fashion. The variation of 1/T1 with field differs for each tissue, and its magnitude at low fields varies by more than a factor of three, far more than does the water content of the tissues. The relevance to contrast in NMR imaging is discussed.

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 diethylenetriaminepentaacetie 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. © 1986 Academic Press, Inc.

The size dependence of the electrical conductivity, and preliminary results for galvanomagnetic effects, in thin, single crystals of high-purity bismuth at 4.2°K are reported for a range of thicknesses comparable to the electron mean free path. The results, when interpreted according to the theory of Ham and Mattis and of Price (in the accompanying papers), show that the scattering of electrons by the surface is specular, and confirm the novel predictions of the theory for the case of specular reflection and anisotropic surfaces of constant energy.

Many experiments show that the long axes of the three extremely prolate ellipsoids which represent the electron Fermi surface in bismuth are tilted \ensuremath{\sim}6\ifmmode^\circ\else\textdegree\fi{} from the trigonal plane of the Brillouin zone; however, the sign of this tilt angle cannot be unambiguously obtained from the literature. We report results obtained from measurements of the de Haas-van Alphen and de Haas-Shubnikov effects and quadratic magneto-resistance which show that the sign of the tilt angle is positive in terms of the usual conventions, which are carefully defined. More explicitly, the long axis of each ellipsoid lies within the \ensuremath{\sim}18\ifmmode^\circ\else\textdegree\fi{} angle made by a bisectrix direction and a $\ensuremath{\Gamma}L$ direction in the Brillouin zone. Our results are shown to agree with the most recent band calculations for Bi. Comparison of de Haas-van Alphen results on Bi and Sb samples, each with the same orientation as determined by x rays, shows that, independently of any convertion, the tilt angles of Bi and Sb have opposite signs. We also clarify the procedures for properly orienting a crystal of bismuth by observing either twinning planes or etch pits in the trigonal plane.

We shall interpret the galvanomagnetic prroperties of bismuth, antimony and the Bi-Sb alloy system in terms of bands of carriers known to exist in the pure semimetals. In our interpretation, the new carriers, presumed holes, observed by several investigators in antimony must have slightly warped oblate ellipsoidal energy surfaces to account for the galvanomagnetic data of Juretschke et al, rather than tilted ellipsoidal surfaces. These new holes, together with the well established holes in bismuth and the Shoenberg electrons in both bismuth and antimony, account for the anomalous Hall coefficients of antimony. Consideration of qualitative features of the nearly free electron model of Jones and Harrison suggests that a linear variation of the spatial potential upon alloying bismuth with antimony is sufficient to account for the considerable changes observed in the transport properties of the alloy system. This model also suggests that the conduction band minima in bismuth are located at different points in the Brillouin zone from those in antimony. Experimental support for these hypotheses is discussed.

Bovine and human B erythrocyte carbonic anhydrases substituted with Co2+ cause an enhancement of the nuclear magnetic relaxation rate of solvent water protons (T1-1) at high pH that is decreased by the addition of carbonic anhydrase inhibitors such as azide and Ethoxzolamide. The part of T1-1 which can be inhibited by Ethoxzolamide is due to exchangeable protons located at the active site of the enzyme. This inhibitable part of T1-1 is pH-dependent with a pK of 7.0 ± 0.2 for the bovine cobalt enzyme and a pK of 8.2 ± 0.2 for the human B cobalt enzyme. From the magnetic field dependence of the inhibitable part of T1-1, a correlation time for the dipolar interaction of a proton with a cobalt electronic spin of 10-11 sec, a proton-cobalt distance of 2.5 to 2.9 A, and for the bovine enzyme a proton residence time, τm, small compared to 10-5 sec are calculated. We conclude from the pH dependence and the proton-cobalt distance that the proton which contributes to the inhibitable part of T1-1 is located either on a hydroxide ion bound to the Co2+ at the active site or on a water molecule with 1 proton hydrogen bonded to a nearby residue. In contrast to the similarity of the inhibitable part of T1-1 for the bovine and human B cobalt enzymes, there is a non-inhibitable, pH-independent contribution resulting from the cobalt substitution, relatively large for the bovine enzyme and very small for the human B enzyme, that is not understood. The contributions of the (diamagnetic) native zinc enzymes to T1-1 have also been measured. As in the case of apotransferrin (Koenig, S. H., and Schillinger, W. E., J. Biol. Chem., 244, 3283 (1969)), the results agree qualitatively but not quantitatively with theory.

