Chromium(III) complexes as intermolecular probes.

Metal ion complexes provide flexible paramagnetic centers that may be used to define intermolecular contacts in a variety of solution phase environments because both the charge and electronic relaxation properties of the complex may be varied. For most complex ions, there are several proton equilibria that may change the effective charge on the complex as a function of pH which in turn affects the efficacy of application for defining the electrostatic surfaces of co-solute molecules. We report here spectrophotometric and nuclear spin relaxation studies on aqueous solutions of chromium(III) complexes of EDTA, DTPA, and bis-amides of both. The effective charges available from these paramagnetic centers range from -3 to +1 and we report the pH ranges over which the effective charge is defined with confidence for application in magnetic relaxation experiments.

Surface nuclear magnetic relaxation and dynamics of water and oil in macroporous media.

Proton nuclear spin-relaxation studies on water- or oil-saturated granular packings and limestone rocks allow estimating surface molecular dynamical parameters. Measurements were performed at various conditions of temperature, magnetic field strengths, and pore size. We show by low field NMR relaxation that changing the amount of surface paramagnetic impurities leads to striking different pore-size dependences of the relaxation times T1 and T2 of liquids in pores. These dependences are well supported by surface-limited or diffusion-limited relaxation models. Surface relaxivity parameters rho(1) and rho(2) are deduced from the pore-size dependence in the surface-limited regime. We evidence the frequency and temperature dependence of the surface relaxivity rho(1) by field cycling NMR relaxation and relevant theoretical models. The typical frequency dependence found allows an experimental separation of the surface and bulk microdynamics in porous media. Several surface dynamical parameters, such as diffusion coefficients, activation energies, time of residence, and coefficient of surface affinity, were therefore determined. The methods presented here give a powerful analysis of the surface microdynamics of confined liquids, which can be applied to the study of oil-bearing rocks.

New ways of probing surface nuclear relaxation and microdynamics of water and oil in porous media.

The microdynamics of water and oil in macroporous media with SiO2 or CaCO3 surfaces has been probed at various temperatures by magnetic field-cycling measurements of spin-lattice relaxation rates. These measurements and an original theory of surface diffusion allow us to obtain surface dynamical parameters such as surface correlation times, residence times and diffusion coefficients. A coefficient of affinity of the liquids for the pore surface is deduced.

Molecular oxygen spin-lattice relaxation in solutions measured by proton magnetic relaxation dispersion.

Proton spin-lattice relaxation rate constants have been measured as a function of magnetic field strength for water, water-glycerol solution, cyclohexane, methanol, benzene, acetone, acetonitrile, and dimethyl sulfoxide. The magnetic relaxation dispersion is well approximated by a Lorentzian shape. The origin of the relaxation dispersion is identified with the paramagnetic contribution from molecular oxygen. In the small molecule cases studied here, the effective correlation time for the electron-nuclear coupling may include contributions from both translational diffusion and the electron T(1). The electron T(1) for molecular oxygen dissolved in several solvents was found to be approximately 7.5 ps and nearly independent of solvent or viscosity.

Experimental characterization of intermolecular multiple-quantum coherence pumping efficiency in solution NMR.

The behavior of intermolecular multiple-quantum coherences in a variety of simple liquids with different chemical and magnetic properties is investigated experimentally and modeled by numerical simulations based on modified Bloch equations. The effects of spin concentration, temperature, intramolecular conformational flexibility, chemical exchange, and spin-spin coupling on the formation of high-order coherences are examined. It is shown that any process that makes the Larmor frequency time-dependent may interfere with the formation of these coherences. Good agreement is achieved between experiments and simulation, using independently known values of the magnetization density, the rate constants for translational diffusion, spin-spin and spin-lattice relaxation, and radiation damping.

Protein-bound water molecule counting by resolution of (1)H spin-lattice relaxation mechanisms.

