The Hb A Variant (β73 Asp→Leu) Disrupts Hb S Polymerization by a Novel Mechanism Kazuhiko Adachi 1 ⁎, Min Ding 1 , Saul Surrey 2 , Maria Rotter 3 Alexey Aprelev 3 , Mikhail Zakharov 3 , Weijun Weng 3 and Frank A. Ferrone 3 1 The Children's Hospital of Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA 2 Cardeza Foundation for Hematologic Research, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA 3 Department of Physics, Drexel University, Philadelphia, PA 19104, USA Polymerization of a 1:1 mixture of hemoglobin S (Hb S) and the artificial mutant HbAβ73Leu produces a dramatic morphological change in the polymer domains in 1.0 M phosphate buffer that are a characteristic feature of polymer formation. Instead of feathery domains with quasi 2-fold symmetry that characterize polymerization of Hb S and all previously known mixtures such as Hb A/S and Hb F/S mixtures, these domains are compact structures of quasi-spherical symmetry. Solubility of Hb S/ Aβ73Leu mixtures was similar to that of Hb S/F mixtures. Kinetics of polymerization indicated that homogeneous nucleation rates of Hb S/ Aβ73Leu mixtures were the same as those of Hb S/F mixtures, while exponential polymer growth (B) of Hb S/Aβ73Leu mixtures were about three times slower than those of Hb S/F mixtures. Differential interference contrast (DIC) image analysis also showed that fibers in the mixture appear to elongate between three and five times more slowly than in equivalent Hb S/F mixtures by direct measurements of exponential growth of mass of polymer in a domain. We propose that these results of Hb S/Aβ73Leu mixtures arise from a non-productive binding of the hybrid species of this mixture to the end of the growing polymer. This “cap” prohibits growth of polymers, but by nature is temporary, so that the net effect is a lowered growth rate of polymers. Such a cap is consistent with known features of the structure of the Hb S polymer. Domains would be more spherulitic because slower growth provides more opportunity for fiber bending to spread domains from their initial 2-fold symmetry. Moreover, since monomer depletion proceeds more slowly in this mixture, more homogeneous nucleation events occur, and the resulting gel has a far more granular character than normally seen in mixtures of non-polymerizing hemoglobins with Hb S. This mixture is likely to be less stiff than polymerized mixtures of other hybrids such as Hb S with HbF, potentially providing a novel approach to therapy. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: hemoglobin; sickle hemoglobin; HbF; anti-HbS polymerization; fiber formation Introduction In sickle cell disease the mutation from GTG to GAG in the triplet code at the sixth position from the N terminus of the β-globin chain results in replacement of negatively charged Glu with the uncharged, hydrophobic Val, which is on the molecular surface of the Hb S molecule and decreases its solubility. When deoxy-Hb S con- centration exceeds its solubility, hemoglobin aggregates and forms long, multi-stranded poly- mers, which are comprised of 14 strands. Because one polymer can nucleate others on its surface, the polymers form in attached arrays, called polymer domains. The domains begin with a Abbreviations used: Hb, hemoglobin; DIC, differential interference contrast; LCR, locus control region. E-mail address of the corresponding author: adachi@email.chop.edu doi:10.1016/j.jmb.2006.07.047 J. Mol. Biol. (2006) 362, 528–538 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.