RUI: Disulfide Bond Reduction: A Powerful Chemical Probe for the Study of Structure-Function Relationships in the Hemocyanins

9972667血蓝蛋白是一种高分子量的含铜蛋白质，在许多节肢动物和软体动物中充当氧载体。 虽然氧被绑定到双核铜中心在这两个蛋白质的四级结构显着不同。 晶体结构分析表明，二硫键是两种血蓝蛋白类型的共同特征。近年来研究表明，鲎血蓝蛋白的二硫键对其氧结合位点的完整性至关重要。这项工作的第一个目标是确定二硫键在维持两类血蓝蛋白中氧结合位点完整性方面的普遍重要性。氧结合功能和活性部位铜的损失通过电化学测定法测定。 将通过凝胶电泳、高效液相色谱、发光光谱和圆二色性分析二硫键断裂导致的结构变化。 这项工作的第二个目标是使用的氧结合功能的减少和恢复后的二硫键作为化学探针，以确定如何intersubunit接触和变构调节剂的氧亲和力影响的构象和稳定性的氧结合位点的节肢动物和软体动物hemocyanins.Most动物生活在一个富氧的环境中，依靠有氧代谢。 绝大多数具有专门用于将氧气运输到内部组织的特定蛋白质。 虽然血红蛋白是研究最广泛的氧转运蛋白，但其他氧转运蛋白也被许多不同的物种使用。 血蓝蛋白是一种高分子量、含铜的蛋白质，在许多节肢动物和软体动物中充当氧载体。 血蓝蛋白引起了研究人员的兴趣，不仅因为它们的生理作用，而且因为它们的结构和功能响应于环境条件的变化而发生的明显改变。 在这个项目中，一个简单的，但功能强大的化学工具，可逆地失去和恢复功能后，还原和恢复蛋白质二硫键的能力，是用来探测内部和环境因素，支配生理上重要的结构-功能关系的血蓝蛋白。 控制血蓝蛋白的组装、稳定性和功能的因素具有普遍意义，因为复杂聚集体的组装、稳定性和功能同样受pH、金属结合、特定离子和代谢物的控制，从而进行许多具有生物重要性的反应。

9972667TophamHemocyanins are high-molecular-weight, copper-containing proteins that serve as oxygen carriers in many arthropods and molluscs. Although oxygen is bound to dinuclear copper centers in both, the quaternary structure of the proteins differ dramatically. Crystal structure analyses show that disulfide bonds are a common feature in both hemocyanin types. Recently, it has been demonstrated that the disulfide bonds of Limulus hemocyanin are essential to the integrity of its oxygen-binding sites. The first objectiveof this work is to determine the general importance of disulfide bonds in maintaining the integrity of the oxygen-binding sites in both classes of hemocyanins. The loss of oxygen-binding function and active-site copper is determined spectrophotometrically. Structural changes that result from the cleavage of the disulfide bonds will be analyzed by gel electrophoresis, high performance liquid chromatography, luminescence spectroscopy, and circular dichroism. A second objective of this work is to use the loss and recovery of oxygen-binding function upon reduction and restoration of the disulfide bonds as a chemical probe to determine how intersubunit contacts and allosteric regulators of oxygen affinity affect the conformation and stability of the oxygen binding sites of arthropod and mollusc hemocyanins.Most animals live in an oxygen-rich environment and rely upon aerobic metabolism. The vast majority have specific proteins specialized for the transport of oxygen to interior tissues. Although hemoglobins are theoxygen transport proteins most extensively studied, other oxygen transport proteins are used by as many different species as the hemoglobins. Hemocyanins are high-molecular-weight, copper-containing proteins that serve as oxygen-carriers in many arthropods and molluscs. The hemocyanins have attracted the interest of researchers, not only because of their physiological role, but also because of the appreciable alterations that occur in their structure and function in response to changes in environmental conditions. In this project, a simple, yet powerful, chemical tool, the ability to reversibly lose and regain function upon thereduction and restoration of protein disulfide bonds, is used to probe the internal and environmental factors that govern physiologically important structure-function relationships in the hemocyanins. The factorsthat govern the assembly, stability, and function of the hemocyanins are of general significance in view of the many reactions of biological importance conducted by complex aggregates whose assembly, stability, and function are similarly controlled by pH, metal binding, specific ions, and metabolites.