Water Protons In Solutions Research Articles

Quantitative theory for the transverse relaxation time of blood water.

An integrative model is proposed to describe the dependence of the transverse relaxation rate of blood water protons (R2blood = 1/T2blood ) on hematocrit fraction and oxygenation fraction (Y). This unified model takes into account (a) the diamagnetic effects of albumin, hemoglobin and the cell membrane; (b) the paramagnetic effect of hemoglobin; (c) the effect of compartmental exchange between plasma and erythrocytes under both fast and slow exchange conditions that vary depending on field strength and compartmental relaxation rates and (d) the effect of diffusion through field gradients near the erythrocyte membrane. To validate the model, whole-blood and lysed-blood R2 data acquired previously using Carr-Purcell-Meiboom-Gill measurements as a function of inter-echo spacing τcp at magnetic fields of 3.0, 7.0, 9.4 and 11.7 T were fitted to determine the lifetimes (field-independent physiological constants) for water diffusion and exchange, as well as several physical constants, some of which are field-independent (magnetic susceptibilities) and some are field-dependent (relaxation rates for water protons in solutions of albumin and oxygenated and deoxygenated hemoglobin, ie, blood plasma and erythrocytes, respectively). This combined exchange-diffusion model allowed excellent fitting of the curve of the τcp -dependent relaxation rate dispersion at all four fields using a single average erythrocyte water lifetime, τery = 9.1 ± 1.4 ms, and an averaged diffusional correlation time, τD = 3.15 ± 0.43 ms. Using this model and the determined physiological time constants and relaxation parameters, blood T2 values published by multiple groups based on measurements at magnetic field strengths of 1.5 T and higher could be predicted correctly within error. Establishment of this theory is a fundamental step for quantitative modeling of the BOLD effect underlying functional MRI.

Nanoparticle assisted nuclear relaxation-based oligonucleotide detection

We present a proof-of-concept “on-off” detection scheme, which uses gadolinium phthalocyanine (GdTcPc)-grafted silica nanoparticles as paramagnetic centers, capable of modifying the transverse relaxation time (T2) of water protons in solution. A DNA strand (as probe) was conjugated to the GdTcPc to act as a recognition element. In the presence of the target DNA, which was complementary to the probe, an increase in the T2 value was detected, with magnitude proportional to the target DNA concentration. The linear range was observed from 30 to 140 nM, with limit of detection of 15 nM. The developed nuclear relaxation-based detection scheme is shown to be a simple, fast and selective method to detect DNA and could be useful in point-of-care diagnostic applications.

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.

Intermolecular protein interactions in solutions of calf lens alpha-crystallin. Results from 1/T1 nuclear magnetic relaxation dispersion profiles

From analyses of the magnetic field dependence of 1/T1 (NMRD profiles) of water protons in solutions of calf lens alpha-crystallin at several concentrations, we find two regimes of solute behavior in both cortical and nuclear preparations. Below approximately 15% vol/vol protein concentration, the solute molecules appear as compact globular proteins of approximately 1,350 (cortical) and approximately 1,700 (nuclear) kD. At higher concentrations, the effective solute particle size increases, reversibly, as evidenced by the appearance of spectra-like 14N peaks in the NMRD profiles and a change in the field and temperature dependence of 1/T1. At these higher concentrations, the profiles are very similar to those of calf gamma II-crystallin, a crystallin that undergoes an analogous transition near approximately 15% protein (Koenig, S. H., C.F. Beaulieu, R. D. Brown III, and M. Spiller, 1990. Biophys. J. 57:461-469). By comparison with recent analyses of NMRD results for solutions of immobilized proteins as models for the transition from protein solutions to tissue (Koenig, S. H., and R. D. Brown III. 1991. Prog. NMR Spectr. 22:487-567), we argue that alpha-crystallin solute behaves as aggregates approximately greater than 50,000 kD as protein concentration is progressively increased above 15%. Finally, the concentration dependence of the NMRD profiles of alpha- and gamma II-crystallin can readily explain recent osmotic pressure data, in particular the intersection of the respective pressure curves at approximately 23% vol/vol (Vérétout, F., and A. Tardieu. 1989. Eur. Biophys. J. 17:61-68).

Nuclear Magnetic Relaxation Dispersion Measurements of Ethylenediamine-Tetraacetatoiron(III) Complexes at High pH and Ionic Strength

Abstract Nuclear magnetic relaxation dispersion measurements are reported for aqueous solutions of iron(III)-EDTA complexes at high pH and high ionic strength where the dominant species is the μ-oxo dimer. Formation of the dimeric species leaves only the outer coordination sphere contribution to the paramagnetic relaxation rate of the water protons in solution. The relaxation efficiency is further decreased by the decrease in the effective magnetic moment due to antiferromagnetic coupling. This result is in distinction to dimeric species that form with Gd(HEDTA) complexes at high pH in which antiferromagnetic coupling has not been observed to decrease the effective magnetic moment.

