Free Heme in Erythrocyte Density Fractions



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Sickle cell anemia has been considered the first molecular disease. A single genetic point mutation, affecting the structure of hemoglobin, leads to deformations in blood cells which can cause cardiovascular crisis. Sickle cell hemoglobin (HbS) polymerizes and forms long rigid fibers, which deform the cell. Free heme has been shown to significantly enhance HbS nucleation, leading to polymerization. In this study, we investigated different density layer fractions of erythrocytes and the concentration of free heme present in the erythrocyte cytosol. Through centrifugation of erythrocytes in Percoll, we were able to form a density gradient of erythrocytes. Using enzyme-linked chemiluminescence, we quantified the concentration of free heme in each erythrocyte density fraction. Two different methods of gathering erythrocyte layers were developed, which generated similar results, but significantly different standard errors. Over both methods, the concentration of free heme in four density layers (1.032, 1.053, 1.071, 1.085 g/mL) were found to be: 1 ± 1 μM, 5 ± 2 μM, 38 ± 12 μM, and 19 ± 17 μM respectively. In the second method of separation developed, the free heme concentration decreases significantly to 3 ± 3 μM.



Heme Erythrocyte