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.

译文

:在温度跃变后,使用DIC显微镜研究了脱氧HbS以及beta4和beta73 HbS变体的纤维形成和结构域形成,以基本了解所涉及的决定因素。过饱和的脱氧HbS生成了许多14股纤维,并形成了卵形的多球状结构域。域名随时间线性增加。过饱和的脱氧α2beta2(E6V,T4S)还生成了时间依赖性的卵形球状结构域,如脱氧形式的HbS和α2beta2(E6V,D73H)。相反,脱氧形式的α2beta2(E6V,T4Y)和HbC-Harlem(alpha2beta2(E6V,D73N))产生时间相关的球形结构域,其中包含许多直的晶体状纤维而无分支迹象。这些结构域中的一些在过夜温育后形成大的针状晶体。 beta4Tyr在HbS中对聚合物形成的抑制作用强于beta4Ser,但弱于beta73Asn或beta73Leu。相反,脱氧-和氧-α2beta2(E6V,T4V)都促进了无序的细小无定形聚集体的形成,而没有像氧-HbS这样的延迟时间,这与针状纤维延迟时间后形成α2β2(E6V)形成对比,D73L)。脱氧-α2beta2(E6V,T4V)的溶解度均与脱氧-α2beta2(E6V,D73H)相似,但比脱氧-HbS的溶解度低约10倍。这些结果表明,脱氧-HbS聚合物中beta4Thr和beta73Asp之间的氢键强度以及氢键和beta6Val疏水相互作用之间的平衡控制了单个域中不同类型纤维的形成,或导致无序,非-有核无定形聚集体。这些结果还导致了一个模型,在该模型中,在单个簇中发生多核而不是单核事件,以生成从单个域生长的大量纤维。

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