Oligomerization and conformation change in solutions of calf lens gamma II-crystallin. Results from 1/T1 nuclear magnetic relaxation dispersion profiles

From analyses of the magnetic field dependence of 1/T1 (nuclear magnetic relaxation dispersion [NMRD] profiles) of water protons in solutions of highly purified calf lens gamma II-crystallin, we find that monomers form oligomers at relatively low concentrations, which increase in size with increasing concentration and decreasing temperature. At approximately 16% by volume and -4 degrees C, the mean oligomeric molecular weight is approximately 120-fold greater than the monomeric value of 20 kD. Below this concentration, there is no indication of any substantive change in conformation of the monomeric subunits. At higher concentrations, the tertiary structure of the monomer appears to reconfigure rather abruptly, but reversibly, as evidenced by the appearance of spectra-like 14N peaks in the NMRD profiles. The magnitudes of these peaks, known to arise from cross-relaxation of water protons through access to amide (NH) moieties of the protein backbone, indicate that the high concentration conformation is not compact, but open and extended in a manner that allows enhanced interaction with solvent. The data are analogous to those found for homogenates of calf and chicken lens (Beaulieu, C. F., J. I. Clark, R. D. Brown III, M. Spiller, and S. H. Koenig. 1988. Magn. Reson. Med. 8:47-57; Beaulieu, C. F., R. D. Brown III, J. I. Clark, M. Spiller, and S. H. Koenig. 1989. Magn. Reson. Med. 10:62-72). This unusually large dependence of oligomeric size and conformation on concentration in the physiological range is suggested as the mechanism by which osmotic equilibrium is maintained, at minimal metabolic expense, in the presence of large gradients of protein concentration in the lens in vivo (cf Vérétout and Tardieu, 1989. Eur. Biophys. J. 17:61-68). Finally, the results of the NMRD data provide a ready explanation of the low temperature phase transition, and "cold-cataract" separation of phases, observed in gamma II-crystallin solutions; we suggest that the phases that separate are the two major conformers detected by NMRD.

Synthesis and characterization of conjugates of poly(α‐amino acids) and manganese (III) protoporphyrin IX as relaxation enhancement agents for MRI

AbstractPolymers are finding increasing use in medicine, and conjugates of paramagnetic species and polymers are now being studied as relaxation enhancement agents to improve contrast in magnetic resonance imaging. To assess the potential of such agents, the enhancement of proton relaxation by paramagnetic species bound to macromolecules has been observed for four conjugates of amino acid polymers and manganese (III) protoporphyrin IX. The conjugates all differ in their abilities to enhance the relaxation of water protons in solution; values of relaxation rates range from 3.2 to 9.1 s−1 mM−1. The rotational correlation times and volumes of the kinetic units within the macromolecules have been determined by fluorescence depolarization in an effort to understand the range of relaxation rates observed for the conjugates. The metalloporphyrins bound to the polymers all have very similar dynamic characteristics.

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

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

Conformation as the determinant of saccharide binding in concanavalin A: Ca2+-concanavalin A complexes.

The existence of two conformational states of concanavalin A (Con A) with different metal ion binding properties has been recently demonstrated (Brown, R. D., Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883). Introduction of Mn2+ to the S1 site and Ca2+ to the S2 site of apo-Con A was shown to induce a conformational change in the protein, ascribed to a cis-trans isomerization of a peptide bond in the secondary structure, which results in extremely tight binding of the metal ions. This induced conformation is referred to as "locked" and the initial conformation as "unlocked". The locked ternary complex is identical with the native protein. In the present paper, we report evidence for the formation of a relatively stable, locked, ternary Ca2+-Con A complex that possesses properties similar to those of native Ca2+-Mn2+Con A. The experimental technique involves measurement of the magnetic field and time dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons in solutions of Ca2+-Con A, after the addition of Mn2+ ion which slowly bind to the protein. The kinetic data can be fit by a model for Ca2+ interactions with Con A which indicates that Ca2+, in the absence of Mn2+, can bind at both the S1 and S2 sites of the protein and, furthermore, can induce the protein to undergo the unlocked to locked conformational transition. In terms of this model, the time-dependent binding of the Mn2+ ions is due to replacement of Ca2+ ions at the S1 sites in the locked protein. The off-rate of Ca2+ from the S2 site of the locked ternary Ca2+-Con A complex is much greater than that from the locked Ca2+-Mn2+-Con A complex. From the effects of added alpha-methyl D-mannopyranoside on the rate of replacement of Ca2+ by Mn2+ at the S1 site of the locked ternary Ca2+-Con A complex, it is concluded that the latter complex binds saccharides as strongly as the locked Ca2+-Mn2+-Con A complex. In addition, analysis of the data indicates that apo-Con A in the locked conformation binds alpha -methyl D-mannopyranoside with approximately 7% of the affinity of the fully metallized locked form of the protein. This strong saccharide-binding activity of locked apo-Con A, compared with that of the unlocked apo-Con A, was further demonstrated by equilibration of unlocked apo-Con A with alpha-methyl D-mannopyranoside, which resulted in the formation of the locked apo-Con A-saccharide complex. These results demonstrate that it is the locked conformation of Con A that is primarily responsible for saccharide-binding activity, and that the function of the bound metals is primarily to maintain the protein in the locked conformation.