We report measurements of the magnetic field dependence of the nuclear magnetic spin lattice relaxation rate T1-1 of solvent protons in solutions of the diamagnetic protein apotransferrin, as a function of temperature, concentration, and pH. The bulk of the data were taken over a range of magnetic field (4 Oe to 1000 Oe) much lower than that traditionally used in nuclear magnetic resonance experiments. The major result in that T1-1 is highly magnetic field-dependent; it is very large below 10 Oe, and decreases monotonically as the magnetic field is increased. The data are compared with the predictions of several models for the interaction of solvent protons with the protein molecules, among them the model (Daszkiewicz, O. K., Hennel, T. W., Lubas, B., and Szczepkowski, T. W., Nature, 200, 1006 (1963)) in which water molecules bind irrotationally to the protein surface. The theory for all of the models is very similar. The conclusions are that proton exchange between the bulk water and the protein surface occurs via the exchange of water molecules in the pH range 5 to 8. At higher pH values, direct proton exchange with basic amino acid residues may be important. The theory shows that exchanging water molecules need not be irrotationally bound to the protein; it is sufficient that the water molecules be restricted to rotate about an axis that maintains a fixed geometry with respect to the protein molecule. The number of water molecules involved is a small fraction of the number usually considered to be in the first hydration shell. From the temperature dependence of the data, it is shown that the variation of T1-1 with magnetic field is related to the rotational diffusion relaxation time of the protein molecules, for all models considered. Additionally, the lifetime on the protein molecules of the exchanging entities, whether entire water molecules or individual protons, is in the range 0.1 to 10 µsec.

The structure factor $S(Q)$ for liquid argon at 85 \ifmmode^\circ\else\textdegree\fi{}K has been determined in a neutron scattering experiment to an accuracy of \ensuremath{\sim}0.01. The problem of obtaining $S(Q)$ from the results of neutron scattering measurements, made as a function of scattering angle using a detector with an energy-dependent efficiency, is considered in detail. Smooth curves for $S(Q)$ and its Fourier transform, the radial distribution function $g(r)$, were obtained from the experimental data by an iterative procedure suggested by Verlet and carried out by Schiff, and are tabulated for convenience. The experimental results are in excellent agreement with the predictions of computer simulations based on either the Lennard-Jones two-body interaction, or on the Barker, Fisher, and Watts potential with corrections for three-body and quantum effects included.

To investigate the role of specifically bound bicarbonate in the metal-complexing function of transferrin, copper transferrin was prepared both in the presence and absence of bicarbonate ion. The magnetic field and temperature dependence of the solvent proton spin-lattice relaxation rate of solutions of the two complexes were determined. The molar relaxivity R (essentially, relaxation rate per protein concentration) for solutions of copper transferrin is very large at low fields (∼20,000 m-1 sec-1 at 4 Oe) and is highly field-dependent, decreasing to almost zero at 12 kOe. At low field, R is almost temperature-independent over the range 0–45°. Comparison of the results with theory indicates that protons are relaxed by a dipolar interaction with the unpaired electronic spin of Cu2+. The observed relaxivity results from the rapid exchange of a water molecule bound sufficiently close to the copper to be an axial ligand. The magnitude of R at low field for copper transferrin-HCO3 is about one-quarter of that for copper transferrin and also disperses almost to zero at high field. Above 25° the low field value of R has a strong positive temperature dependence. There is no significant variation in R over the pH range 7 to 9.5. The data suggest that, as in the absence of bicarbonate, the protons of water molecules are relaxed by a dipolar interaction with the electron spin of Cu2+, and that bicarbonate binding alters the water exchange rate.

