Heterogeneity of Band 3, the Major Intrinsic Protein of Human Erythrocyte Membranes: Studies by Crossed Immunoelectrophoresis and Crossed Immuno-Affinoelectrophoresis1

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Abstract
The binding of the human erythrocyte membrane major intrinsic protein (Band 3) to concanavalin A (Con A), Ricinus communis agglutinin (RCA), and pokeweed mitogens (PWM) has been studied by crossed immunoelectrophoresis and crossed immuno-affinoelectrophoresis. Although pure by the criterion of polyacrylamide gel electrophoresis, Band 3 was resolved by crossed immunoelectrophoresis into three peaks. A major peak was split on both the anodic and cathodic sides, and consisted of at least two components. A smaller additional peak was also observed. By introducing Con A in the first dimension of the run, the main peak was dissociated into 2 peaks of different heights. Partial binding to Con A was confirmed by introducing Con A Sepharose 4B in an intermediate gel in the second dimension of the electrophoresis. When the first dimension gel of the crossed immunoelectrophoresis contained RCA, or when RCA Sepharose 4B was present in an intermediate gel in the second dimension of the run, the major peak was far smaller because of binding to that lectin. In experiments with a first dimension gel containing pokeweed mitogens, the major peak was higher but narrower than in the control experiments, presumably due to selective binding of some constituents of Band 3. Immuno-affinoelectrophoretic methods therefore show that Band 3 is heterogeneous and that its components bind differently to the above lectins.

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