Nuclear Magnetic Resonance Studies of the Complexation of Trimethyllead by Glutathione in Aqueous Solution and in Intact Human Erythrocytes

The complexation of trimethyllead (TML), (CH₃)₃Pb^IV, by glutathione (y-L-glutamyl-L-cysteinylglycine (GSH)) has been studied in aqueous solution and in intact human erythrocytes by nuclear magnetic resonance spectroscopy. The deprotonated sulfhydryl group is shown to be the strongest binding site for TML. Formation constants, including microscopic formation constants for sulfhydryl complexes in which the amino group is protonated and deprotonated, have been determined from the dependence of the chemical shift of the exchange-averaged resonance for the methyl protons of TML on solution conditions. To determine if TML is also complexed by GSH in a more complex biological system, we have measured spin-echo Fourier transform (SEFT) NMR spectra for intact erythrocytes to which TML has been added. Resonances are observed for the naturally occurring GSH and several other small molecules, including glycine, alanine, ergothionine, creatine, and lactic acid. Of these potential ligands, TML is found to be complexed only by the intracellular GSH, and the complex is identical with that which forms in much simpler aqueous solutions. The NMR results on the erythrocytes also indicate that TML, added as trimethyllead acetate, rapidly crosses the erythrocyte membrane and that the TML-poisoned erythrocytes continue to metabolize glucose. Measurements have also been made on the competitive complexation of TML in intact erythrocytes by the naturally present GSH and added penicillamine, a molecule which has been tried as a treatment for alkyllead poisoning. The results obtained in this study demonstrate that detailed information about the complexation of metals in intact cellular systems such as erythrocytes can be obtained by ¹H NMR spectroscopy.