Proton Transverse Relaxation Rate Research Articles

Nuclear magnetic relaxation in aqueous solutions of syndiotactic, methylene deuterated poly (methacrylic acid)

Proton and deuteron longitudinal and transverse relaxation rates were determined for methyl protons and carboxylic deuterons in solutions of poly(methacrylic acid) in D2O. The frequency-dependence of these rates, as obtained in a range of 4–60 and 3·4–9·2 MHz respectively, is analysed in terms of an anisotropic rotational diffusion model. The parameters involved are determined and their temperature-dependence is reported. The results are discussed, together with the methylene deuteron parameters, previously obtained from experiments on H2O solutions of the same polymer.

The determination of molecular-motion parameters from proton-relaxation-enhancement measurements in a number of Gd(III) - antibody-fragment complexes. A comparative study.

Longitudinal and transverse proton relaxation rates for water in the hydration spheres of Gd(III) bound to the non-immune rabbit IgG fragments Fc (C-terminal half of heavy-chain dimer), pFc' (C-terminal quarter of heavy-chain dimer) and Fab (N-terminal half of heavy and light chain) have been measured at a number of frequencies and temperatures using pulsed nuclear magnetic resonance spectrometry. For the fragments Fc and pFc', a full computer analysis showed that the results could be fitted by parameters of similar magnitude to those found previously for IgG. In contrast to the results for the other complexes the Fab -Gd(III) complex showed no slow exchange contribution to the relaxation rates. Under these circumstances it was found possible to obtain an accurate value for the hydration number (q) from measurements of the longitudinal and transverse relaxation rates at a chosen frequency such that the product of the nuclear Larmor frequency (omega1) and the correlation time for the dipolar relaxation processes (tauc) was approximately unity. Water-proton relaxation rates were also determined for the complex of Gd(III) with the Fv fragment of the mouse myeloma protein MOPC 315. A computer analysis of the results revealed a slow exchange contribution to the rates and this gave errors in the variable parameters similar to those observed previously for IgG, Fc and pFc'. The conclusions drawn from the different systems are discussed in terms of the present state of application of the proton relaxation enhancement technique in biology.

Application of nuclear magnetic resonance spectroscopy to proteins

The active sites of enzymes can be studied in great detail using nuclear magnetic resonance spectroscopy. The determination of pKa values of active site histidine residues in bovine pancreatic ribonuclease and the characterization of the binding of peptide hormones to carrier proteins are two such examples. The study of the active site of staphylococcal nuclease is another example and is presented in detail in this paper. The structure of 3'5'-thymidine diphosphate bound in the active site of staphylococcal nuclease has been studied by measuring the relaxation rate enhancement of substrate analog nuclei by a paramagnetic metal ion. The lanthanide ion, Gd(III), was substituted for Ca(II) in the formation of the ternary complex of nuclease: Gd(III) : 3'5'-thymidine diphosphate. Measurements were made of the transverse relaxation rates of protons and the longitudinal and transverse relaxation rates of the phosphorus nuclei of bound nucleotide. Internuclear distances between the metal ion and atoms of the 3'5'-thymidine diphosphate nucleotide were determined from these data by using the Solomon-Bloembergen equation. In general, these distances corresponded closely to those determined by previous X-ray crystallography of the thymidine diphosphate complex. These internuclear distances were also used with a computer program and graphics display to solve for metal : nucleotide geometries which were consistent with the experimental data. A geometry similar to the structure of the metal : nucleotide complex bound to nuclease determined by X-ray analysis was one of the solutions to this computer modeling process. For staphylococcal nuclease the NMR and X-ray methods yield compatible high resolution information about the structure of the active site.

Nuclear-magnetic-resonance studies of carboxypeptidase B. Binding of inhibitors to the manganese enzyme.

Longitudinal and transverse proton relaxation rates of water in solutions of porcine manganese carboxypeptidase B have been measured in the presence of various competitive inhibitors by pulse nuclear magnetic resonance (NMR) spectrometry. The inhibition constant of Mn-carboxypeptidase activity by L-argininic acid and acetyl-L-arginine was in agreement with the equilibrium constant obtained by the NMR method, indicating similar and specific binding of the inhibitors to the active site of the manganese enzyme. Titration of the water boound to the metal ion revealed the presence of one water molecular which could be displaced from the sphere of the managenese ion by various inhibitors. The structural features of the inhibitors required for this displacement as well as the mode of interaction is described.