Dispersion curves for phonons propagating in the trigonal direction in bismuth at room temperature and at 75°K have been obtained in a neutron inelastic scattering experiment. Observed frequencies (units 1013 rad/sec) at 75°K are as follows: at the zone center, ωTO = 1.40 ± 0.02, ωLO = 1.89 ± 0.02; at the zone boundary in the trigonal direction, ωTA = 0.73 ± 0.01, ωLA = 1.12 ± 0.02, ωTO = 1.91 ± 0.02, ωLO = 2.03 ± 0.02. At room temperature, the observed frequencies were about 1.5 percent lower. Data were also obtained for longitudinal phonons propagating in the binary direction at 75°K. It is interesting to note that the splitting between the zone boundary frequencies for the optical and acoustic branches for each polarization is quite large. This splitting is difficult to understand if bismuth is thought of as a slightly distorted simple cubic lattice. The experimental results may be qualitatively understood if bismuth is considered to be made up of a series of double layers normal to the trigonal axis. The atoms in each double layer form a crinkled hexagonal net with strong, probably covalent, bonds between atoms. The forces between atoms on adjacent double layers are relatively weak. This model is consistent with the easy cleavage of bismuth normal to the trigonal axis. Analysis of the trigonal dispersion curves in terms of a linear chain model indicates that there are significant forces connecting a given atom with atoms situated on the four planes on either side of it.

It is pointed out that if the Kohn effect is looked for, not as an obvious kink in the phonon dispersion curves of metals as originally suggested, but as a damped sinusoidal variation of the interplanar force constants with separation, then (1) the effect has been observed in many metals and (2) for Na, which is very close to a free-electron metal, the measured force constants oscillate with the expected wavelength ($\frac{\ensuremath{\pi}}{{k}_{F}}$). From the data for Al and Pb, it would appear that even though the Fermi surface is quite close to a free-electron sphere in the extended zone, electrons do not scatter isotropically around this surface, but presumably stay within their respective Brillouin zones even at temperatures of the order of the Debye $\ensuremath{\theta}$.

The ratio of the $g$ value of potassium ${\mathrm{K}}^{39}$ in the ground state to the gyromagnetic ratio of the proton in mineral oil has been measured in an experiment combining atomic beam and nuclear resonance techniques. It is found to be $\frac{{g}_{J}(\mathrm{K})}{{g}_{p}}=658.2274\ifmmode\pm\else\textpm\fi{}0.0023$. This result, when combined with the measurement of $\frac{{g}_{J}(\mathrm{H})}{{g}_{p}}$ by Koenig, Prodell, and Kusch, yields $\frac{{g}_{J}(\mathrm{K})}{{g}_{J}(\mathrm{H})}=1.000016\ifmmode\pm\else\textpm\fi{}0.0004 \mathrm{percent}.$ This is to be compared with the recent direct measurement by Pohlman, Bederson, and Eisinger: $\frac{{g}_{J}(\mathrm{K})}{{g}_{J}(\mathrm{H})}=1.00016\ifmmode\pm\else\textpm\fi{}0.006 \mathrm{percent}$. The present experiment is of superior precision and is in agreement with independent experimental evidence. The present result is found to agree with the theoretically predicted value.

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.

