Abstract
The addition of hemoglobin to isolated membrane ghosts of human erythrocytes followed by catalytic oxidation with Cu2+-o-phenanthroline results in the covalent attachment of hemoglobin to the membrane. A decrease in the mobility of Band 3 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to an increase in molecular weight of approximately 16,000 suggests that Band 3 is a site for the covalent attachment of hemoglobin to the membrane under these conditions. This conclusion was confirmed using two-dimensional gels in which the covalent linkage was cleaved with 2-mercaptoethanol in the second dimension. 14C-labeled hemoglobin was used to show that the new band was composed of one globin monomer per Band 3 monomer. Additionally, it was demonstrated that only the beta chain of hemoglobin is covalently bound to the Band 3 protein.
Highlights
The addition of hemoglobin to isolated membrane hemoglobin for binding to the membrane as measured by ghosts ofhumanerythrocytes followed by catalytic fluorescence energy transfer [28, 29]and by kinetic measureoxidation with Cu’+-o-phenanthrolinreesults in theco- ments of CO binding to membrane-bound hemoglobin [30]
IDENTIFICATION OF BAND 3 AS A SITE FOR HEMOGLOBIN BINDING IN Cu*+-o-PHENANTHROLINE CATALYZED CROSS-LINKING*
A the binding of 4,4’-bis(isothiocyano)-2,2”stilbenedecrease in the mobility of Band 3 in sodium dodecyl disulfonate to whole erythrocytes, known to bind to Band 3 sulfate-polyacrylamidegel electrophoresis corresponding to an increase in molecwuleairght of approximately 16,000 suggests that Band 3 is a site for the covalent attachment of hemoglobintothemembraneunder these conditions
Summary
Val. 256, No 24, Issue of December 25, pp. 13152-13158, 1981 Printed m U.S.A. Cross-linkingof Hemoglobin to the Cytoplasmic Surfaceof Human Erythrocyte Membranes. IDENTIFICATION OF BAND 3 AS A SITE FOR HEMOGLOBIN BINDING IN Cu*+-o-PHENANTHROLINE CATALYZED CROSS-LINKING*
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