Nuclear magnetic resonance studies of aqueous solutions of alkylureas: Proton and carbon-13 chemical shifts

Aqueous solutions of methylated and ethylated derivatives of urea have been investigated by proton and carbon-13 magnetic resonance technique. The chemical shifts of water protons in solution of alkylureas, relative to that of pure water, are as a measure of the hydrogen-bonding reinforcing or weakening ability of these substances. The dependence of this effect on the concentration and on the temperature has also been studied. The results obtained indicate that different contrasting effects act in these solutions, among then the water structuring effect of the nonpolar surface of the alkylureas and the effect of the volume of the solute.13C chemical shifts of the carbonyl groups of the alkylureas give useful information about the interactions between water and the polar part of the ureas. Carbonyl-water interactions exist but are partially destroyed by increasing concentrations of the solute.

Nuclear Magnetic Resonance Investigation of the State of Water in Protein Solution<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

The line width of the NMR signal of water protons in solutions of native actomyosin and actomyosin denatured by heat, acetone or urea was measured over the temperature range from -10 degrees to below the freezing point. The line widths of the water band which increased exponentially with decreasing temperature were compared with each other and also with those of the corresponding control solution without actomyosin. The line broadening observed for native actomyosin solution on lowering the temperature was significantly smaller than that for heat-denatured actomyosin solution. This difference implies that this signal is sensitive to conformational perturbations of the protein. In addition, the temperature dependence of the line width for heat-, acetone-, or urea-denatured actomyosin solution was similar to that for the corresponding control solution. These phenomena can be interpreted in terms of the state of water associated with the hydrophobic and hydrophilic residues. Similar NMR studies of actomyosin solution containing dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) showed that DMSO and DMF prevent the formation of ice crystals until about -70 degrees, suggesting that the cryoprotective effects of DMSO and DMF are due to the change in the state of water described above. These differences in temperature dependence between the sample and control solutions are well-correlated with the viscosity of the solution. This correlation is useful for elucidation of the mechanism of the protein denaturation.

Collagen hydration: Pulsed nuclear magnetic resonance studies of structural transitions

The technique of pulsed NMR (spin-echo) spectroscopy was used to measure the longitudinal ( T 1) and transverse ( T 2) relaxation times, and the spin-diffusion coefficient ( D), of water protons in solutions and gels of native and denatured collagen in vitro. Both T 1 and T 2 were decreased compared to water, and these parameters decreased progressively with increasing collagen concentration; in contrast, D was only slightly reduced. Upon heat-denaturation of collagen gels and solutions, both T 1 and T 2 increased irreversibly, while D remained essentially constant. The increase in relaxation times upon denaturation may reflect an actual loss of “bound” water. Additionally, these results may reflect a sharp decrease in the extent of long-range order experienced by the water molecules. The irreversibility of denaturation as observed by NMR, when contrasted with its reversibility as observed by optical rotation, suggests that an interpretation solely in terms of hydration changes would be incomplete.

Magnetic-field-dependent water proton spin-lattice relaxation rates of hemoglobin solutions and whole blood

The spin-lattice relaxation rates of solvent water protons in solutions of normal and sickle cell hemoglobin, normal red blood cells, and whole blood have been measured at magnetic fields between 5 Oe and 12 kOe. The field-dependent data have been analyzed to obtain the rotational tumbling time of the hemoglobin molecules and a measure of water-protein interaction. Comparison of the protein tumbling time in concentrated hemoglobin solutions to the tumbling time of hemoglobin inside red blood cells indicates that encapsulation of hemoglobin within the cell membrane has little effect on its molecular motion. The relaxation rates of solutions of deoxygenated sickle cell hemoglobin indicate that when the solution forms a gel at 10°C, approximately 90% of the hemoglobin molecules remain in solution and are freely tumbling, but at 35°C, virtually all of the hemoglobin has been incorporated into the gel structure.

