Dallas L. Rabenstein

Simplification and assignment of carbon-13 NMR spectra with spin-echo fourier transform techniques

Abstract A series of spin-echo Fourier transform (SEFT) experiments are described with which 13 C NMR spectra can be simplified and resonances assigned to methyl, methylene, methine, and quaternary carbon sites. The second half of the echo from a standard two-pulse spin-echo sequence is collected as the free-induction decay. Gated broadband proton decoupling is used to modulate the phase and intensity of the 13 C resonances. The assignment of resonances to carbon sites is done with three SEFT spectra measured with the decoupler gated off during one of the two evolution periods. The first spectrum, measured with an evolution time of 1/ J sec between the 90 and 180° pulses, provides all the chemical shift information of the standard broadband decoupled spectrum and in addition distinguishes methyl and methine carbons from methylene and quaternary carbons. The other two SEFT spectra identify quaternary carbons and distinguish methine carbons from methyl carbons. Since the resonances are decoupled to singlets, assignments for large molecules can be made with more certainty than with off-resonance decoupled spectra. Subspectra containing only resonances from methine and methyl carbons or quaternary and methylene carbons, 1 H coupled or decoupled, are obtained by spin-echo difference or addition experiments. The fully coupled subspectra are shown to be of use for the measurement of 13 C 1 H coupling constants. The estimation of one-bond coupling constants by analysis of the intensity modulation of 13 C resonances in SEFT spectra measured as a function of the length of the evolution period is also described. The SEFT experiments are demonstrated with spectra for cholesterol.

Determination of cysteine in plasma and urine and homocysteine in plasma by high-pressure liquid chromatography

Abstract New methods are described for the determination of reduced and total cysteine in urine and the nonprotein fraction of plasma, and total homocysteine in the nonprotein fraction of plasma. The method for reduced cysteine involves separation by high performance liquid chromatography (hplc), followed by detection with a mercury-based electrochemical detector. The detector has a detection limit of ca, 10 −6 m cysteine, and is selective for sulfhydryl components of the biological fluids analyzed. Sample preparation involves centrifugation and filtration, and the hplc analysis time is approximately 8 min. Total cysteine and homocysteine are determined by electrolytic reduction of their disulfides to the sulfhydryl form prior to the hplc analysis. Results are presented which suggest that cysteine disulfide exchange reactions are a source of error in methods which employ derivatization with iodoacetate for the determination of cysteine in plasma.

1H NMR methods for the noninvasive study of metabolism and other processes involving small molecules in intact erythrocytes

1H NMR methods are described with which resolved resonances can be obtained for many of the small molecules in intact erythrocytes. In one method, the more intense hemoglobin resonances are suppressed by transfer of saturation throughout the hemoglobin spin system by cross relaxation following a selective saturation pulse. In a second method, the hemoglobin resonances are eliminated with the spin-echo pulse sequence by using a between-pulse delay time long enough for complete elimination of the hemoglobin resonances by spin-spin relaxation. Selected examples of the study of erythrocyte biochemistry by 1H NMR are discussed.

Carbon-13 chemical shift parameters for amines, carboxylic acids, and amino acids

Abstract Chemical shifts are reported for the alkyl carbons and the carboxyl carbons of a number of amines, carboxylic acids, and amino acids in aqueous solution. Spectra were obtained for the fully protonated, the fully deprotonated and, in the case of the amino acids, the zwitterion forms of the various molecules. The important structural features which determine alkyl carbon chemical shifts in these molecules have been incorporated into a single linear parameterization scheme for which parameters have been derived by stepwise multiple regression analysis. With the exception of the alpha carbon of alpha amino acids, for which empirical pair-interaction correction terms are necessary, the effects of the different substituents and structural features on alkyl carbon chemical shifts are additive. The dependence of the chemical shift on the protonation state of nearby amino and carboxyl groups is accounted for by having separate substituent parameters for the protonated and deprotonated forms of amino and carboxyl substituents. A similar linear parameterization scheme and parameters are presented for the carboxyl carbon chemical shifts.

A proton nuclear magnetic resonance study of the binding of methylmercury in human erythrocytes

The binding of methylmercury, CH3Hg(II), by small molecules in the intracellular region of human erythrocytes has been studied by 1H-NMR spectroscopy. To suppress or completely eliminate interfering resonances from the much more abundant hemoglobin protons, spectra were measured by a technique based on the transfer of saturation throughout the envelope of hemoglobin resonances following a selective presaturation pulse or by the spin-echo Fourier transform method. With these techniques, 1H-NMR spectra were measured for the more abundant intracellular small molecules, including glycine, alanine, creatine, lactic acid, ergothioneine and glutathione, both intact and hemolyzed erythrocytes to which CH3Hg(II) had been added. The results for intact erythrocytes indicate that part of the CH3Hg(II) is complexed by intracellular glutathione. These results also indicate that exchange of CH3Hg(II) among glutathione molecules is fast, with the average lifetime of a CH3Hg(II)-glutathione complex estimated to be less than 0.01 s. From exchange-averaged chemical shifts of the resonance for the proton on the alpha-carbon of the cysteine residue of glutathione, it is shown that, in hemolyzed erythrocytes, the sulfhydryl group of glutathione binds CH3Hg(II) more strongly than the sulfhydryl groups of hemoglobin.

