Role of the β4Thr−β73Asp Hydrogen Bond in HbS Polymer and Domain Formation from Multinucleate-Containing Clusters

Abstract

Fiber formation and domain formation from deoxy-HbS as well as from beta4 and beta73 HbS variants were investigated after temperature jump using DIC microscopy to gain a basic understanding of the determinants involved. Oversaturated deoxy-HbS generated numerous 14-stranded fibers and formed ovoid-shaped, multispherulitic domains. Domain number increased linearly as a function of time. Oversaturated deoxy-alpha2beta2(E6V,T4S) also generated time-dependent, ovoid-shaped spherulitic domains like HbS and alpha 2beta2(E6V,D73H) in the deoxy form. In contrast, alpha 2beta2(E6V,T4Y) and HbC-Harlem (alpha2beta2(E6V,D73N)) in the deoxy form generated time-dependent, ball-shaped domains containing many straight, crystalline-like fibers without evidence of branching. Some of these domains formed large needlelike crystals after overnight incubation. The inhibitory effect on polymer formation by beta4Tyr in HbS was stronger than that by beta4Ser but weaker than that by beta73Asn or beta73Leu. In contrast, both deoxy- and oxy-alpha2beta2(E6V,T4V) promoted formation of tiny, disordered amorphous aggregates without a delay time like oxy-HbS, which is in contrast to formation after a delay time of needlelike fibers for alpha 2beta2(E6V,D73L). Solubilities for both deoxy- and oxy-alpha 2beta2(E6V,T4V) were similar to that of deoxy-alpha 2beta2(E6V,D73H) but approximately 10-fold lower than that of deoxy-HbS. These results suggest that the strength of the hydrogen bond between beta4Thr and beta73Asp and the balance between the hydrogen bond and beta6Val hydrophobic interactions in deoxy-HbS polymers control formation of different types of fibers in a single domain or lead to formation of disordered, non-nucleated amorphous aggregates. These results also lead to a model in which multinucleation rather than a single-nucleation event occurs in a single cluster to generate numerous fibers growing from a single domain.

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

“…Recombinant HbSβ4Val (α 2 β 2 E6V, T4V ), HbAβ4Val (α 2 β 2 T4V ), HbSβ73His (α 2 β 2 E6V, D73H ) and HbAβ73His (α 2 β 2 D73H ) were expressed in bacteria after subcloning the corresponding cDNAs into pHE2, which co-expresses α- and β-globin chains as well as methionine aminopeptidase under transcriptional control of a ptac promoter [16, 24]. β-globin chain variants were constructed by site-specific mutagenesis of the normal β chain using recombination/PCR, as described [22, 25], and were co-expressed with α chains to form tetrameric HbS and HbA variants [22].…”

“…Aggregate, fiber and/or crystal formation for deoxy-Hb and oxy-Hb in 1.0 M phosphate buffer, pH 7.4, were evaluated by DIC microscopy and by turbidity using a temperature-jump method as described [16, 22]. Deoxy-Hb was generated by the addition of sodium dithionite in 3M phosphate buffer, pH 7.4, at 4° C to oxy-HbS solutions so that the final concentration of sodium dithionite was 100 mM in 1.0 M phosphate buffer, pH 7.4 [22].…”

“…Deoxy-Hb was generated by the addition of sodium dithionite in 3M phosphate buffer, pH 7.4, at 4° C to oxy-HbS solutions so that the final concentration of sodium dithionite was 100 mM in 1.0 M phosphate buffer, pH 7.4 [22]. One or 2 μl of solution was inserted into a glass capillary tube (30 μm path length × 0.3 mm width with 50 mm length) (Dynamics, Inc., Rockaway, NJ) by capillary action [16]. Both ends of the tube were sealed by Mount-Quick solution (Daido Sangyo Co., LTD., Tokyo, Japan) [22], and the tube was mounted on the glass slide after a 1-h incubation on ice.…”

“…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.…”

“…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.…”