LI-7 NMR RELAXATION STUDY OF LI+ BINDING IN HUMAN ERYTHROCYTES

We used Li-7 NMR spin-lattice (T1) and spin-spin (T2) relaxation time measurements to investigate the binding of Li+ in human red blood cell (RBC) suspensions. In RBCs containing 1.4 mM Li+, the intracellular L i-7 NMR T2 relaxation value (0.30 +/- 0.03 s) was much smaller than th e corresponding T1 value (6.0 +/- 0.1 s), yielding a ratio of T1 to T2 of 20. For 1. 5 mM LiCl solutions whose viscosities were adjusted to 5 cP with glycerol, the values of the T1/T2 ratios were as follows: 49 for unsealed RBC membrane (2.0 mg of protein/mL); 4.4 for spectrin (1 .9 mg/mL); 1.5 for 5.4 mM 2,3-bisphosphoglycerate (BPG); 2.2 for 2.7 m M carbonmonoxyhemoglobin (COHb); 1.6 for 2.0 mM ATP; and 1.2 for a 50/ 50% (v/v) glycerol-water mixture. Intracellular viscosity and the elec tric field gradients experienced by Li+ when traversing the spectrin-a ctin network therefore are not responsible for the large values of the T1/T2 ratios observed in Li+-loaded RBCs. We conclude that the RBC me mbrane is the major Li+ binding site in Li+-loaded RBCs (K(b) = 215 +/ - 36 M-1) and that the binding of Li+ to COHb, BPG, spectrin-actin, or ATP is weak. Partially relaxed Li-7 NMR spectra of Li+-containing RBC membrane suspensions indicated the presence of two relaxation compone nts, one broad and one narrow. At the same extravesicular Li+ and prot ein concentrations, the T1 values for right-side-out RBC vesicle suspe nsions were at least 2-fold larger than those for inside-out RBC vesic le suspensions; the inner layer of the RBC membrane, which has a large r percentage of anionic phospholipids than the outer layer, contribute s mostly to Li+ binding.