Identification of plasma Hpr as a hemoglobin-interacting protein by hemoglobin-affinity precipitation. (A) Immunoblot with an anti-Hp antibody (recognizing both Hp and Hpr) of proteins eluted from hemoglobin-coupled Sepharose (Hb), BSA-coupled Sepharose (BSA), or underivatized Sepharose (blank) after incubation with serum from a person (Hp phenotype 2-1) with a normal Hp level (0.9 g/L) and from a patient with Hp-deficient sickle cell disease. (B) Immunoblotting (reducing 12%-15% SDS-PAGE) of the hemoglobin-binding serum proteins from a person with the Hp phenotype 2-1 (same material as shown in Figure 2A) to identify the different α-chains of the Hp1, Hp2, and Hpr proteins. Lanes 2, 3, and 4 show silver-stained reducing (12%-15% gradient) SDS-polyacrylamide gel of Hp1-1 purified from human plasma, Hp2-2 purified from human plasma, and purified recombinant Hpr, respectively, demonstrating the distinct migration patterns of the Hp1, Hp2, and Hpr α-chains. The identity of the bands from the purified proteins was verified by mass spectrometry (data not shown). (C) Silver-stained (8%-16% gradient) SDS-polyacrylamide gel of proteins from normal human serum bound to hemoglobin-coupled Sepharose or underivatized Sepharose. The identity of the protein bands representing apoL-I and Hpr was determined by mass spectrometry and amino-terminal sequencing (as indicated).