Kinetics of sickle haemoglobin polymerization in single red cells

Kinetic studies on solutions of purified haemoglobin S indicate that the rate of intracellular polymerization is an important variable in the pathophysiology of sickle cell disease. Until now, however, no experimental technique has been available to measure directly the kinetics of intracellular polymerization. Indirect methods, which use visual determination of cellular shape changes or changes in filterability of red cell suspensions, have given apparently conflicting results. Here we report our initial results on the application of a laser-photolysis, light scattering technique to measure directly the kinetics of haemoglobin S polymerization in single red cells. In our experiment, deoxyhaemoglobin S is rapidly formed by photolysing the carbon monoxide complex with an argon ion laser focused inside the cell, and the change in scattered light is used to detect the appearance of polymer. We find a very wide distribution of delay times, ranging from 1 ms to greater than 100 s, indicating that the polymerization inside red cells proceeds by the same nucleation and growth mechanism as in solutions of purified haemoglobin S.