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Aggregation of Normal and Sickle Hemoglobin in High Concentration Phosphate Buffer

Kejing Chen ^*, Samir K. Ballas , Roy R. Hantgan  and Daniel B. Kim-Shapiro ^*

^* Departments of Physics and Biochemistry, Wake Forest University, Winston-Salem, North Carolina; and The Cardeza Foundation, Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania

Correspondence: Address reprint requests to Roy Hantgan, Tel.: 336-716-4675; Fax: 336-716-7671; E-mail: rhantgan{at}wfubmc.edu.

Correspondence: Address reprint requests to Daniel Kim-Shapiro, Tel.: 336-758-4993; Fax: 336-758-6142; E-mail: shapiro{at}wfu.edu

Sickle cell disease is caused by a mutant form of hemoglobin, hemoglobin S, that polymerizes under hypoxic conditions. The extent and mechanism of polymerization are thus the subject of many studies of the pathophysiology of the disease and potential treatment strategies. To facilitate such studies, a model system using high concentration phosphate buffer (1.5 M–1.8 M) has been developed. To properly interpret results from studies using this model it is important to understand the similarities and differences in hemoglobin S polymerization in the model compared to polymerization under physiological conditions. In this article, we show that hemoglobin S and normal adult hemoglobin, hemoglobin A, aggregate in high concentration phosphate buffer even when the concentration of hemoglobin is below the solubility defined for polymerization. This phenomenon was not observed using 0.05 M phosphate buffer or in another model system we studied that uses dextran to enhance polymerization. We have used static light scattering, dynamic light scattering, and differential interference contrast microscopy to confirm aggregation of deoxygenated and oxygenated hemoglobins below their solubility and have shown that this aggregation is not observable using turbidity measurements, a common technique for assessing polymerization. We have also shown that the aggregation increases with increasing temperature in the range of 15°–37°C and that it increases as the concentration of phosphate increases. These studies contribute to the working knowledge of how to properly apply studies of hemoglobin S polymerization that are conducted using the high phosphate model.