Relationship between β4 Hydrogen Bond and β6 Hydrophobic Interactions during Aggregate, Fiber or Crystal Formation in Oversaturated Solutions of Hemoglobin A and S

The 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, multi-stranded 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 crystals after a long incubation time in high phosphate buffers [7, 8]. In fact, despite the polymeric nature of deoxy-HbS, its assembly behaves thermodynamically like crystallization, with a well-defined solubility as measured by classic sedimentation experiments [9]. Moreover, the kinetics of deoxy-HbS polymerization is related to solubility in which the rate is proportional to the super-saturation point raised to a high power [2]. Previously we found that the solubility of hemoglobin is not unique and depends on experimental conditions and type of polymers generated [8, 10]. Growth of domains, development of deoxy-HbS polymers and their structure as well as oxy-HbC crystallization were studied by differential interference contrast (DIC) microscopic methods [11–13]. Deoxy-HbS polymerization kinetics observed by DIC microscopy indicated that domain structure depended on the kinetics of nuclei formation [12]. More recently, DIC microscopic examination of deoxy-HbS fibers after laser photolysis and temperature-jump conditions showed that domains containing fibers produced time-dependent, multi-sperulitic domains made from homogeneous nuclei by formation of dense liquid clusters during a phase transition [14–16]. These studies suggested that homogeneous nucleation prior to HbS polymer formation occurs in a single liquid cluster, which represents a precursor phase during a liquid-to-solid-phase transition. In addition, Galkin et al. showed that formation of deoxy-HbS fibers is a first-order phase transition, similar to gas-solid phase transition, and this permits the application of thermodynamic and kinetic rules of phase transition to the analysis of HbS polymerization [15]. Galkin et al. also reported that nucleation of deoxy-HbS polymers proceeds via a precursor involving a meta-stable mesoscopic cluster of a dense liquid phase in crowded HbS solution [15]. They suggested that homogeneous nucleation of HbS polymers occurs within a cluster as a precursor phase [15, 17]. Nucleation prior to fiber formation of deoxy-HbS also was suggested to proceed by a two-step process like formation of crystalline nuclei: a droplet of dense liquid forms within which a crystalline nucleus appears due to the ordering of a certain number of molecules [18, 19]. Intracellular polymers or fibers of Hb S cause a significant reduction in red blood cell deformability (sickling), leading to obstruction of flow in the microcirculation. Each polymer of Hb S assembles into 14-stranded fibers, which then form a viscous gel [1, 2]. Further analysis of the fibers demonstrated that each is composed of seven half-staggered double strands [2], suggesting that the double-stranded structure of deoxy Hb S crystals may be relevant components of fibers within cells. An important feature of the double strand is that the contact between two molecules in adjacent strands (i.e., the so-called “lateral” contact) involves β6Val and β4Thr in hydrophobic and hydrogen bond interactions, respectively. Specifically, β85Phe and β88Leu are involved with β6Val and β73Asp is involved with β4Thr in a hydrogen bond in the E and F helices of the β chain [20, 21]) as schematically shown in Fig. 1. Figure 1 Schematic diagram showing some of the lateral Hb tetramer hydrophobic and hydrogen bond interactions during deoxy-Hb S polymer formation Morphological, kinetic and thermodynamic studies of fiber/domain formation using engineered HbS and HbA β4 and β73 variants suggest the importance of the β4 hydrogen bond interaction with β73Asp in deoxy-HbS fiber/domain formation [16, 22]. However, the relationship between the hydrophobic and hydrogen bond interactions on the surface of the A-helix of the β chain of hemoglobin leading to formation of amorphous aggregates, fibers and crystals of oversaturated HbA and HbS during a liquid- to solid-phase transition is not completely understood. In this report, we focus on evaluation of polymerization properties of oversaturated HbAβ4Val (α2β2T4V) and HbAβ73His (α2β2D73H) compared to those of HbA, HbS, HbSβ4Val (α2β2E6V, T4V) and HbSβ73His (α2β2E6V, D73H) in order to obtain a better understanding of the relationship between β4/β73 hydrogen bond and β6 amino acid hydrophobicity in the A-helix of hemoglobin in promoting amorphous aggregate versus fiber/domain formation following crystallization in oversaturated hemoglobin solutions. These amino acid variants were selected since β4Val maps close to β6Val in HbS and should create a region of hydrophobicity similar to the normally mutated β6Glu to Val site in HbS. Furthermore, substitution of β4Thr to Val also should disrupt the normal β4Thr-β73Asp hydrogen bond and perturb polymer formation of HbS. In addition, β73His should constrain/inhibit formation of the normal β73Asp-β4Thr hydrogen bond. Results from these variants therefore are important in helping to define determinants critical for aggregate versus crystal or fiber formation, and may help in design of targeted inhibitors of HbS polymer formation in vivo.