Nucleation-controlled aggregation of deoxyhemoglobin S effect of organic phosphates on the kinetics of aggregation of deoxyhemoglobin S in concentrated phosphate buffer

Abstract

The kinetics of the aggregation of deoxyhemoglobin S in concentrated phosphate buffer were studied turbidimetrically in the presence of organic phosphates. The addition of inositol hexaphosphate shortened the delay time by 35–80%, the range depending on the hemoglobin concentration. The logarithmic plot of delay time versus hemoglobin concentration in 1.85 M potassium phosphate buffer, pH 7.34, showed a linear relationship with a slope ( n value) of 2.5 ± 0.14 in the absence of inositol hexaphosphate, while the slope was decreased to 2.1 in the presence of inositol hexaphosphate. The turbidity ( A 700) of aggregates per g deoxy Hb S was found to be lower in the presence of inositol hexaphosphate than in the absence of inositol hexaphosphate. The solubility of deoxy Hb S was decreased by inositol hexaphosphate. 2,3-Diphosphoglyceric acid also showed an effect similar to that of inositol hexaphosphate. However, if the pentacyclohexylammonium salt of diphosphoglyceric acid were used, the delay time was increased significantly. The opposite effect of the pentacyclohexylammonium salt of diphosphoglyceric acid on the delay time was found to be attributed to the anti-gelation effect of the pentacyclohexoxylammonium ion. The difference in the effect of inositol hexaphosphate and diphosphoglyceric acid on the delay time appears to be related to their effect on the solubility of deoxy Hb S.