Relationship between β4 hydrogen bond and β6 hydrophobic interactions during aggregate, fiber or crystal formation in oversaturated solutions of hemoglobin A and S

Abstract

Oversaturated deoxy-α 2 β 2 T4V aggregated instantly without a delay time, which is in contrast to the delay time before the generation of fibers of deoxy-HbS and deoxy-α 2 β 2 E6V, D73H . Solubility of deoxy-α 2 β 2 T4V was ~10-fold lower than that of deoxy-HbS and was similar to oxy-and deoxy-α 2 β 2 E6V,T4V . These results indicate that β4Val in HbA in the oxy and deoxy forms with or without β6Val facilitates hydrophobic interaction of the A-helix with the EF helix of adjacent molecules without forming a β4/β73 hydrogen bond. Deoxy-HbA generated crystals following aggregation as does HbC-Harlem(α 2 β 2 E6V,D73N ), while α 2 β 2 T4V and α 2 β 2 D73H as well as HbS, α 2 β 2 E6V,D73H and α 2 β 2 E6V,T4V in the oxy and deoxy forms did not form crystals, indicating in addition to the strength of β6 amino acid hydrophobicity that the synergism between the β4Thr hydrogen bond and β6 hydrophobic interaction free energies on the A-helix play a critical role in formation of fibers versus crystalline nuclei during phase transition. KeywordsHemoglobin; Polymerization; Fiber formation; Crystallization; Hydrogen bond; Hydrophobic interaction; Sickle hemoglobin; Phase transitionThe single amino acid replacement of β6Glu with a hydrophobic β6Val in the β chain of hemoglobin (HbS) leads to decreased solubility and formation of gels including long, multistranded fibers when HbS is deoxygenated [1,2]. HbS fibers appear stable but over longer periods in vitro form crystals with a lower solubility [3,4]. In addition, the single amino acid replacement of β6Glu to β6Lys (HbC) leads to crystallization in the oxy-form in vitro and in vivo [5]. Intracellular polymers or fibers which go on to form gels and crystals from soluble hemoglobin cause a significant reduction in RBC deformability and leads to membrane damage, which can obstruct flow in the microcirculation in patients with a sickle cell disorder [1,2,6]. Furthermore, oxy-and deoxy-HbA formed amorphous aggregates when oversaturated in vitro even though HbA has a high solubility. In addition, oversaturated deoxy-HbA generated Please address all correspondence to: Kazuhiko Adachi, Ph.D., Division of Hematology, The Children's Hospital of Philadelphia, 34th St. & Civic Center Blvd., Philadelphia, PA 19104, Tel: 215-590-3576; Fax: 215-590-4834, e-mail: adachi@email.chop.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript crystals after a long incubation time in high phosphate buffers [7,8]. In fact, despite the polymeric nature of deoxy-HbS...

Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility

“…The hydrophobicity and the size of the side chain of the amino acid at ␤ S 2 6 or the ␤ S 2 4 position affects the solubility of the resulting mutants (13)(14)(15). Solubility can be viewed as the concentration of hemoglobin that remains in solution after completion of polymerization.…”