Temperature- and concentration-dependence of urea permeability of human erythrocytes determined by NMR

The temperature- and concentration-dependence of [13C]urea self-exchange across the human red cell membrane has been determined by NMR measurements of T1 (spin-lattice) relaxation times. T1 for intracellular label is 17 s, which is much longer than the urea exchange time across the cell membrane (about 0.5 s). T1 for urea in extracellular solution is quenched with 17 mM of impermeable Mn2+ in less than 2 ms. Hence the observed T1 (corrected for intracellular decay) is a measure of urea exchange across the cell membrane. The method is tested by showing both PCMBS and increasing concentrations of urea lengthen T1. Urea exchange permeability, defined as Purea = flux/conc, can be described by Purea = Vmax/(K1/2 + conc). Studies of temperature-dependence showed that activation energies were strongly dependent on both temperature and concentration. However, this apparently anomalous behavior was resolved into two well-behaved functions, K1/2 and Vmax, with linear Arrhenius plots and apparent 'activation energies' of 15.5 and 12.4 kcal/mol, respectively. These were used to construct an equation for calculating Purea at any concentration and temperature. Assuming a simple channel model with single binding, K1/2 becomes the dissociation equilibrium constant for the site with delta H degree = 15.5 kcal/mol and delta S degree = 51.8 cal/(mol.deg); dissociation is entropically driven.