Diffusion-limited reaction of free nitric oxide with erythrocytes

Abstract

Concentration changes of nitric oxide (NO) were monitored using an NO-sensitive electrode in phosphate-buffered saline (PBS) with either free oxyhemoglobin (oxyHb) or red blood cells (RBCs). In aerated PBS, the half-life of 0.9 microM NO is greater than 4 min. NO is undetectable (<50 nM) when added to a solution of oxyHb because the reaction of NO with oxyHb is rapid. The disappearance rate of NO in PBS containing RBCs is rapid, compared with PBS, but it is much slower (by a factor of approximately 650) than with an equivalent solution of free oxyHb. The half-life of NO is inversely proportional to the concentration of RBCs, independent of oxyHb concentration inside RBCs, and the disappearance rate of NO is first order in NO concentration and first order in the concentration of RBCs. After all the oxyHb reacts with NO to form methemoglobin, the disappearance rate of NO slows greatly. These data indicate that the reaction of NO with oxyhemoglobin within RBCs is limited by the diffusion of NO into the cell, which has also been shown previously for the reaction of O2 with deoxyhemoglobin. Experimental data show that the half-life of NO in the presence of 2.1 x 10(6) RBCs/ml is 4. 2 s. From this value, we estimate that the half-life of NO in whole blood (5 x 10(9) RBCs/ml) will be 1.8 ms. A simple analytical expression for the half-life of NO in PBS with RBCs was derived in this study based on a spherical diffusion model. The calculated half-life of NO from the expression is in good agreement with the experimental values.