Water Proton Relaxation Rate Research Articles

Transition from Lorentzian to Gaussian line shape of magnetization transfer spectrum in bovine serum albumin solutions.

Magnetization transfer experiments using an off-resonance irradiation technique were performed on bovine serum albumin solutions by varying the irradiation frequency and the concentration. A transition of the magnetization transfer spectrum from Lorentzian to Gaussian line shape was observed around the critical concentration of 6.2 mmoles of protein to kg of solution. Observed magnetizations were well expressed by the rate equations of populations for spins below and above the transition, which yielded the magnetization transfer rates, the intrinsic relaxation rates of both protein and water protons, and the effective tumbling time and the rigid line width of the protein. The result showed that the estimates of the values for magnetization transfer rate do not change once the critical concentration is reached.

Dynamic properties of bound water studied through macroscopic water relaxations in concentrated protein solutions

Magnetization transfer experiments using an off-resonance irradiation technique were performed on bovine serum albumin solutions by varying the irradiation frequency and concentration. Observed macroscopic magnetizations of water protons were well expressed by the rate equations of populations for spins, which gave the tumbling time of protein protons and the intrinsic relaxation rates of water and protein protons. These parameters conformed to a model of rapid-exchange water system with bound water molecules in the interior of the protein that interact with protein protons. Analysis of the data enabled the separation of relaxation rates into the respective contributions by the interior bound water and water in the hydration layer at tbe protein surface, and determined the amounts and the average correlation times of these water fractions. The average residence time of the interior bound water with respect to exchange with the bulk water was found to be (5 ± 2). 10 -6 s. The estimation of the hydration layer showed excellent agreement with the amount measured by a thermodynamical method.

Dual divalent cation requirement of the MutT dGTPase. Kinetic and magnetic resonance studies of the metal and substrate complexes.

Kinetic analyses of both the Mn(2+)- and Mg(2+)-activated hydrolysis of dGTP by MutT show the requirement for two divalent cations. Whereas Mn2+ supports a 20-fold lower kcat (0.19 s-1) than Mg2+ (4.0 s-1), the Km of Mn2+.dGTP (6.3 microM) is 45-fold lower than that of Mg2+.dGTP (284 microM). Adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPCPP) is a linear competitive inhibitor with respect to dGTP with a Ki for Mg2+.AMPCPP (42 microM) which is 57-fold lower than the Ki of Mg2+.AMPCPP (2.4 mM). Such tightening suggests that a metal-bridge E.M2+.NTP.M2+ complex is the catalytically active species. The 12 dissociation constants describing the quaternary MutT.M2+.AMPCPP.M2+ complex were evaluated for both Mn2+ and Mg2+, using EPR and NMR methods. MutT binds a single Mn2+ with a Kd of 130 +/- 40 microM in reasonable agreement with the kinetically determined activator constant of Mn2+ of 230 +/- 72 microM. The MutT.AMPCPP complex binds two Mn2+ ions, the weaker of which has a Kd of 16 +/- 2 microM in agreement with the kinetically determined KmMn2+ of 26 +/- 10 microM. MutT.Mn2+ binds Mn2+.AMPCPP with Kd of 16 +/- 4 microM, whereas MutT alone binds Mn2+.AMPCPP with a Kd of 135 +/- 30 microM. The 17-fold enhanced paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons found with the binary MutT.Mn2+ complex decreases to 4.7-fold upon binding of AMPCPP and to 8.7-fold upon binding of Mn2+.AMPCPP, further supporting a metal-bridge MutT.M2+.NTP.M2+ complex. By competition with Mn2+ MutT binds Mg2+ at one site with a Kd of 7.5 mM, and MutT.AMPCPP binds Mg2+ at two sites, the weaker of which has a Kd of 0.9 mM. These values are comparable to the kinetically determined KaMg of 15 +/- 7 mM and KmMg of 1.7 +/- 0.7 mM, respectively. Studies with the racemic, substitution-inert beta, gamma-bidentate tetraamminecobalt (III)-beta,gamma-phosphate-ATP (Co3+(NH3)4ATP) complex show that MutT slowly hydrolyzes only the lambda stereoisomer but requires Mg2+ or Mn2+ to do so, confirming a dual metal ion requirement.

