Briehl R W
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461.
J Mol Biol. 1995 Feb 3;245(5):710-23. doi: 10.1006/jmbi.1994.0057.
Pathogenesis in sickle cell disease depends on polymerization and gelation of deoxyhemoglobin S. Under the double nucleation model, polymerization is initiated by homogeneous nucleation, followed by heterogeneous nucleation on pre-existing fibers. Fibers grow by non-cooperative addition of hemoglobin. The model derives from macroscopic results rather than direct observation of individual events. We observe individual events and structures by differential interference contrast (DIC) microscopy to show consistency with the model, to define structure and development of gel domains and their relation to kinetics, and to demonstrate the mechanism of fiber melting. Kinetics were controlled by producing deoxyhemoglobin by photolysis of CO hemoglobin under DIC observation. The first visible polymers appeared randomly and were usually linear aggregates less than 1 micron long, consistent with homogeneous nucleation and immediate post-nucleation aggregates. Aggregates then branched extensively, consistent with heterogeneous nucleation. This branching of new fibers was also induced at countable rates on isolated single fibers. Branching and fiber growth rapidly produced dense domains. Changes in photolytic intensity altered domain growth rates and domain structure. At low intensity and slow growth, fibers grew radially without branching. Domains lacked cross-links and polymer density was low. High intensity produced faster growth, much heterogeneous nucleation and highly cross-linked, dense, domains. At still higher intensity, homogeneous nucleation was very rapid, producing many small domains. These results show a hierarchy of processes: as deoxyhemoglobin concentration increases, growth occurs without observable nucleations, and then heterogeneous and finally homogeneous nucleation become dominant. This is consistent with the double nucleation model under which the concentration dependence of growth is low, and that of heterogeneous and homogeneous nucleation successively higher. Under decreased photolysis, fiber ends melted continuously without fiber breakage; increased photolysis reversed this, producing growth. Isolated fibers melted and grew at both ends. The results are consistent with a fiber melting mechanism that is the reverse of growth.
镰状细胞病的发病机制取决于脱氧血红蛋白S的聚合和凝胶化。在双核化模型中，聚合由均匀成核引发，随后在预先存在的纤维上发生异相成核。纤维通过血红蛋白的非协同添加而生长。该模型源于宏观结果而非对单个事件的直接观察。我们通过微分干涉对比（DIC）显微镜观察单个事件和结构，以显示与模型的一致性，定义凝胶域的结构和发育及其与动力学的关系，并证明纤维熔化的机制。在DIC观察下，通过CO血红蛋白的光解产生脱氧血红蛋白来控制动力学。最初可见的聚合物随机出现，通常是长度小于1微米的线性聚集体，这与均匀成核和核后立即形成的聚集体一致。聚集体随后广泛分支，这与异相成核一致。新纤维的这种分支也以可计数的速率在孤立的单根纤维上诱导产生。分支和纤维生长迅速产生致密域。光解强度的变化改变了域的生长速率和域结构。在低强度和缓慢生长时，纤维径向生长而不分支。域缺乏交联且聚合物密度低。高强度导致生长更快、大量异相成核以及高度交联、致密的域。在更高强度下，均匀成核非常迅速，产生许多小域。这些结果显示了一系列过程：随着脱氧血红蛋白浓度增加，生长在没有可观察到的成核情况下发生，然后异相成核，最终均匀成核占主导。这与双核化模型一致，在该模型下生长的浓度依赖性较低，而异相和均匀成核的浓度依赖性依次更高。在光解减少的情况下，纤维末端连续熔化而不发生纤维断裂；光解增加则逆转了这种情况，导致生长。孤立的纤维两端同时熔化和生长。结果与纤维熔化机制一致，该机制与生长相反。