Water proton spin-lattice relaxation is studied in dilute solutions of bovine serum albumin as a function of magnetic field strength, oxygen concentration, and solvent deuteration. In contrast to previous studies conducted at high protein concentrations, the observed relaxation dispersion is accurately Lorentzian with an effective correlation time of 41 +/- 3 ns when measured at low proton and low protein concentrations to minimize protein aggregation. Elimination of oxygen flattens the relaxation dispersion profile above the rotational inflection frequency, nearly eliminating the high field tail previously attributed to a distribution of exchange times for either whole water molecules or individual protons at the protein-water interface. The small high-field dispersion that remains is attributed to motion of the bound water molecules on the protein or to internal protein motions on a time scale of order one ns. Measurements as a function of isotope composition permit separation of intramolecular and intermolecular relaxation contributions. The magnitude of the intramolecular proton-proton relaxation rate constant is interpreted in terms of 25 +/- 4 water molecules that are bound rigidly to the protein for a time long compared with the rotational correlation time of 42 ns. This number of bound water molecules neglects the possibility of local motions of the water in the binding site; inclusion of these effects may increase the number of bound water molecules by 50%.

Decay of dipolar order in diamagnetic and paramagnetic proteins and protein gels.

Magnetic relaxation in solids may be complicated by the creation and loss of dipolar order at finite rates. In tissues the molecular and spin dynamics may be significantly different because of the relatively high concentration of water. We have applied a modified Jeneer-Broekaert pulse sequence to measure dipolar relaxation rates in both dry and hydrated protein systems that may serve as magnetic models for tissue. In lyophilized and dry serum albumin, the dipolar relaxation time, T(1D) is on the order of 1 ms and is consistent with earlier reports. When hydrated by deuterium oxide, the dipolar relaxation times measured were on the order of tens of microseconds. When paramagnetic centers are included in the protein, the Jeneer-Broekaert echo decay times became the order of the decay time for transverse magnetization, i.e., the order of 10 micros or less. In the hydrated or paramagnetic systems, the dipolar relaxation times are too short to require inclusion in the quantitative analysis of magnetization transfer experiments.

Comparisons of pressure and temperature activation parameters for amide hydrogen exchange in T4 lysozyme.

Activation enthalpies and entropies are reported for proton-deuteron exchange at 42 amide sites in T4 lysozyme and compared with activation volumes for the same residues obtained earlier [Hitchens, T. K., and Bryant, R. G. (1998) Biochemistry 37, 5878-5887]. There is no correlation found between activation volume and activation entropy or activation enthalpy. The activation enthalpy is linearly related to the activation entropy in part as a consequence of a relatively narrow sampling window for the rate constants that corresponds to a narrow range of activation free energy. A consequence of the entropy-enthalpy compensation is preservation of rank order of proton exchange. Variations in DeltaH, DeltaS, and DeltaV for residues that are structurally close together in the folded protein suggest that there may be a variety of energetically distinct pathways for the access of solvent to these structurally related exchange sites.

Lanthanide chelates as bilayer alignment tools in NMR studies of membrane-associated peptides.

Theequimolar complex, consisting of the lipid-like, amphiphilic chelating agent 1,11-bis[distearylamino]-diethylenetriamine pentaacetic acid (DTPA-18) and Tm(3+), is shown by deuterium ((2)H) NMR to be useful in aligning bicelle-like model membranes, consisting of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC). As shown previously (1996, R. S. Prosser et al., J. Am. Chem. Soc. 118, 269-270), in the absence of chelate, the lanthanide ions bind loosely with the lipid phosphate groups and confer the membrane with a sufficient positive magnetic anisotropy to result in parallel alignment (i.e., average bilayer normal along the field). Apparently, DTPA-18 sequesters the lanthanide ions and inserts into the phospholipid bilayer in such a manner that bilayer morphology is preserved over a wide temperature range (35-70 degrees C). The inherent paramagnetic shifts and line broadening effects are illustrated by (2)H NMR spectra of the membrane binding peptide, Leu-enkephalin (Lenk-d(2), Tyr-(Gly-d(2))-Gly-Phe-Leu-OH), in the presence of varying concentrations of Tm(3+), and upon addition of DTPA-18. Two conclusions could be drawn from this study: (1) The addition of Tm(3+) to the bicelle system is consistent with a conformational change in the surface associated peptide, and this effect is shown to be reversed by addition of the chelate, and (2) The paramagnetic shifts are shown to be significantly reduced by addition of chelate.

High-resolution magnetic relaxation dispersion measurements of solute spin probes using a dual-magnet system.

