Biochimica et Biophysica Acta, 696 (1982) 53--64 53 Elsevier Biomedical Press BBA 11003 A PROTON NUCLEAR MAGNETIC RESONANCE STUDY OF THE BINDING OF METHYLMERCURY IN HUMAN ERYTHROCYTES DALLAS L. RABENSTEIN, ANVARHUSEIN A. ISAB and R. STEPHEN REID Department of Chemistry, University of Alberta, Edmonton, AIherta T6G 2G2 (Canada) (Received April 8th, 1981) (Revised manuscript received September 24th, 1981) Key Words: 1H-NMR; Methylmercurv binding; Glutathione; Hemoglobin; (tIuman ervthrocyte) The binding of methylmercury, CH3Hg(II), by small molecules in the intracellular region of human erythrocytes has been studied by IH-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, IH-NMR spectra were measured for the more abundant intracellular small molecules, including glycine, alanine, creatine, lactic acid, ergothioneine and glutathione, in 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 a-carbon of the cysteine residue of glutathione, it is shown that, in hemolyzed erythrocytes, the suifhydryl group of glutathione binds CH3Hg(II) more strongly than the sulfhydryl groups of hemoglobin. Introduction It has been estimated from studies of the metabolism of orally administered tracer doses of methylmercury in humans that from 5 to 10% of the body burden of methylmercury is in the blood, the amount decreasing from the higher to the lower level with time following exposure [1]. In blood, the methylmercury is distributed between the plasma and the erythrocytes, with 90% or more of the total in the erythrocytes [1-4]. Because of the high affinity of mercury for sulfur, it is gener- ally assumed that the methylmercury in erythro- cytes and other tissues is complexed by sulfhydryl groups [4-6]. The two most abundant sulfhydryl- containing molecules in erythrocytes are hemo- 0167-4889/82/0000-0000/$02.75 ~ 1982 Elsevier Biomedical Press globin and glutathione; the membrane contains less than 6% of the total sulfhydryl content of human erythrocytes [5]. In this paper, we report the results of an in vitro study by ~H-NMR spectroscopy of the binding of methylmercury in human erythrocytes. IH-NMR spectra were measured for the more abundant small molecules of the intracel!ular region of intact erythrocytes by a technique based on selective saturation [7], or by the spin-echo Fourier trans- form NMR technique [8,9], to either suppress or completely eliminate interfering resonances from the hemoglobin protons [10]. The results for erythrocytes to which methylmercury had been added indicate that part of the added methyl- mercury is complexed by the intracellular glutath-