Band 3 diffusion on healthy and diseased red blood cells and implications for RBC membrane structure

Gayani Chandima Kodippili, Purdue University

Abstract

Band 3 which is the one of the most abundant membrane protein in red blood cell membrane. It is bound to the spectrin network via other proteins such as ankyrin, 4.1 or junctional complex. In addition to that literature suggests the presence of different sub populations of band 3. The spectrin mesh leads to the hexagonal compartments in red blood cell membrane. The arrangement of this skeleton network affects the stability and the size of the compartments. We discovered that spectrin compartment size and the band3 diffusion vary from normal to disease patient’s blood such as HbSS (Sickle cell anemia), HbSC (Sickle hemoglobin C disease), HbSB° (Sickle cell zero-beta-thalasamia), HbSB+ (Sickle cell beta-plus-thalasamia), HS (Hereditary Spherocytosis), and HPP (Hereditary Pyropoikilocytosis). HbSS has comparatively smaller compartment size and slow diffusion coefficient than normal blood cells. We have been able to show that by Single particle tracking (SPT) studies, which is investigated by video-enhanced TIR microscopy with 8ms temporal resolution, by labeling the band 3 with DIDS-Biotin linker bound to streptavidin Q-dots. Specificity and binding ability of linker to band 3 were investigated; (i) whole red blood cells were labeled with a synthesized compound called DIDS-biotin linker (Figure 1). Labeled red blood cells were co-precipitated with avidin beads to indicate the presence of band3. (ii) Fluorescence microscopy images were collected by incubating the cells in the presence and the absence of linker. RBC appears only in the fluorescents field of the specimen which is incubated with linker. (iii) Flow cytometry study of binding assay indicates the concentration dependency of DIDS-biotin linker to red blood cells. The 50% saturation was attained at ∼1-2E-5M. (iv) The cells were labeled with 1E-11M concentration of linker to obtain the one or few Q-dots (0-3) per cell. The SPT data suggest the presence of mobile and immobile populations of band 3 in red blood cell membranes. Based on these studies variation of distribution of these populations on different blood samples suggest the changes in structural attachment of band 3 to the skeleton. This may also help to explain why the HS and HPP red blood cells are fragile compared to rigid sickle cells.

Degree

Ph.D.

Advisors

Low, Purdue University.

Subject Area

Biochemistry|Physical chemistry|Biophysics

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