PH dependence of the nuclear magnetic relaxation rate of solvent water protons in solutions of bovine superoxide dismutase

The nuclear magnetic relaxation rate of solvent water protons has been studied in solutions of the copper enzyme bovine superoxide dismutase at pH values between pH 6 and 13. An increase of relaxivity is observed between pH 10 and 12.5, followed by a steep decrease above pH 12.5. The first effect is reversible, while the second one is not. Comparison with theory and supplementary measurements suggests that the relaxivity increase is due to a decrease of the copper-proton internuclear distance ν, which occurs at the active site of the enzyme in the corresponding pH region. The results are discussed in view of the parallel changes of the EPR spectrum and of the enzyme activity. A deprotonation of the copper-bound H 2O to OH − appears to be a likely explanation of the observed facts.

H 2 CO 3 as substrate for carbonic anhydrase in the dehydration of HCO 3 .

Carbonic anhydrase, a metalloenzyme containing one zinc atom per protein molecule of molecular weight 30,000, catalyzes the interconversion of CO(2) and HCO(3) (-) in solution. The rate of catalysis, among the fastest known, is pH-dependent, with a pK(Enz) near neutral. Arguments are presented to show that: (i) only the high-pH form of the enzyme is active both for the hydration and dehydration reactions (ii) at high pH there is an H(2)O ligand on the metal (not an OH(-) as is often argued), and (iii) the substrate for the dehydration reaction is the neutral H(2)CO(3) molecule. The arguments are based on data in the literature on the nuclear relaxation rates of Cl(-) ions and water protons in solutions of carbonic anhydrase, on strict application of the principle of microscopic reversibility, and on kinetic considerations. It has been argued that H(2)CO(3) cannot be the substrate for the dehydration reaction because the observed CO(2) production rate is somewhat faster than the maximum rate at which H(2)CO(3) molecules can diffuse to the active site of the enzyme. However, current models that consider HCO(3) (-) as the substrate implicity require that protons diffuse to the enzyme at an even greater rate, well outside the limitations imposed by diffusion. We consider two mechanisms to obviate the diffusion limitation problem, and conjecture that at high substrate concentration, H(2)CO(3) reaches the active site by collision with the enzyme molecule, and subsequent surface diffusion to the active site. At lower substrate concentrations, corresponding to [HCO(3) (-)] <1 mM, generation of H(2)CO(3) molecules near the enzyme by the recombination reaction H(+) + HCO(3) (-) --> H(2)CO(3) can supply an adequate flux of substrate to the active site.

Properties of micellar solutions. Part 3.—Spin lattice relaxation times of water protons in solutions of sodium alkyl sulphates

Properties of micellar solutions. Part 3.—Spin lattice relaxation times of water protons in solutions of sodium alkyl sulphates

Properties of micellar solutions. Part 2.—N.m.r. chemical shift of water protons in solutions of sodium alkyl sulphates

Properties of micellar solutions. Part 2.—N.m.r. chemical shift of water protons in solutions of sodium alkyl sulphates

On the T1/T2 Ratio for Protons in Aqueous Mn++ Solutions

The T1≠T2 anomaly for water protons in solutions containing Mn++ has been investigated. At 40 Mc (proton frequency), T1/T2=9.5(±0.5) for Mn++ solutions. When the Mn++ is complexed by the hexadentate ligand, ethylenediaminetetraacetate (MnY—2), or by the tetradentate ligand, nitrilotriacetate (MnX—1), T1 is increased slightly and T2 is increased markedly, so that T1=T2 and the anomaly disappears. For MnX—1, there are probably still 2 H2O molecules complexed to Mn++. EPR studies show that the paramagnetic relaxation time is reduced by complexing (from 3×10—9 sec for Mn++ to 3×10—10 sec for MnX—1). The results therefore support the suggestion by Bloembergen that the anomalous T2 broadening is caused by an AI·S coupling between the electron spin, S, of Mn++ and the proton spins, I, in the coordination sphere plus a long paramagnetic relaxation time. All features of the variation of T1 and T2 with frequency are explained rather well by consideration of the combined effects of the I·S interaction and the dipole interaction. The experimental results are Mn++ MnY−2 MnX−1MnX2−4NT1 M sec1.6×10−43.05×10−42.35×10−4NT2 M sec1.65×10−52.6×10−42.25×10−47.4×10−4T1/T29.5(±0.5)1.15(±0.12)1.05(±0.15)