Measurements of the magnetic field dependence of the longitudinal magnetic relaxation rates (NMRD profiles) of solvent protons and deuterons led to the discovery of two classes of solvent binding sites in Ca2+-Mn2+-concanavalin A (CMPL) [Koenig, S. H., Brown, R. D., III,Brewer, C. F. (1985) Biochemistry (second of three papers in this issue)]. In this paper, we compare proton and deuteron NMRD profiles of Ca2+-Mn2+-lentil lectin (CMLcH) and Ca2+-Mn2+-pea lectin (CMPSA) with those of CMPL. All three metalloproteins are D-mannose/D-glucose-specific lectins that have a high degree of structural similarity and require the metal ions for their biological activities. We have developed a method for the preparation of fully active metal ion derivatives of lentil lectin (LcH) and pea lectin (PSA), including the diamagnetic derivatives Ca2+-Zn2+-LcH and Ca2+-Zn2+-PSA [Bhattacharyya, L., Brewer, C. F., Brown, R. D., III,Koenig, S. H.(1984) Biochem. Biophys. Res. Commun. 124, 857-862]. The behavior of these two lectins with regard to their NMRD profiles is essentially identical, for both the paramagnetic and diamagnetic forms. Together with CMPL, all three lectins have a common paramagnetic contribution with a negative temperature dependence of the rates, while CMPL contributes an additional component with a positive temperature dependence. The common contribution derives from the class of fast exchanging water molecules observed in the proton NMRD profile of CMPL (Koenig et al., 1985); their protons are calculated to be relatively remote from the Mn2+ ions (4.4 A for CMPL and 5.5 A for LcH and PSA).(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of Mn2+ aquo ions with native horse-liver alcohol dehydrogenase demetalized specifically at the catalytic sites has been investigated by studying the magnetic field dependence and time dependence of the magnetic spin-lattice relaxation rate of solvent water protons. We find no detectable binding of Mn2+ ions to the catalytic sites in times on the order of hours; however, we do find that these ions bind to the enzyme at two previously unreported types of sites: one, characterized by a low dissociation constant (0.01 mM at pH 7.7, 5 degrees C), low relaxivity, and a stoichiometry of one per two catalytic sites, and a second, with a high dissociation constant (1.5 mM at pH 7.7, 5 degrees C) and high relaxivity. The stoichiometry of the second type of site could not be determined because of the relatively weak bindng of Mn2+ ions to these sites. Both Zn2+ and Cd2+ ions bind to the newly found tight-binding sites, displacing Mn2+ ions and thereby altering the relaxation rates of solvent protons. By monitoring the return to equilibrium of these altered rates, we find that Zn2+ ions enter the catalytic sites from the new tight-binding sites with an on-rate of approximately 0.1 M-1 s-1. It is not clear whether binding to these new sites is an obligatory intermediate for reintroduction of Zn2+ ions into the catalytic sites, but a small excess of Zn2+ ions beyond one per monomer causes the protein to precipitate. Cd2+ ions, by contrast, enter the catalytic sites at least 1 order of magnitude more rapidly than do Zn2+ ions, a rate too rapid to observe by our techniques. However, once the catalytic sites are filled, Cd2+ ions displace Mn2+ ions at the new sites as do Zn2+ ions.

Gd(DTPA)2− (diethylenetriaminepentaacetic acid) and the polyaza macrocyclic Gd(DOTA)− (1,4,7,10-tetraazacyclododecane-N, N′,N″,N′″) are paradigms of general purpose paramagnetic complexes useful for enhancing contrast in magnetic resonance imaging (MRI). It is of both fundamental and practical interest to determine how one might modify the chemical structure of these chelate complexes to improve their utility for MRI in specific circumstances. In the present work, we investigated polyaza methylene phosphonate complexes of Gd3+ ions to compare their NMRD profiles with those of their carboxylate analogs and with Gd(DTPA)2−. We find that the number q of exchangeable water molecules coordinated directly to the Gd3+ ions tends to be smaller in the phosphonates, in principle reducing their utility in MRI. However, these phosphonates have a tendency to oligomerize, and the resulting decrease in rotational mobility of the paramagnetic oligomers increases their relaxivity at higher fields, offsetting the effect of decreases in q. In particular, Gd(DOTRP)3− (1,5,9-triazacyclododecane N, N′,N″,-tris (methylenephosphonic acid) would be an increasingly effective contrast agent above ∼ 10 MHz if the oligomerization was stable in vivo (and the Gd3+ ions were sufficiently well bound). At lower fields, the relaxivity of these small chelate complexes is dominated by τso, the relaxation time of the spin moments of the paramagnetic ions. We find this to be favorably long for complexes of Gd3+ with the macrocyclic phosphonate ligands, as was found earlier by us for Gd( DOTA)−. This situation, ostensibly related to the relatively high symmetry and rigidity of the macrocyclic complexes, can increase the low-field relaxivity of the phosphonates almost a factor of 2 beyond that of Gd(DTPA)2−. © 1989 Academic Press, Inc.