NMR relaxometric studies of water accessibility to haem cavity in horse heart and sperm whale myoglobin

AbstractThe measurement of longitudinal relaxation rates of water protons in solutions containing horse heart and sperm whale metmyoglobins at various pH values and temperatures shows that the two species differ essentially in the exchange lifetime, τM, of the water molecule in the haem cavity. The 1/T1 nuclear magnetic relaxation dispersion dispersion profiles support this view whereas 17O NMR spectroscopy shows that the exchange mechanism involves the whole water molecule. The lack of any 1H/2H isotope effect on the exchange rate suggests that the displacement of the coordinated water molecule from Fe(III) is not the rate‐determining step in the overall exchange process. In contrast to the parent species, no difference is detectable from the relaxometric investigations on the corresponding fluorometmyoglobin derivatives.

Mutational tests of the NMR‐docked structure of the staphylococcal nuclease–metal–3′,5′‐pdTp complex

In the X-ray structure of the staphylococcal nuclease-Ca(2+)-3',5'-pdTp complex, the conformation of the inhibitor 3',5'-pdTp is distorted by Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5:183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3',5'-pdTp in the staphylococcal nuclease-metal-pdTp complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D.J., Serpersu, E.H., Gittis, A.G., Lattman, E.E., Mildvan, A.S. (preceding paper)]. In the NMR-docked structure, the 5'-phosphate of 3',5'-pdTp overlaps with that in the X-ray structure. However, the 3'-phosphate accepts a hydrogen bond from Lys-49 (2.89 A) rather than from Lys-84 (8.63 A), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 A) which does not occur in the X-ray structure (5.28 A). These interactions have been tested by binding studies of 3',5'-pdTp, Ca2+, and Mn2+ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca2+ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While the K84A mutation did not significantly weaken 3',5'-pdTp binding to the enzyme (1.5 +/- 0.7 fold), the K49A mutation weakened 3',5'-pdTp binding to the enzyme by the factor of 4.4 +/- 1.8-fold. Similarly, the Y115A mutation weakened 3',5'-pdTp binding to the enzyme 3.6 +/- 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca(2+)-3',5'-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3',5'-pdTp than by the X-ray structure.

Voltage-sensitive magnetic gels as magnetic resonance monitoring agents.

Many polymer gels undergo a volume phase transition in electric fields. We report here the synthesis of a polyelectrolyte gel that incorporates magnetic particles of iron oxide into the polymer network; these particles couple the gel volume transition to the NMR relaxation times of the surrounding water. We find that the water proton relaxation rates (T2(-1)) in aqueous suspensions of particles of the magnetic gel increase significantly in an electric field. Such changes can also be induced by the hyperpolarization of red blood cells in the suspension, presumably as a result of the electric fields at the cell membrane surfaces. These gels may play a part in magnetic resonance imaging analogous to that of voltage-sensitive dyes in optical imaging.

Comparative study of the reduction rates of various types of imidazoline radicals in tissues

During recent years aminoxyls have been studied as perspective contrast agents for NMR imaging in biological systems. We report a study of 30 low-molecular-weight imidazoline radicals in which we have compared the initial rates of reduction of these compounds by rat liver microsomes and ascorbate. The nuclear magnetic relaxation rate of water protons in microsomes was determined in the presence of a number of aminoxyls and it was found that the rates depend on the substituents. In microsomes the weakest oxidants among these aminoxyls are imidazolines. This type of radical is also the most resistant reduction by ascorbate.

The cooperative binding of phenylalanine to phenylalanine 4-monooxygenase studied by 1H-NMR paramagnetic relaxation. Changes in water accessibility to the iron at the active site upon substrate binding.