The magnetic field dependence of the nuclear spin-lattice relaxation rate provides a detailed report of the spectral density functions that characterize the intra- and intermolecular fluctuations that drive magnetic relaxation. We have addressed the difficult sensitivity and resolution problems associated with low magnetic field strengths by using two magnets in close proximity and shielded from each other. The sample is stored in the high magnetic field, pneumatically driven to the variable satellite field, then returned to the high field for detection at high resolution. A magnetic shield effectively decouples the two magnets so that varying the satellite field strength has minimal effect on the field strength and shim of the high field magnet. The disadvantage of the sample-shuttle magnet-pair system is the restriction imposed on the relaxation times by the finite shuttle times. Experiments not described here have shown this rate maximum to be about 20 s(-1) for most practical solutions. However, we demonstrate here that the sensitivity gains over switched-current magnet systems permit characterization of solute inter- and intramolecular dynamics over the time scale range from tens of microseconds to less than a picosecond. This range permits investigation of a number of crucial chemical dynamics questions, while high sensitivity permits examination of a variety of solute spins. Representative data are presented for (1)H, (111)Cd, and (7)Li.

Consequences of (129)Xe-(1)H cross relaxation in aqueous solutions.

We have investigated the transfer of polarization from (129)Xe to solute protons in aqueous solutions to determine the feasibility of using hyperpolarized xenon to enhance (1)H sensitivity in aqueous systems at or near room temperatures. Several solutes, each of different molecular weight, were dissolved in deuterium oxide and although large xenon polarizations were created, no significant proton signal enhancement was detected in l-tyrosine, alpha-cyclodextrin, beta-cyclodextrin, apomyoglobin, or myoglobin. Solute-induced enhancement of the (129)Xe spin-lattice relaxation rate was observed and depended on the size and structure of the solute molecule. The significant increase of the apparent spin-lattice relaxation rate of the solution phase (129)Xe by alpha-cyclodextrin and apomyoglobin indicates efficient cross relaxation. The slow relaxation of xenon in beta-cyclodextrin and l-tyrosine indicates weak coupling and inefficient cross relaxation. Despite the apparent cross-relaxation effects, all attempts to detect the proton enhancement directly were unsuccessful. Spin-lattice relaxation rates were also measured for Boltzmann (129)Xe in myoglobin. The cross-relaxation rates were determined from changes in (129)Xe relaxation rates in the alpha-cyclodextrin and myoglobin solutions. These cross-relaxation rates were then used to model (1)H signal gains for a range of (129)Xe to (1)H spin population ratios. These models suggest that in spite of very large (129)Xe polarizations, the (1)H gains will be less than 10% and often substantially smaller. In particular, dramatic (1)H signal enhancements in lung tissue signals are unlikely.

Distance estimates from paramagnetic enhancements of nuclear relaxation in linear and flexible model peptides.

The distance dependence of electron-nuclear dipole-dipole coupling was tested using a series of poly-L-proline based peptides of different length. The poly-proline based peptides were synthesized with a nitroxide spin label on the N-terminus and a tryptophan on the C-terminus, and paramagnetic enhancements of nuclear spin-lattice relaxation rates were measured for the aromatic protons on the tryptophan as a function of the number of proline spacers in the sequence. As expected, paramagnetic enhancements decrease with distance, but the distances deduced from the NMR relaxation rates were shorter than expected for every peptide studied compared to a rigid linear poly-L-proline type II helix structure. Calculations of cross-relaxation rates indicate that this difference is not the result of spin-diffusion or the creation of a spin-temperature gradient in the proton spins caused by the nitroxide. Molecular dynamics simulations were used to estimate dynamically averaged value of (2). These weighted average distances were close to the experimentally determined distances, and suggest that molecular motion may account for differences between the rigid linear models and the distances implied by the NMR relaxation data. A poly-L-prolone peptide synthesized with a central glycine hinge showed dramatic relaxation rate enhancements compared to the peptide of the same length lacking the hinge. Molecular dynamics simulations for the hinged peptide support the notion that the NMR data is a representation of the weighted average distance, which in this case is much shorter than that expected for an extended conformation. These results demonstrate that intermoment distances based on NMR relaxation rates provide a sensitive indicator of intramolecular motions.

An outbreak of Escherichia coli O157:H7 infection from unpasteurized commercial apple juice.

