Polymorphism in the M(r) 32,000 Rh protein purified from Rh(D)-positive and -negative erythrocytes

A M(r) 32,000 integral membrane protein has previously been identified on erythrocytes bearing the Rh(D) antigen and is thought to contain the antigenic variations responsible for the different Rh phenotypes. To study it on a biochemical level, a simple large-scale method was developed to purify the M(r) 32,000 Rh protein from multiple units of Rh(D)-positive and -negative blood. Erythrocyte membrane vesicles were solubilized in NaDodSO₄, and a tracer of immunoprecipitated ¹²⁵I surface-labeled Rh protein was added. The Rh protein was purified to homogeneity by hydroxylapatite chromatography followed by preparative NaDodSO₄/PAGE. Approximately 25 nmol of pure Rh protein was recovered from each unit of Rh(D)-positive and -negative blood. Rh protein purified from both Rh phenotypes appeared similar by one-dimensional NaDodSO₄/PAGE and the N-terminal amino acid sequences for the first 20 residues were identical. Rh proteins purified from Rh(D)-positive and -negative blood were compared by two-dimensional iodopeptide mapping after ¹²⁵I-labeling and α-chymotrypsin digestion. The peptide maps were very similar; however, at least two additional iodopeptides were consistently noted in the Rh proteins purified from Rh(D)-positive erythrocytes. These data indicate that a similar core Rh protein (or group of related proteins) exists in both Rh(D)-positive and -negative erythrocytes, and the Rh proteins from erythrocytes with different Rh phenotypes contain distinct structural polymorphisms.