Anvarhusein A. Isab

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.

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.

The incorporation of 2h-labelled glycine into the glutathione of intact human erythrocytes studied by 1h spin-echo fourier transform NMR

1. Introduction Although the concentration of glutathione (y-L- glutamyl-L-cysteinyl-glycine, GSH) in human erythrocytes is generally found to be -2 mM [ 1,2], it is in a dynamic state, continually being degraded to and resynthesized from its constituent amino acids. The details of the degradation and synthesis reactions are the subject of considerable research [3,4], as is the mechanism by which the GSH level is regulated [l]. Much of this research has focussed on the indi- vidual steps in the overall process using enzyme pre- parations, although GSH dynamics in whole cells have also been studied by following the incorporation of radioactively-labelled amino acids [5,6]. GSH is considered to be essential for maintenance of the structural integrity of the erythrocyte [7], being involved in the protection of protein thiol groups against oxidation, the catalysis of sulfhydryl- disulfide exchange reactions, and the detoxification of foreign compounds [4]. In assessing the GSH status of the cell, not only is the GSH level of interest but also the capacity of the cell for GSH synthesis. Although this information can be obtained from whole cell studies using radioactively labelled amino acids, rather elaborate separations are necessary prior to the measurement step. Here we show that intra- cellular GSH dynamics can be monitored directly by ‘H NMR spectroscopy by following the incorporation

A 1H nmr study of the interaction of aurothiomalate (“Myocrisin”) with human red blood cells in vitro

The results of 1H spin-echo Fourier transform (SEFT) nuclear magnetic resonance (nmr) experiments suggest that some aurothiomalate binds intracellular glutathione (GSH) when added to suspensions of red cells in vitro. When added to red cell lysates, a specific binding of gold to cysteine of GSH is observed together with release of thiomalate. Gold binding to GSH can be reversed by addition of dimercaptopropanol sulfonate. Spectra are compared to those of an aurothiomalate-GSH model system. The relationship of these findings to the mechanism of action of Myocrisin is discussed.

1H nmr study of the effectiveness of various thiols for removal of methylmercury from hemolyzed erythrocytes

The effectiveness of eight thiol ligands for removing methylmercury (CH3Hg(II)) from its glutathione and hemoglobin complexes in hemolyzed erythrocytes has been studied by 1H nuclear magnetic resonance spectroscopy. These complexes are the predominant methylmercury species in human erythrocytes. The effectiveness was determined from the exchange-averaged chemical shift of the resonance for the proton on the alpha-carbon of the cysteinyl residue and from the intensity of the resonance for the methylene protons of the glycine residue of reduced glutathione (GSH), both of which provide a measure of the amount of glutathione in the CH3Hg(II)-complexed form. The thiol ligands were found to release GSH from its CH3Hg(II) complex in the order 2, 3-dimercaptosuccinic acid greater than mercaptosuccinic acid greater than cysteine greater than mercaptoacetic acid greater than D-penicillamine greater than 2,3-dimercaptopropanesulfonic acid greater than N-acetyl-D,L-penicillamine greater than D,L-homocysteine.