Viscosity studies of deoxyhemoglobin S: evidence for formation of microaggregates during the lag phase

To look for evidence for the formation of deoxy S microaggregates during the lag phase, the kinetics of gelation of purified deoxy S in dilute phosphate buffer were continuously monitored by a Wells-Brookfield cone and plate microviscometer. Three separate manipulations were performed during the lag phase. (1) The rotating cone of the viscometer was stopped for varying intervals and then restarted. (2) Samples were withdrawn from runs in mid-to-late lag phase ("poised") and reintroduced early in the lag phases of other runs with appropriate volume, temperature, and concentration adjustments. (3) Deoxy S was partially replaced in mid-lag phase by solutions with equivalent volumes, temperatures, and concentrations of deoxy A, F, or C or deoxygenated albumin. The results support the conclusions that (1) irreversible microaggregates are formed early in the lag phase, (2) their formation is favored by stirring, and (3) they are stable if the shearing is stopped for intervals up to six times the total lag times of control runs. Replacement of deoxy S by an equivalent concentration of deoxygenated nonsickle protein during the lag phase does not change the lag time, whereas mixtures of sickle hemoglobin and other proteins made before the reaction is initiated by temperature jump have markedly prolonged lag times. This suggests that once microaggregates of deoxy S are formed, the excluded volume effect of deoxygenated nonsickle hemoglobins or albumin supports polymerization at the same rate as sickle hemoglobin. These results are consistent with the proposed equilibrium nucleation/irreversible growth model of nucleation.