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

Abstract

The temperature- and concentration-dependence of [ 13C]urea self-exchange across the human red cell membrane has been determined by NMR measurements of T 1 (spin-lattice) relaxation times. T 1 for intracellular label is 17 s, which is much longer than the urea exchange time across the cell membrane (about 0.5 s). T 1 for urea in extracellular solution is quenched with 17 mM of impermeable Mn 2+ in less than 2 ms. Hence the observed T 1 (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 T 1. Urea exchange permeability, defined as P urea = flux/ conc, can be described by P urea = V max / (K 1 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, K 1 2 and V max, 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 P urea at any concentration and temperature. Assuming a simple channel model with single binding, K 1 2 becomes the dissociation equilibrium constant for the site with ΔH° = 15.5 kcal/mol and ΔS° = 51.8 cal/(mol·deg); dissociation is entropically driven.