Single Molecule Studies of the Diffusion of Band 3 in Sickle Cell Erythrocytes.
Sickle cell disease (SCD) is caused by an inherited mutation in hemoglobin that leads to sickle hemoglobin (HbS) polymerization and premature HbS denaturation. Previous publications have shown that HbS denaturation is followed by binding of denatured HbS (a.k.a. hemichromes) to band 3, the consequent clustering of band 3 in the plane of the erythrocyte membrane that in turn promotes binding of autologous antibodies to the clustered band 3, and removal of the antibody-coated erythrocytes from circulation. Although each step of the above process has been individually demonstrated, the fraction of band 3 that is altered by association with denatured HbS has never been determined. For this purpose, we evaluated the lateral diffusion of band 3 in normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and hemoglobin SC erythrocytes (HbSC) in order to estimate the fraction of band 3 that was diffusing more slowly due to hemichrome-induced clustering. We labeled fewer than ten band 3 molecules per intact erythrocyte with a quantum dot to avoid perturbing membrane structure and we then monitored band 3 lateral diffusion by single particle tracking. We report here that the size of the slowly diffusing population of band 3 increases in the sequence: normal cells<HbSC<RSC<ISC. We also demonstrate that the size of the compartment in which band 3 is free to diffuse decreases roughly in the same order, with band 3 diffusing in two compartments of sizes 35 and 71 nm in normal cells, but only a single compartment in HbSC cells (58 nm), RSC (45 nm) and ISC (36 nm). These data suggest that the mobility of band 3 is increasingly constrained during SCD progression, suggesting a global impact of the mutated hemoglobin on erythrocyte membrane properties.
Department of Physics, Purdue University, West Lafayette, IN, 47907, United States of America.
Jeff
J
Spector
Department of Chemistry, Purdue University, West Lafayette, IN, 47907, United States of America.
Gayani C
GC
Kodippili
Department of Physics, Purdue University, West Lafayette, IN, 47907, United States of America.
Ken
K
Ritchie
Department of Chemistry, Purdue University, West Lafayette, IN, 47907, United States of America.
Philip S
http://orcid.org/0000-0001-9042-5528
PS
Low
D000755:Anemia, Sickle Cell / Q000097:blood* / Q000473:pathology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D004058:Diffusion; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004913:Erythrocytes, Abnormal / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism; D006451:Hemoglobin, Sickle / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015335:Molecular Probes / Q000737:chemistry; D045663:Quantum Dots / Q000737:chemistry; D000072760:Single Molecule Imaging / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
D001457:Anion Exchange Protein 1, Erythrocyte; D006420:Hemeproteins; D006451:Hemoglobin, Sickle; D015335:Molecular Probes; C001332:hemichrome