MOLECULAR AND RED CELL DETERMINANTS OF SICKLE CELL DISEASE

该项目的总体目标是描述结构的特征， 脱氧硫代血红蛋白聚合物和凝胶在结构上的组装机制 在红细胞内。 具体目标是：1）探索性质， 聚合物水隔室（PWC）的功能后果。这个想法 是基于模型研究预测的体积变化与 聚合法使用C-70 kD葡聚糖， 聚合物Cp计算为54.7 g/dl。该值低于 红细胞中的Hb浓度（136 g/dl），并意味着存在 聚合物缔合水。有人提出，这与 红细胞成分的区室化。尤其是布钦博士 想要研究有机磷酸盐的再分配，鉴于 它们对增强凝胶化的报道效果，并评估是否改变 在代谢活动中与脱氧HbS的凝胶化有关， 红细胞2)在单独的脱氧HbS和脱氧HbS中测试Cp的可变性。 与化学修饰或变体的其他血红蛋白的混合物 通过重组技术生产。这个想法是基于初步的数据 其中HbS和HbA或HbC或HbF的1：1混合物显示出 Csat. 3)在葡聚糖存在下进行Csat测定， 并将其与从微 Ferrone博士的动力学测定。4)测试膜相关的作用 在HbS的异质成核中的位置。在这些研究中， 将制备100个RBC膜，内翻（IOV）和正面朝外 所有的细胞膜成分都被保留下来，细胞骨架， 释放细胞骨架蛋白的游离IOV，和 杂种红细胞脱氧。5)识别内部和 纤维中的内部接触点，将集中在 容易受到化学修饰。各种化学品 修饰，无论是特定的或通过半合成引入 提出的方法为a）特定Glu残基的酰胺化，B） 嵌合血红蛋白的构建c）接触的光亲和标记 区域d）交联的不对称杂化物的制备。

The overall goal of this project is to characterize the structure and assembly mechanisms of the deoxy HbS polymer and gel in structure in within the red cells. Specific aims are: 1) Exploring the properties and functional consequences of the polymer water compartment (PWC). The idea is based on model studies predicting a volume shift associated with polymerization. Using C-70kD dextran the concentration of deoxy-HbS in the polymer, Cp, was calculated to be 54.7 g/dl. This value is lower than the concentration of Hb in the red cells (136 g/dl), and implies the presence of polymer associated water. It is proposed that this is associated with compartmentalization of red cell components. In particular, Dr. Bookchin wants to investigate the redistribution of organic phosphates, in view of their reported effect on enhancing gelation, and to assess whether changes in metabolic activity are associated with gelation of the deoxy HbS in the red cells. 2) Testing the variability of Cp in deoxy HbS alone and in mixture with other hemoglobins either chemically modified or variants produced by recombinant techniques. The idea is based on preliminary data in which 1:1 mixtures of HbS and HbA or HbC or HbF show an increase in Csat. 3) Implementing the Csat determination in the presence of dextran to microquantities and correlate it with the values obtained from the micro- kinetic assay of Dr. Ferrone. 4) Testing the role of membrane associated sites in the heterogenous nucleation of HbS. For these studies three types of RBC membranes will be prepared, inside out (IOVs) and right side out ghosts in which all the membrane components are preserved, cytoskeleton- free IOVs from which the cytoskeletal proteins have been released, and hybrid erythrocytes deoxygenation. 5) The identification of intra and inter contact points in the fibers, will be done focussing on sites which are susceptible to chemical modifications. A large variety of chemical modifications, either specific or introduced through a semisynthetic approach are proposed as a) amidation of specific Glu residue, b) construction of chimeric hemoglobins c) photoaffinity labeling of contact regions d) preparation of crosslinked asymmetric hybrids.