Translational diffusion of glycine in erythrocytes measured at high resolution with pulsed field gradients

Abstract

In the past we used relaxation measurements of [ 2,13C]glycine to estimate its intracellular translational diffusion coefficient and the viscosity of the cytoplasm in erythrocytes (I, 2). Here we report on a more direct approach to these measurements using pulsed field gradient (PFG) NMR (3) in a high-resolution mode at 9.4 T with low concentrations of solute. The results indicate that the intracellular glycine diffusion coefficient is one-third of the free solution value of 1.18 X 10 -9 m 2 s-l. The use of relaxation time measurements to estimate macroviscosity via the Debye equation (4) and thence translational diffusion coefficients using the Stokes-Einstein equation (5, 6) requires that the solvent mo lecules be “seen” as a cont inuum by the probe mo lecule. For a probe mo lecule in the cytoplasm of a red cell, the “solvent” mo lecules are hemoglobin and H20. Herrmann and M iiller ( 7) showed that the intracellular macroviscosity is almost exclusively determined by the hemoglobin concentration. Our results indicate that the glycine mo lecule does not see the solvent as a continuum, which is consistent with it being three orders of magn itude smaller than hemoglobin. Human blood was obtained from the Red Cross Blood Transfusion Service, Sydney, NSW. [ 2,‘3C]Glycine was from Merck Sharp and Dohme, Montreal, Canada. The blood was washed twice in saline ( 154 mmo l/liter of NaCl, 10 mmo l/liter of glucose, 277 K) and then bubbled with carbon monoxide. The cells were centrifuged to give a pellet of hematocrit -0.9; 5 m l of the pellet was then added to 5 m l of solution containing [ 2,13C]glycine ( 100 mmo l/liter) in saline ( 125 mo l/liter ofNaC1 and 20 mmo l/liter ofglucose in 20/80 ‘H20/ ‘H*O, pH 7.4) which had been bubbled with carbon monoxide. The cell suspension was sealed in a centrifuge tube and incubated at 3 10 K for 5 h. Following the incubation the cell suspension was saturated with carbon monoxide and centrifuged. The cells were then resuspended in saline ( 154 mmo l/liter of NaCl-saturated with carbon monoxide) constituted in 100% 2H20 and the cells were centrifuged to give a pellet with a hematocrit greater than 0.9. Lysates of the glycine-incubated cells were made by sonication with a Branson B 12 sonifier (Branson Sonic Power Company, Danbury, Connecticut). Packed cells ( -0.5 m l) were transferred to flat-bottom (9 m m o.d.) NMR tubes and a Teflon vortex plug was inserted. These tubes were placed in 10 m m o.d. tubes