A proton nuclear magnetic resonance study of the interaction of mercury with intact human erythrocytes.

The binding of mercuric ion (Hg(II)) by small molecules in the intracellular region of intact human erythrocytes has been studied by 1H-NMR spectroscopy. HgCl2 added to intact erythrocytes in saline-glucose suspension is found to cross the membrane and reach an equilibrium distribution among the molecules of the erythrocyte within 4 min. In the intracellular region Hg(II) reacts with GSH and hemoglobin to form the ternary mixed-ligand complex GSH-Hg(II)-hemoglobin. The analogous complex with ergothioneine is formed after all the GSH is complexed. 1H-NMR spectra show that the GSH-Hg(II)-hemoglobin complex also forms in simpler solutions containing HgCl2, GSH and hemoglobin, whereas the complex Hg(GSH)2 predominates in solutions of GSH and HgCl2. The lifetime of the GSH in the GSH-Hg(II)-hemoglobin complex is shown to be less than 30 s, which provides direct evidence for the first time that Hg(II) complexes in biological systems are quite labile, even though their thermodynamic stability is large. The effectiveness of eight sulfhydryl-containing ligands, some of which have been used as antidotes for Hg(II) poisoning, for releasing GSH from its Hg(II) complex in hemolyzed erythrocytes was also studied. Dithiol ligands were found to be more effective than monothiols, with dithioerythritol the most effective of the dithiols.

A proton nuclear magnetic resonance study of the interaction of cadmium with human erythrocytes

The binding of Cd2+ by molecules in the intracellular region of human erythrocytes has been studied by 1H-NMR spectroscopy. From changes in spin-echo Fourier transform NMR spectra for both intact and hemolyzed erythrocytes to which CdCl2 was added, direct evidence was obtained for the binding of Cd2+ by intracellular glutathione and hemoglobin. Time-courses were measured by 1H-NMR for the uptake of Cd2+ by intact erythrocytes in saline/glucose solution and in whole blood. In both cases, the uptake, as indicated by changes in the 1H-NMR spectrum for intracellular glutathione, plateaus after about 30 min. The effectiveness of the disodium salt of EDTA and of various thiol-chelating agents for releasing glutathione from its Cd2 + complexes in hemolyzed erythrocytes was also studied. EDTA was found to be more effective than thiols, and dithiols more effective than monothiols.

Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 26. Mixed ligand complexes of cadmium, nitrilotriacetic acid, glutathione, and related ligands

The complexation of glutathione and related ligands by the nitrilotriacetic acid complex of Cd2+ (Cd(NTA)-) has been investigated by 1H NMR as a model for the coordination chemistry of Cd2+ and GSH in biological systems. Related ligands included glycine, glutamic acid, cysteine, N-acetylcysteine, penicillamine, N-acetylpenicillamine, mercaptosuccinic acid, and the S-methyl derivative of glutathione. The nature of the complexes formed was deduced from 1H NMR spectra of Cd(NTA)- and the ligands. Mixed ligand complexes (Cd(NTA)L) and single ligand complexes (CdLx) are formed with the thiol ligands, whereas only mixed ligand complexes form with glycine, glutamic acid and S-methylglutathione. Formation constants of the mixed and the single ligand complexes were determined from NMR data. The results indicate that formation constants for binding of a thiolate donor group by Cd2+, either as the free ion or in a coordinately unsaturated complex, are in the range 10(5)-10(6).

Determination of the intracellular pH of intact erythrocytes by 1H NMR spectroscopy

A method is described for determining the intracellular pH of intact erythrocytes by /sup 1/H NMR. The determination is based on the pH dependence of the chemical shifts of resonances for carbon-bounded protons of an indicator molecule (imidazole) in intact cells. The imidazole is introduced into the erythrocytes by incubation in an isotonic saline solution of the indicator. The pH dependence of the chemical shifts of the imidazole resonances is calibrated from /sup 1/H NMR spectra of the imidazole-containing red cell lysates whose pH is varied by the addition of acid or base and measured directly with a pH electrode. To reduce in intensity or eliminate the much more intense envelope of resonances from the hemoglobin, the /sup 1/H NMR measurements are made by either the spin-echo Fourier transform technique or by the transfer-or-saturation by cross-relaxation method.

NUCLEAR MAGNETIC RESONANCE STUDIES OF THE SOLUTION CHEMISTRY OF METAL COMPLEXES. X. DETERMINATION OF THE FORMATION CONSTANTS OF THE METHYLMERCURY COMPLEXES OF SELECTED AMINES AND AMINOCARBOXYLIC ACIDS

Abstract The aqueous solution chemistry of the methylmercury complexes of a series of amines and amino acids has been investigated by proton magnetic resonance spectroscopy. Methylmercury-amine complexes form at intermediate pH values; in acidic solution the complex is dissociated due to protonation of the amine while in basic solution the complex dissociates through formation of methylmercuric hydroxide. Formation constants of the complexes were determined from the pH-dependence of the chemical shift of the methyl group of methylmercury, from the pH-dependence of the mercury-proton spin-spin coupling constant of methylmercury, and from the pH-dependence of the chemical shift of the carbon-bonded ligand protons in solutions containing amine and methylmercury at a molar ratio of either one or two. The nature of the complex formed by the amino acids is pH dependent, with methylmercury binding to the carboxylate group at low pH and to the amino group at higher pH. Formation constants were determined for bind...