The effect of the paramagnetic high-spin Fe(III) ion in phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1) on the water proton longitudinal relaxation rate has been used to study the environment of the iron center. The relaxation rate was measured as a function of the concentration of enzyme, substrate (phenylalanine), inhibitor (noradrenaline) and activator (lysolecithin), as well as of the temperature (18-40 degrees C) and the external magnetic field strength (100-600 MHz). From the frequency dependence of the relaxation rate, an effective correlation time (tau c) of 4.2(+/- 0.5) x 10(-10) s was calculated for the enzyme-substrate complex, which most likely represents the electron spin relaxation rate (tau s) for Fe(III) (S = 5/2) in this complex. The relaxation rate was proportional to the concentration of enzyme (0.04-1 mM) both in the absence and presence of phenylalanine, but the paramagnetic molar relaxivity at 400 MHz and 22 degrees C decreased from 2.2(+/- 0.05) x 10(3) s-1.M-1 in the enzyme as isolated to 1.2(+/- 0.06) x 10(3) s-1.M-1 in the presence of saturating concentrations of the substrate. The activation energy of the relaxation rate also decreased from 11.3 +/- 0.8 kJ/mol to -1.5 +/- 0.2 kJ/mol upon incubation of the enzyme with 5 mM phenylalanine. The results obtained can be interpreted in terms of a slowly exchanging water molecule coordinated to the catalytic paramagnetic Fe(III) in the native and resting enzyme, and that this water molecule seems to be displaced from coordination on the binding of substrate or inhibitor. Moreover, the effect of increasing concentrations of phenylalanine and noradrenaline on the water proton relaxation rate and on the hydrophobic surface properties of the enzyme indicate that substrate and inhibitor induce a similar cooperative conformational change upon binding at the active site. By contrast, the activator lysolecithin does not seem to affect the interaction of water with the catalytic Fe(III).

NMR relaxation in binary aqueous mixtures of acetone and tetrahydrofurane

NMR proton relaxation rates of normal and 17O-enriched water and deuteron relaxation rates of heavy water were measured in mixtures with acetone and tetrahydrofurane at different compositions. The 17O-induced proton relaxation rate was extracted and from this the rotational correlation time of water was determined. Using these correlation times the composition-dependence of the deuteron quadrupole coupling constant of water was derived. A strong variation was found for the system acetone-water, whereas little variation was observed for tetrahydrofurane-water.

Synthesis, characterization, and 1/T1 NMRD profiles of gadolinium(III) complexes of monoamide derivatives of DOTA-like ligands. X-ray structure of the 10-[2-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-1-[(phenylmethoxy)methyl]-2-oxoethyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-gadolinium(III) complex

The synthesis of two novel DOTA-like ligands (5a,b) containing a polyhydroxy(benzyloxy)propionamide substituent and their Gd(III) complexes (6a,b) is reported. Debenzylation by hydrogenolysis of the latter complexes in the presence of Pd/C leads to the corresponding derivatives (7a,b) with a primary alcoholic function. Water proton relaxation rates of aqueous solutions of 6a,b and 7a,b strongly suggest that these complexes contain only one water molecule in their inner coordination sphere, as was previously found for the parent DOTA complex

Relaxation enhancement of water protons by manganese(III) porphyrins: influence of porphyrin aggregation

The ability of eight manganese (III) porphyrins to enhance the longitudinal relaxation rate of water protons in aqueous solution has been studied by nuclear magnetic resonance dispersion (NMRD). In NMRD, the relaxation rates of solvent water protons are measured as a function of proton Larmor frequency from 0.01 to 50 MHz (field strengths from 0.23 mT to 1.17 T); a plot of relaxation rate versus frequency is called an NMRD profile. To enable comparison of the data, the observed rates are expressed as relaxivities by normalizing the rates to the concentration of manganese (III) porphyrin

Magnetically coupled paramagnetic relaxation agents.

Measurements of the spin-lattice relaxation rates of water protons made over a wide frequency range have demonstrated that the effects of paramagnetic relaxation agents may be considerably enhanced when the paramagnetic center is incorporated into a compact structure macromolecule such as a protein that is rotationally constrained. The immobilization of the macromolecule profoundly changes the nature of the magnetic field dependence of the relaxation rate for both the diamagnetic and the paramagnetic samples. The immobilization also amplifies the effect of the paramagnetic center as a water-proton relaxation agent. The direct exchange of labile water molecules or protons is not a requirement for the high efficiency of this class of magnetic relaxation agents.

