MOLECULAR AND RED CELL DETERMINANTS OF SICKLE CELL DISEASE

这个项目的总体目标是描述结构和 脱氧HBS聚合物和凝胶在体内的组装机理 在红血球里。具体目标是：1)探索物业和 聚合物水舱(PWC)的功能后果。这个想法 是基于模型研究，预测了与 聚合反应。用C-70kD葡聚糖测定脱氧HBS的浓度 聚合物Cp为54.7g/dl。该值低于 红细胞中Hb的浓度(136g/dl)，表明存在 聚合物伴生水的浓度。据建议，这与以下情况有关 红血球成分的划分。尤其是，布钦博士 想要调查有机磷酸盐的再分布，鉴于 它们在增强凝胶方面的报道效果，并评估变化 代谢活性与脱氧HBs的凝胶化有关 红血球。2)检测脱氧HBS和脱氧HBS中CP的变异性 与其他化学修饰或变种的血红蛋白的混合物 通过重组技术生产。这个想法是基于初步数据 其中，HBS和HBA或HbC或HbF的1：1混合物表现出增加 CSAT。3)在葡聚糖存在的情况下实施Csat测定 并将其与从微观量获得的值相关联 费龙博士的动态分析。4)检测膜的缔合作用 HbS异相成核中的位置。对于这些研究，有三种类型 的红细胞膜将被制备，内向外(IOV)和右向外 所有膜成分都保存下来的幽灵，细胞骨架- 释放细胞骨架蛋白的游离IOV，以及 混合红细胞脱氧作用。5)Intra和Intra的识别 纤维中的相互接触点，将集中在以下位置 对化学修饰很敏感。各种各样的化学物质 修饰，具体的或通过半合成的 方法被提出为a)特定谷氨酸残基的酰胺化，b) 嵌合血红蛋白的构建c)接触光亲和标记 D)制备交联型不对称杂交物。

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.