Temperature and frequency dependence of solvent proton relaxation rates in solutions of manganese(II) carbonic anhydrase

Longitudinal and transverse proton relaxation rates of water in solutions of manganese(II) bovine carbonic anhydrase have been measured by pulsed nuclear magnetic resonance spectrometry as a function of temperature (2-35 degrees), frequently (5-100 MHz) and pH. The pH dependence of the longitudinal relaxation rate was fitted to a sigmoidal curve with a pK value at 7.8, while the esterase activity of the manganese(II) enzyme in the hydrolysis of p-nitrophenyl acetate revealed an inflection point at pK = 8.2. The hydration number of manganese(II) carbonic anhydrase could be derived using either the frequency dependence of T1p or the T1p/T2p ratio at only one (high) frequency. Both treatments are in agreement with a model in which one water molecule is bound to the metal at high pH. At low pH the relaxation data imply that no-H20 exists in the first coordination sphere of the manganese ion. The various parameters which are responsible for the proton relaxation mechanisms have been evaluated and are compared to other manganese(II) enzyme systems. The pH dependence of the binding constant of manganese to apocarbonic anhydrase is also reported.

Proton and deuterium magnetic resonance study of the aqueous nickelous complex

The absorption signals of protons and deuterons in bound and nonbound water molecules in the aqueous nickelous system were observed. From the PMR and DMR measurements, the hyperfine coupling constants were calculated and found to be (1.3±0.1)×105 and (2.0±0.2)×104 Hz, respectively. The spin density at the proton (and deuteron) nuclei was calculated to be (2.9±0.3)×10−5 electrons/a.u.3, in accordance with theoretical expectations. The rate constant at 298°K for the exchange of water was found to be 3×104 sec−1, and the activation energy 12 kcal/mole. The relaxation time of the electron at 243°K, assuming T1e =T2e was found to be 1.9×10−12 sec and the activation ernergy 0.8 kcal/mole. The temperature dependence of the proton transverse relaxation rate was studied. It is suggested that in the absence of chemical exchange the transverse relaxation rate is governed both by the relaxation of the nickelous unpaired electrons and by the diffusional motion of the water molecules. Methods of estimating the diffusional correlation time are discussed.

The active site of staphylococcal nuclease: paramagnetic relaxation of bound nucleotide inhibitor nuclei by lanthanide ions.

The structure of 3',5'-thymidine diphosphate bound in the active site of staphylococcal nuclease (EC 3.1.4.7) was studied by measuring the relaxation rate enhancement of substrate analog nuclei by a paramagnetic metal ion. The lanthanide ion, Gd(III), was substituted for Ca(II) in the formation of the ternary complex of nuclease-Gd(III)-3',5'-thymidine diphosphate. Measurements were made of the transverse relaxation rates of protons and the longitudinal and transverse relaxation rates of the phosphorus nuclei of the bound nucleotide. Internuclear distances between the metal ion and atoms of the 3',5'-thymidine diphosphate nucleotide were determined from these data by the Solomon-Bloembergen equation. In general, these distances corresponded closely to those determined by previous x-ray crystallography of the thymidine diphosphate complex.These internuclear distances were also used with a computer program and graphics display to solve for metal-nucleotide geometries, which were consistent with the experimental data. A geometry similar to the structure of the metal-nucleotide complex bound to nuclease determined by x-ray analysis was one of the solutions to this computer modeling process. For staphylococcal nuclease, the nuclear magnetic resonance and x-ray methods yield compatible high resolution information about the structure of the active site. However, differences of uncertain significance exist between the two structures.

Monomethylammonium Ion as a Magnetic Resonance Probe for Monovalent Cation Activators: THE MONOVALENT CATION IN PYRUVATE KINASE CATALYSIS

