The objective of this project is to test the hypothesis that interaction of mutant and unpaired Hb chains with specific membrane components affects KCI cotransport and deformability of red cells (RBC).
Two Specific Aims will be pursued: 1) to determine the extent to which mutant unpaired Hb chains modify KCI co-transport, membrane deformability and mechanical strength, and 2) to identify membrane components that interact with Hb to mediate these effects. To define (IOV) and resealed membranes in the presence of normal Hb vs mutant Hb and excess alpha and beta globin chains. Differential extraction and readdition of selected membrane skeletal components will test for medication of Hb effects via the membrane skeleton, and maneuvers to block association of Hb chains with integral membrane proteins will test for medication by B3 and glycophrin. Differential effects of various type of Hb on membrane deformability and mechanical strength will be assessed by comparative assays of resealed membranes containing various concentrations and types of Hb, using the ektacytometer, a diffraction viscometer. To identify specific membrane components that interact with Hb to modulate these functions, RBC will be treated with variety of cross-linking agents at low concentrations to stabilize Hb/membrane associations in the intact cell and prevent their disruption during analysis. Hb cross-linking to specific membrane components will be detected by separating membrane proteins by polyacrylamide gel electrophoresis, preparing immunoblots and probing them with anti-Hb and antibodies against membrane components. Apparent complexes between Hb and membrane components will be confirmed by immunoprecipitation with antibody against the membrane component, and probing of immunoblots of the immunoprecipitate using anti-Hb. To probe for interaction of Hb with the protein that mediates KCI cotransport, rabbit polyclonal antibodies against the protein will be prepared, making use of the differential expression of the antigen in high K and low K sheep RBC. It is expected that information derived from these studies will provide new information about the effects of HbS and HbC on membrane function that may be useful in understanding the variable pathophysiologic manifestations in the sickling disorders.
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