MOLECULAR CONTROLS OF HEMOGLOBIN FUNCTION

我们的工作假设是，成功的基于Hb的血液替代品可能 通过微调的交联化和巯基修饰生成 是通过定点突变实现的，这种突变是由 Hb功能的分子调控。这一假设得到了我们的 最近发现无细胞Hb的血管收缩活性， 归因于没有清除作用，可以通过其亚硝化反应来控制 硫醇基团。分子控制功能对血液设计至关重要 替代品包括修饰的HBS有助于氧化的能力 毒性，氧化还原循环，无清除/捐献反应。我们建议 通过光谱测量来评估这些关键功能 血红素和巯基配体结合的动力学和平衡及新发现 测定血红素氧化还原电位的光谱电化学法。 这些互补的技术将并行使用来量化 正常HBS和改良HBS之间的差异。我们的创新 光谱电化学方法使我们能够直接比较氧化和 配体结合曲线，并将有助于血液替代品的设计 区分那些响应的功能方面 活性位铁原子的氧化还原势及其影响因素 由速记功能控制。选定用于研究的HB系统将使我们能够 为了单独测试修改血红素口袋的效果， 巯基和中心空腔。主要关注的将是 习惯于定义由以下因素导致的功能修改范围 羟基改性与化学交联法相结合。我们的 目标是追踪来自特定部位的分子途径 它们在配体结合中的最终功能表达的修饰 氧化毒性、氧化还原循环的平衡和动力学 无细胞外Hb清除/捐献反应。

Our working hypothesis is that successful Hb-based blood substitutes may be generate by cross linking and sulfhydryl modification, with fine-tuning achieved by site-directed mutagenesis that is guided by knowledge of the molecular controls of HB function. This hypothesis is support by our recent discovery that the vasoconstrictive activity of cell-free Hb, attributed to NO scavenging, can be controlled by nitrosylation of its thiol groups. Molecular control functions critical to the design of blood substitutes include the ability of modified Hbs to contribute to oxidative toxicity, redox cycling and NO scavenging/donating reactions. We propose to evaluate these critical functions by spectroscopic measurements of the kinetics and equilibria of heme-and sulfhydryl- ligand binding and novel spectroelectrochemical methods for measuring the heme redox potential. These complementary techniques will be used in parallel to quantify differences between normal and modified Hbs. Our innovative spectroelectrochemical methods allow us to directly compare oxidation and ligand-binding curves and will aid in the design of blood substitutes by differentiating between those aspects of function that are responsive to the redox potential of the active-site iron atoms and those aspects governed by steno features. Hb systems selected for study will enable us to separately test the effects of modifications of the heme pocket, the sulfhydryl groups, and the central cavity. Primary attention will be given to defining the range of functional modifications that result from sulfhydryl modification in combination with chemical cross-linking. Our goal is to trace the molecular pathway from specific sites of modifications to their ultimate functional expression in ligand binding equilibria and kinetics that underlie oxidative toxicity, redox cycling and NO scavenging/donating reactions of cell-free hb.