BACKGROUND: Escherichia coli O157:H7 infections have traditionally been associated with animal products, but outbreaks associated with produce have been reported with increasing frequency. In fall 1996, a small cluster of E. coli O157:H7 infections was epidemiologically linked to a particular brand (brand A) of unpasteurized apple juice. OBJECTIVE: To define the extent of the outbreak, confirm the source, and determine how the apple juice became contaminated. DESIGN: Descriptive epidemiologic study and traceback investigation. SETTING: Western United States and British Columbia, Canada. PATIENTS: Patients with E. coli O157:H7 infection who were exposed to brand A apple juice. MEASUREMENTS: Clinical outcome and juice exposure histories of case-patients, pulsed-field gel electrophoresis of case and juice isolates, and juice production practices. RESULTS: Seventy persons with E. coli O157:H7 infection and exposure to brand A unpasteurized apple juice were identified. Of these persons, 25 (36%) were hospitalized, 14 (20%) developed the hemolytic uremic syndrome, and 1 (1%) died. Recalled apple juice that was produced on 7 October 1996 grew E. coli O157:H7 with a pulsed-field gel electrophoresis pattern indistinguishable from that of case isolates. Apple juice produced on 7 October 1996 accounted for almost all of the cases, and the source of contamination was suspected to be incoming apples. Three lots of apples could explain contamination of the juice: Two lots originated from an orchard frequented by deer that were subsequently shown to carry E. coli O157:H7, and one lot contained decayed apples that had been waxed. CONCLUSIONS: Standard procedures at a state-of-the-art plant that produced unpasteurized juices were inadequate to eliminate contamination with E. coli O157:H7. This outbreak demonstrated that unpasteurized juices must be considered a potentially hazardous food and led to widespread changes in the fresh juice industry.

Anomalous surface diffusion of water compared to aprotic liquids in nanopores.

1H nuclear magnetic relaxation dispersion experiments show remarkable differences between water and acetone in contact with microporous glass surfaces containing trace paramagnetic impurities. Analyzed with surface relaxation theory on a model porous system, the data obtained for water show that proton surface diffusion limited by chemical exchange with the bulk phase permits long-range effectively one-dimensional exploration along the pores. This magnetic-field dependence coupled with the anomalous temperature dependence of the relaxation rates permits a direct interpretation in terms of the proton translational diffusion coefficient at the surface of the pores. A universal rescaling applied to these data collected for different pore sizes and on a large variety of frequencies and temperatures, supports this interpretation. The analysis demonstrates that acetone diffuses more slowly, which increases the apparent confinement and results in a two-dimensional model for the molecular dynamics close to surface relaxation sinks. Surface-enhanced water proton diffusion, however, permits the proton to explore a greater spatial extent of the pore, which results in an apparent one-dimensional model for the diffusive motions of the water that dominate nuclear spin relaxation.

Magnetic relaxation contrast agents in magnetization transfer imaging.

RATIONALE AND OBJECTIVES: The effects of magnetic relaxation agents are explored in the context of magnetization transfer pulse sequences using cross-linked protein gels as modeled tissue systems. METHODS: Magnetization transfer pulse sequences were used to study contrast agents that are designed to bind to rotationally immobilized protein targets. RESULTS: The dynamic range available from contrast agents, used in conjunction with magnetization transfer pulse sequences, is comparable with or better than that based on spin-echo imaging sequences with short repetition times. Furthermore, useful changes in the intensity of water resonances may be achieved by using this combined approach even though the paramagnetic metal center may not have a free coordination position in the chelate complex for water molecule exchange. CONCLUSIONS: The inclusion of magnetization transfer acquisition protocols in the context of magnetic imaging with contrast agents presents new opportunities for control of the information content of the image and for new classes of contrast agent structure and delivery.

Magnetic relaxation dispersion of 7Li. II. Complex formation with nitroxides in the aqueous phase.

Measurements of 7Li nuclear spin-lattice relaxation times are made at applied magnetic field strengths from 0.25 mT to 7.05 T, in order to determine directly the form of the frequency-dependent spectral densities that modulate relaxation. This magnetic resonance dispersion (MRD) technique provides detailed information regarding molecular dynamics down to the picosecond time scale. 7Li MRD measurements made on aqueous lithium ion in the presence of small concentrations of nitroxide free radicals give direct evidence supporting the formation of a coordination complex. The dipole-dipole electron-nuclear coupling is modulated by both translational and rotational diffusive motions, and both of these contributions are resolved. However, scalar coupling arising from the presence of paramagnetic electron spin density at the nucleus dominates the nuclear relaxation. Changes in the pH and free radical moiety are compared with dynamical variables, geometric constraints, and formation constants obtained from a model of nuclear relaxation. The calculated bimolecular formation constants are on the order of 2 x 10(-3) M-1, and the relative accuracy of this parameter is tested.