Metal ion specificity at the catalytic site of yeast enolase.

A new, more gentle enzyme purification for yeast enolase was developed. A series of kinetic experiments was performed with yeast enolase where the concentration of Mg(II) is kept constant and at the Km' level; the addition of Mn(II), Zn(II), or Cu(II) gives a hyperbolic decrease in the enzyme activity. The final velocity of these mixed-metal systems is the same as the velocity obtained only with Mn(II), Zn(II), or Cu(II), respectively. The concentration of the second metal that gives half-maximal effect in the presence of Mg(II) is approximately the same as the apparent Km (Km') value measured for that cation alone. Direct binding of Mn(II) to apoenolase in the absence and presence of Mg(II) shows that Mn(II) and Mg(II) compete for the same metal site on enolase. In the presence of D-2-phosphoglycerate (PGA) and Mg(II), only a single cation site per monomer is occupied by Mn(II). Water proton relaxation rate (PRR) studies of enzyme-ligand complexes containing Mn(II) and Mn(II) in the presence of Mg(II) are consistent with Mn(II) binding at site I under both conditions. PRR titrations of ligands such as the substrate PGA or the inhibitors orthophosphate or fluoride to the enolase-Mn(II)-Mg(II) complex are similar to those obtained for the enolase-Mn(II) complex, also indicating that Mn(II) is at site I in the presence of Mg(II). High-resolution 1H and 31P NMR was used to determine the paramagnetic effect of enolase-bound Mn(II) on the relaxation rates of the nuclei of the competitive inhibitor phosphoglycolate. The distances between the bound Mn(II) and the nuclei were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear magnetic spin-lattice relaxation of water protons caused by metal cage compounds.

The nuclear magnetic spin-lattice relaxation rates of water protons are reported for solutions of manganese(II), copper(II), and chromium(III) cage complexes of the sarcophagine type. As simple aqueous solutions, the complexes are only modest magnetic relaxation agents, presumably because they lack protons on atoms in the first-coordination-sphere protons that are sufficiently labile to mix the large relaxation rate at the metal complex with that of the bulk solvent. The relaxation is approximately modeled using spectral density functions derived for translational diffusion of the interacting dipole moments with the modification that the electron spin relaxation rate is directly included as a contribution to the correlation time. In all cases studied, the electron spin relaxation rate is sufficiently large that it contributes directly to the water-proton spin relaxation process. The poor relaxation efficiency of the cage compound may, however, be improved dramatically by binding the complex to a protein. The efficiency is improved even further if the rotational motion of the protein is reduced drastically by an intermolecular cross-linking reaction. The relaxation efficiency of the cross-linked protein-cage complexes rivals that of the best first-coordination-sphere relaxation agents like [Gd(DTPA)(H2O)]2- and [Gd(DOTA)(H2O)]-.

NMR proton relaxation and chemical exchange in the system H162O/H172O-[2H6]dimethylsulphoxide

NMR proton relaxation rates of normal and 17O enriched water in a mixture of 68 mol% water and 32 mol% [2H6]dimethylsulphoxide were measured for temperatures between 298 K and 183 K. In the range between 240 K and 204 K the limit of fast proton-proton exchange between H16 2O and H17 2O is not obeyed, and relaxation curves deviate from mono-exponential behaviour. By fitting the relaxation curves to a model of NMR two-phase relaxation the proton-proton exchange rate within the aqueous component could be obtained. With decreasing temperature, proton-proton exchange slows down and a residence time of about 125 ms at 215 K is found, but it becomes faster again for still lower temperatures. From the phase-averaged relaxation rates of water in the 17O enriched mixtures, the 17O induced proton relaxation rate was derived as a function of temperature. This yields the rotational correlation times of the water molecule in the mixture and the dipolar spin-lattice coupling parameter. The latter is considerably lower t...

Water-proton nuclear magnetic relaxation in heterogeneous systems: hydrated lysozyme results.

