HEMICHROME BINDING TO ERYTHROCYTE BAND 3: IMPLICATIONS IN RED CELL AGING (SICKLE CELL, HEINZ BODY)

STEPHEN MILES WAUGH, Purdue University

Abstract

Hemichromes, the precursors of red cell Heinz bodies, were prepared by treatment of native hemoglobin with phenylhydrazine, and their interaction with the cytoplasmic surface of the human erythrocyte membrane was studied. Hemichromes were shown to bind with high affinity to the cytoplasmic domain of the integral membrane protein, band 3. Furthermore, the isolated cytoplasmic domain of band 3 interacted tightly with hemichromes, rapidly forming a water-insoluble copolymer of defined stoichiometry upon mixing the two proteins in aqueous solution. Copolymer formation was highly dependent upon pH and ionic strength forming best at low pH (pH (DBLTURN) 6.0) and low ionic strength. However, the copolymer became more stable at higher pH values as the hemichrome concentration was increased. The copolymer was highly specific for hemichromes since it did not form when the cytoplasmic domain of band 3 was mixed with oxyhemoglobin, carbonmonoxyhemoglobin, deoxyhemoglobin or methemoglobin. The affinity of the cytoplasmic domain of band 3 was likely much greater for hemichromes than for native hemoglobin since a 20-fold molar excess of native hemoglobin was required to reduce copolymerization by 50%. In addition, the cytoplasmic domain of band 3 readily formed copolymer with a hemichrome derivative which was covalently crosslinked between (beta) subunits, resulting in a blocked 2,3-diphosphoglycerate binding cavity. This same crosslink prevented band 3 binding with native hemoglobin (Walder et al., 1984). Heinz body binding to the membrane of sickle cells was shown to induce intramembrane clustering of band 3. Furthermore, conditions which caused band 3 clustering in normal cells also promoted IgG binding to these cells. From these results a potential mechanism for red cell aging was proposed in which Heinz body induced clustering of band 3 generates a site for autologous IgG binding. In this mechanism, the surface-bound IgG targets the senescent erythrocyte for phagocytosis.

Degree

Ph.D.

Subject Area

Biochemistry

COinS