The carbonic anhydrases (EC 4.2.1.1), zinc metallo-enzymes found, for example, in mammalian erythrocytes, catalyze the reversible hydration-dehydration reaction CO2 + H2O ⇌ H+ + HCO 3 − (cf. [8, 30, 40] for reviews).

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.

Earlier studies of the magnetic field dependence of the nuclear spin magnetic relaxation rate of solvent protons in solutions of diamagnetic proteins have indicated that this dependence (called relaxation dispersion) is related to the rotational Brownian motion of solute proteins. In essence, the dispersion is such that 1/T1 (the proton spin-lattice relaxation rate) decreases monotonically as the magnetic field is increased from a very low value (approximately 10 Oe); the dispersion has a point of inflection at a value of magnetic field which depends on protein size, shape, concentration, temperature, and solvent composition. The value of the proton Larmor precession frequency nu(c) at the inflection field appears to relate to tau (R), the rotational relaxation time of the protein molecules. We have measured proton relaxation dispersions for solutions of various proteins that span a three-decade range of molecular weights, and for one sample of transfer ribonucleic acid. We have also measured deuteron relaxation dispersions for solutions of three proteins: lysozyme, carbonmonoxyhemoglobin, and Helix pomatia hemocyanin with molecular weight 900 000. A quantitative relationship between both proton and deuteron dispersion data and protein rotational relaxation is confirmed, and the point is made that magnetic dispersion measurements are of very general applicability for measuring the rotational relaxation rate of macromolecules in solution. It has been previously shown that the influence of proton motion on the relaxation behavior of the solvent is not due to exchange of solvent molecules between the bulk solvent and a hydration region of the protein. In the present paper, we suggest that the interaction results from a long range hydrodynamic effect fundamental to the situation of large Brownian particles in an essentially continuum fluid. The general features of the proposed mechanism are indicated, but no theoretical computations are presented.

A CONTROVERSIAL question in the chemistry of the transferrins is whether the two specific sites of the proteins bind metal ions pairwise or randomly. Equilibrium dialysis studies by Aasa et al.1, in which care was taken to show that true equilibrium had been achieved, indicated that the stability constants for the sites were virtually identical; this suggested that the binding sites were equivalent and non-interacting. This work was corroborated by studies using the techniques of moving boundary electrophoresis2 and isoelectric focusing3. In each of these studies, three species of transferrin were recognized and it was suggested that these represented molecules containing nought, one or two iron atoms. This assignment has been challenged by Wood-worth et al.4, who argued that the species with intermediate electrophoretic mobility or intermediate isoelectric point was really a dimer composed of molecules devoid of iron and molecules containing two iron atoms. This alternative interpretation was taken as supporting the earlier contention of Woodworth based on kinetic studies that iron atoms bind to transferrin only pairwise, and that solutions of transferrin less than fully saturated with iron would contain only two separable species of protein molecules, those without any iron and those with two iron atoms per molecule5.

The inner-sphere water coordination number for Eu3+ and Gd3+ complexed with five DTPA analogs, in which one or two terminal carboxylate groups are functionalized as propyl amides or propyl esters, have been studied using phosphorescence lifetime and nuclear magnetic relaxation dispersion (NMRD) measurements. Both methods show that the water coordination number does not increase above that observed for the analogous DTPA complexes. The phosphorescence lifetime results indicate that all five Eu3+ complexes have one inner-sphere water molecule at 25 degrees C. The NMRD profiles for three of the Gd3+ complexes at 25 degrees C are also consistent with one inner-sphere water molecule, whereas two complexes have profiles consistent only with a mixture of complexes, 50% containing a single water molecule and 50% with none. Lowering the temperature alters the population of these species such that all five Gd3+ complexes have significantly less bound water on average at 5 degrees C. These results explain the anomalous temperature dependencies of the NMRD curves reported previously for the Gd(DTPA)-protein conjugates. We suggest that the Gd(DTPA)-conjugate systems have a fluxional coordination sphere whereby the amount of inner-sphere coordinated water varies from near zero at 5 degrees C to a high of two near 37 degrees C.