Spin-lattice relaxation rates of water protons in hydrated immobilized lysozyme are measured as a function of magnetic field strength. The dependence of water relaxation versus hydration is presented from 35 to 55% by weight water content. The water-proton relaxation is directly coupled to that of the protein and the coupling exists in the absence of chemical exchange. A model is applied where relaxation within the two proton phases is coupled through a dipolar cross-relaxation mechanism as well as chemical exchange. The observed amplitudes of the water-proton relaxation profiles scale with the ratio of protein to water protons as well as the protein-proton relaxation rate. The field dependence of the protein-proton spin-lattice relaxation is presented in the presence of D2O where a cross-relaxation coupling is absent. The coupled relaxation model accounts well for the NMR relaxation data as a function of magnetic field strength which is similar to measurements on other heterogeneous systems such as tissues.

Nature of lysozyme–water interactions by proton NMR

Proton spin-lattice relaxation measurements were performed in 10 mM lysozyme solution as a function of temperature and degree of substitution of solvent H2O with D2O. The results show that in the temperature range from 274 to 323 K, the intermolecular lysozyme proton water proton coupling contributes appreciably to the observed water proton relaxation rate. In this system exchange between water protons and labile protein protons does not dominate the behaviour with temperature of the water-lysozyme intermolecular contribution to the spin-lattice relaxation.

Multinuclear NMR studies of water in solutions of simple carbohydrates.

The dependence of water proton and deuterium transverse relaxation rates on pulse spacing is used to investigate the hydrogen exchange kinetics and water reorientational dynamics in glucose solutions over a wide concentration range. A complementary T 1ρ dispersion analysis is also undertaken. The results show that within the pH range 4–8 proton exchange between glucose and water occurs primarily by a non-ionic, cyclic transfer mechanism involving the interaction of two water molecules per glucose hydroxyl group. Interaction with the glucose introduces a slight anisotropy into the reorientational motion of the water. The two correlation times characterizing this anisotropic motion are estimated from the deuterium relaxation data. Similar phenomena are expected in all aqueous carbohydrate systems having accessible hydroxyl groups.

Site-specific water proton relaxation enhancement of iron(III) chelates noncovalently bound to human serum albumin.

Binding of potential blood pool and hepatobiliary paramagnetic iron(III) contrast agents, rac- and meso-Fe(5-Br-EHPG)- (iron(III) N,N'-ethylenebis [(5-bromo-2-hydroxyphenyl)glycinate]) and Fe(5-Br-HBED)- (iron(III) N,N'-bis-(5-bromo-2-hydroxybenzyl)ethylenediaminediacetic acid) to human serum albumin (HSA) has been studied using the proton relaxation enhancement (PRE) effect on solvent protons. These chelates bind avidly to multiple sites on HSA with binding constants on the order of 10(4) to 10(5) M-1. Interestingly, binding results in a decrease in the diamagnetic component of the water relaxivity due to HSA, while the expected enhancement of the paramagnetic component of water proton relaxation rates occurs due to the increase in the rotational correlation times of the protein-bound agents. These relaxation enhancements are variable, depending upon the site on the protein to which these chelates are bound, and can be as high as approximately 7 mM-1 s-1 at 5 degrees C and approximately 5 mM-1 s-1 at 37 degrees C at 20 MHz (enhancements of approximately 2-5). Change of temperature from 5 to 37 degrees C also appears to switch the relative affinities of these chelates for their primary and secondary binding sites. It is found that the important HSA binding site for the heme breakdown product, bilirubin-IX alpha, is a target for these agents and is the site of highest relaxivity for all the agents.

Water proton relaxation rate enhancements as a function of magnetic field strength and nature and size of paramagnetic solutes

The theoretical expectations for nuclear longitudinal relaxation rate enhancements for water protons sensing paramagnetic metal ions are shown. The cases of S = 1 2 (Cu 2+ and VO 2+), S = 1 (Ni 2+), S = 5 2 (Mn 2+ and Fe 3+), S = 7 2 (Gd 3+) are considered by including some factors which split the S manifold at zero field and the effect of the rotation.