Abstract Pyruvate kinase, which has an absolute requirement for both a monovalent and a divalent cation for activity, can be activated by the monomethylammonium cation at a rate 0.5% that measured in the presence of K+. The kinetically determined dissociation constant for the monovalent activator in the presence of saturating substrates (K1 = 85 mm) agrees with that measured for K+ and the dissociation constant measured in the presence of saturating substrates and the divalent activator Mn2+, (K4 = 20.5 ± 3.3 mm) is somewhat larger than that for K+. The presence of carbonbound protons on the movovalent activator permits the investigation of the interaction of the monovalent activator with the enzyme-Mn2+ complexes by proton nuclear magnetic resonance techniques. The presence of the enzyme has no effect on the longitudinal (1/T1) and transverse (1/T2) relaxation rates of the carbon-bound protons. The formation of the enzyme-Mn2+ complex increases the relaxation rates due to the electron-nuclear dipolar interaction of the paramagnetic Mn2+ and the protons. In the presence of the substrate phosphoenolpyruvate, the relaxation rates are even further increased. A measure of the relaxation rates at two frequencies allows the evaluation of the correlation time for the electron-nuclear interaction, τc by several methods. No significant change in the correlation time is seen when the substrate complex is formed. The electron-nuclear distance can thus be evaluated from the correlation time, τc, and from the values measured for 1/T1. In the absence of substrate the protons of the methyl group are 8.3 ± 0.6 A from the enzyme-bound Mn2+. Upon formation of the pyruvate kinase-Mn2+-phosphoenolpyruvate complex, the protons move to 6.6 ± 0.2 A of the Mn2+. This result shows that the substrate affects the conformation of the active site by bringing the monovalent and divalent cation activators in closer proximity and that the monovalent activator can indeed bind in the active site of pyruvate kinase. The Mn2+-methyl proton distance measured is that predicted if the monovalent cation acts by binding P-enolpyruvate via the carboxyl group to form the active complex (Nowak, T., and Mildvan, A. S. (1972) Biochemistry 11, 2819). Monomethylammonium ion can thus serve as a useful probe in studying the interactions of monovalent cations with many biological systems with relative ease and should lead to a greater understanding of monovalent cation activation and function in biology.

The Mechanism of Aconitase Action: III. DETECTION AND PROPERTIES OF ENZYME-METAL-SUBSTRATE AND ENZYME-METAL-INHIBITOR BRIDGE COMPLEXES WITH MANGANESE(II) AND IRON(II)

Abstract A magnetic resonance study of the paramagnetic contribution to the longitudinal (1/T1) and the transverse (1/T2) relaxation rates of the carbon-bound protons of citrate and trans-aconitate has been made utilizing Mn(II), Fe(II), and aconitase. The width of the methylene resonances in the nuclear magnetic resonance (NMR) spectrum of citrate (an AB pattern) increases linearly with Mn(II) concentration from 0 to 200 µm due to a paramagnetic effect on 1/T2 of these protons. Aconitase enhances the effect of Mn(II) on the 1/T1 and 1/T2 relaxation rates of the methylene protons of citrate and enhances the effect of Fe(II) on both relaxation rates of the —CH2— and —CHD—protons of pro-R monodeuterocitrate. From the Solomon-Bloembergen equation for 1/T1, the Mn(II) to proton distance in the aconitase-manganese-citrate ternary complex is 4.3 ± 0.5 A, and the Fe(II) to proton distances in the aconitase-iron-monodeuterocitrate ternary complex are 3.8 ± 0.2 A (—CH2—) and 3.1 ± 0.2 A (—CHD—). The absolute and relative values of these distances are in good agreement with crystallographic distances in the binary Mn(II)-citrate chelate complex. Hyperfine coupling of the unpaired electrons of enzyme-bound Fe(II) with the protons of citrate is detected. The data are, therefore, consistent with direct coordination of the substrate, citrate, by the metal ion on aconitase. In contrast with citrate, the NMR spectrum of the inhibitor trans-aconitate shows a nonlinear dependence of the width of the coupled methylene and methine resonances on the concentration of Mn(II) below 30 µm due to chemical exchange spin decoupling (Frankel, L. S. (1969) J. Mol. Spectrosc. 29, 273). Above 30 µm Mn(II), the resonance widths increase linearly with Mn(II) due to a simple paramagnetic effect on 1/T2. A ternary aconitase-manganese-trans-aconitate bridge complex is detected since aconitase enhances the effect of Mn(II) on the 1/T2 relaxation rate of the protons of trans-aconitate. From 1/T1, Mn(II) to proton distances in this ternary complex are 4.8 ± 0.5 A (—CH2—) and 5.3 ± 0.6 A (—CH—). As shown by NMR, both citrate and trans-aconitate compete for the metal site on aconitase at concentrations consistent with their respective dissociation constants as determined kinetically. From 1/T2, the aconitase-Fe(II)-citrate complex forms and dissociates rapidly enough to participate in enzyme catalysis. Hence the metal bridge complexes detected by NMR may be equated with the enzymatically active substrate complex and the enzymatically inactive inhibitor complex observed kinetically. Three mechanisms for the role of Fe(II) in the catalysis, consistent with the data, the forward and reverse ferrous wheel mechanisms, and the Bailar twist mechanism, are discussed.