Waugh S M, Low P S
Biochemistry. 1985 Jan 1;24(1):34-9. doi: 10.1021/bi00322a006.
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 approximately 2.5 hemichrome tetramers (or approximately 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 microscope. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
高铁血红素是红细胞海因茨小体的前体，通过用苯肼处理天然血红蛋白制备而成，并研究了它们与人红细胞膜细胞质表面的相互作用。发现高铁血红素与渗漏红细胞血影的结合是双相的，表现出高亲和力和低亲和力位点。高亲和力位点位于带3的细胞质结构域上，因为：（i）甘油醛-3-磷酸脱氢酶是带3的已知配体，能与高铁血红素竞争其结合位点；（ii）通过蛋白水解切割去除带3的细胞质结构域会导致高亲和力位点丧失；（iii）分离出的带3细胞质结构域与高铁血红素紧密相互作用，在水溶液中混合后迅速形成pH依赖性的水不溶性共聚物。由于高铁血红素与带3细胞质结构域的共聚物易于分离，因此对其性质进行了部分表征。结果表明，该共聚物具有确定的化学计量比，每个带3二聚体含有约2.5个高铁血红素四聚体（或约5个高铁血红素二聚体），与初始反应溶液中高铁血红素与带3的比例无关。发现该共聚物具有宏观尺寸，产生的颗粒无需使用显微镜即可轻松观察到。这种共沉淀对高铁血红素也具有高度选择性，因为在与天然血红蛋白的混合溶液中，分离出的沉淀中只观察到高铁血红素。此外，将带3的细胞质结构域与氧合血红蛋白、脱氧血红蛋白、（碳氧）血红蛋白或高铁血红蛋白混合时，从未观察到沉淀。（摘要截断于250字）
