Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 Received June 28, 1984 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. Binding of hemichromes to leaky red cell ghosts was found to be biphasic, exhibiting both high-affinity and low-affinity sites. The high-affinity sites were shown to be located on the cytoplasmic domain of band 3, since (i) glyceraldehyde-3-phosphate dehydrogenase, a known ligand of band 3, competes with the hemichromes for their binding sites,(ii) removal of the cytoplasmic domain of band 3 by proteolytic cleavage causes loss of the high-affinity sites, and (iii) the isolated cytoplasmic domain of band 3 interacts tightly with hemichromes, rapidly forming a pH-dependent, water-insoluble copolymer upon mixing in aqueous solution. Since the copolymer of hemichromes with the cytoplasmic domain of band 3 was readily isolatable, a partial characterization of its properties was conducted. The copolymer was shown to be of defined stoichiometry, containing~ 2.5 hemichrome tetramers (or~ 5 hemichrome dimers) per band 3 dimer, regardless of the ratio of hemichrome: band 3 in the initial reaction solution. The copolymer was found to be of macroscopic dimensions, generating particles which could be easily visualized without use of a mi-croscope. The coprecipitation was also highly selective for hemichromes, since, in mixed solutions with native hemoglobin, only hemichrome was observed in the isolated pellet. Furthermore, no precipitate was ever observed upon mixing the cytoplasmic domain of band 3 with oxyhemoglobin, deoxyhemoglobin,(carbonmonoxy) hemoglobin, or methemoglobin. The affinity of the cytoplasmic domain of band 3 was likely much higher for hemichromes than for native hemoglobin, since a 20-fold molar excess of hemoglobin was required to reduce copolymerization by 50%. We suggest that the copolymerization of band 3 and hemichromes in vivo can explain the aggregation of Heinz bodies on the erythrocyte membrane and the resulting hemolysis observed in numerous hemoglobinopathies. e interaction of native hemoglobin (Hb) with the human erythrocyte membrane has recently received considerable attention. Two classes of binding sites have been identified, and one of these, the higher affinity class, has been located on the cytoplasmic domain of the major erythrocyte protein, band 3 (Shaklai et al., 1977a, b; Salhany et al., 1980; Sayare f This work was supported in part by Grant GM 24417 from the National Institutes of Health.

& Fikiet, 1981). Thisinteraction has been shown to be electrostatic in nature with the affinity of Hb for the mem-brane increasing as pH and ionic strength decrease (Shaklai et al., 1977a, b; Fung, 1981; Fischer et al., 1975). A similar pH dependence of Hbbinding to the membrane has also been reported for intact cells (Eisinger et al., 1982). The isolated cytoplasmic domain of band 3 has also been shown to bind two molecules of hemoglobin, supporting the identification of band 3 as a major membrane attachment site for soluble