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Liquid-Liquid Phase Separation in Hemoglobins: Distinct Aggregation Mechanisms of the ß6 Mutants

Qiuying Chen ^*, Peter G. Vekilov , Ronald L. Nagel ^*  and Rhoda Elison Hirsch ^* 

^* Department of Medicine, Division of Hematology, Albert Einstein College of Medicine, Bronx, New York; Department of Chemical Engineering, University of Houston, Houston, Texas; Department of Physiology and Biophysics, and Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York

Correspondence: Address reprint requests to Dr. Rhoda Elison Hirsch, Dept. of Medicine and Dept. of Anatomy and Structural Biology, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-3604; Fax: 718-824-3153; E-mail: rhirsch{at}aecom.yu.edu.

Reversible liquid-liquid (L-L) phase separation in the form of high concentration hemoglobin (Hb) solution droplets is favored in an equilibrium with a low-concentration Hb solution when induced by inositol-hexaphosphate in the presence of polyethylene glycol 4000 at pH 6.35 HEPES (50 mM). The L-L phase separation of Hb serves as a model to elucidate intermolecular interactions that may give rise to accelerated nucleation kinetics of liganded HbC (ß6 Lys) compared to HbS (ß6 Val) and HbA (ß6 Glu). Under conditions of low pH (pH 6.35) in the presence of inositol-hexaphosphate, COHb assumes an altered R-state. The phase lines for the three Hb variants in concentration and temperature coordinates indicate that liganded HbC exhibits a stronger net intermolecular attraction with a longer range than liganded HbS and HbA. Over time, L-L phase separation gives rise to amorphous aggregation and subsequent formation of crystals of different kinetics and habits, unique to the individual Hb. The composite of R- and T-like solution aggregation behavior indicates that this is a conformationally driven event. These results indicate that specific contact sites, thermodynamics, and kinetics all play a role in L-L phase separation and differ for the ß6 mutant hemoglobins compared to HbA. In addition, the dense liquid droplet interface or aggregate interface noticeably participates in crystal nucleation.

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