A novel method for solubility and copolymerization measurements of sickle hemoglobin

Mark K. Fugate

doi:10.17918/00010896

The polymerization of sickle hemoglobin (HbS) in red blood cells is the inciting event in the pathology of sickle cell disease, so understanding the polymerization process and how to disrupt it is of great scientific and medical interest. The stability of the polymer is quantified by a measurement of solubility, generally achieved via sedimentation. In this work, we present the first results of a novel optical micro-method for making solubility measurements. By growing aligned HbS polymers along an HbS concentration gradient set up inside a 20 [mu]m thick glass microcapillary and measuring polarized absorption parallel and perpendicular to the capillary axis, we can observe polymers by their linear dichroism (LD) and calculate total concentration as the average absorption. The concentration at which the LD signal disappears is identified as the solubility. Our measurements are generally consistent with those reported by other means, while using only micrograms of hemoglobin per sample. This technique should prove useful for the characterization of modified hemoglobins and the effects of pharmaceuticals. We also show how the same technique can be adapted to measure simultaneously the solubility and the hemoglobin composition of polymers formed in mixed samples. This is achieved by replacing the native heme of one species with a nickel or cobalt protoporphyrin-IX, imbuing it with a distinct absorption spectrum which manifests in the LD measurements. Our technique offers the first direct measurement of polymer composition in mixed samples. We use this method to investigate whether fetal hemoglobin (HbF) participates in polymer formation with HbS. Although existing literature on the subject offers conflicting evidence, it is commonly held that HbF does not copolymerize. Our measurements definitively show, however, that HbF copolymerizes to a significant extent, leading us to calculate copolymerization probabilities of 0.28 and 0.38 for hybrid F/S and pure F tetramers, respectively. At the same time, our solubility measurements are consistent with there being zero copolymerization, clarifying how the disagreement in published works came to be. We propose that the copolymerization model must be incomplete, and suggest some directions for future research, such as the possibility of structural heterogeneity in HbS aggregates. These results may also suggest that there may be even more effective treatment strategies for sickle cell disease beyond inducing HbF production.

A novel method for solubility and copolymerization measurements of sickle hemoglobin

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A novel method for solubility and copolymerization measurements of sickle hemoglobin

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