Pressure dependence of amide hydrogen-deuterium exchange rates for individual sites in T4 lysozyme.

We report measurements of the pressure dependence of rate constants for the exchange of amide residue protons with solvent deuterium for T4 lysozyme. Data obtained at nine pressures from 0.1 to 200 MPa are analyzed using an elementary kinetic model and the formalism of transition state theory which yield activation volumes for the exchange process. Resolution of individual amide sites was accomplished using the HSQC two-dimensional (2D) NMR experiment on uniformly (15)N-labeled protein. The observed activation volumes span the range from 2.75 to -25.1 mL/mol at 22 degreesC and pH* 7.5. When corrected for the pressure dependence of the ionic product for water and for the reported activation volume for the amide exchange reaction in model compounds, the portion of the activation volume associated with the accessibility of the solvent or catalyst to the amide sites ranges from -15.1 to 12.8 mL/mol. There is no simple correlation between the activation volumes and the protection factors for amide hydrogen exchange. The activation volumes for residues in close proximity in either the primary sequence or the folded structure may differ considerably. There is no trivial correlation between the activation volume and the secondary structural unit in which a residue is located, and activation volumes for residues that are apparently structurally coupled may be very different. The modest sizes of the activation volumes obtained under these conditions are in contrast to large values reported for bovine pancreatic trypsin inhibitor at more extreme conditions of 60 degreesC and pH* 8 where major unfolding events or structural rearrangements may dominate the mechanism [Wagner, G. (1983) Q. Rev. Biophys. 16, 1-57].

Water translational motion at the bilayer interface: an NMR relaxation dispersion measurement.

Nuclear magnetic relaxation rates for water protons in aqueous palmitoyloleoylphosphatidylcholine vesicle suspensions containing different nitroxide free radical spin labels are reported as a function of magnetic field strength corresponding to proton Larmor frequencies from 10 kHz to 30 MHz. Under these conditions the water proton relaxation rate is determined by the magnetic coupling between the water protons and the paramagnetic nitroxide fixed on the phospholipid. This coupling is made time-dependent by the relative translational motion of the water proton spins past the nitroxide radical. Using theories developed by Freed and others, we interpret the NMR relaxation data in terms of localized water translational motion and find that the translational diffusion constant for water within approximately 10 A of the phospholipid surface is 6 x 10(-10) m2 s(-1) at 298 K. Similar results are obtained for three different nitroxide labels positioned at different points on the lipid. The diffusion is a thermally activated process with an activation energy only slightly higher than that for bulk water.

MR imaging and spectroscopy using hyperpolarized 129Xe gas: preliminary human results.

Using a new method of xenon laser-polarization that permits the generation of liter quantities of hyperpolarized 129Xe gas, the first 129Xe imaging results from the human chest and the first 129Xe spectroscopy results from the human chest and head have been obtained. With polarization levels of approximately 2%, cross-sectional images of the lung gas-spaces with a voxel volume of 0.9 cm3 (signal-to-noise ratio (SNR), 28) were acquired and three dissolved-phase resonances in spectra from the chest were detected. In spectra from the head, one prominent dissolved-phase resonance, presumably from brain parenchyma, was detected. With anticipated improvements in the 129Xe polarization system, pulse sequences, RF coils, and breathing maneuvers, these results suggest the possibility for 129Xe gas-phase imaging of the lungs with a resolution approaching that of current conventional thoracic proton imaging. Moreover, the results suggest the feasibility of dissolved-phase imaging of both the chest and brain with a resolution similar to that obtained with the gas-phase images.

Cholera from raw seaweed transported from the Philippines to California.

In March 1994, a California woman without any recent travel developed acute, profuse, watery diarrhea. Her astute physician diagnosed cholera after ordering the appropriate stool culture, and the patient improved on an oral antibiotic. Epidemiologic investigation implicated seaweed from the Philippines that was transported by a friend to California and subsequently eaten raw as the vehicle of infection.