MAGNETIC SUSCEPTIBILITY STUDIES OF METALLOPROTEINS

金属蛋白的磁化率研究

• 批准号: 3281184
• 负责人: EDMUND P DAY
• 金额: $ 10.65万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1987-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3281184
• 关键词: Escherichia coli; Mossbauer spectrometry; cytochrome oxidase; electron spin resonance spectroscopy; magnetism; metalloproteins; molecular biology; oxidation reduction reaction; photolysis; sulfite reductase

Magnetic susceptibility studies of metalloproteins are potentially of great value. Few other techniques can measure the magnetic properties of integer spin states and of other paramagnetic states which are EPR-silent for reasons such as relaxation broadening. The difficulty with susceptibility studies of metalloproteins has been the lack of sensitivity of susceptometers leading to a requirement for highly concentrated samples (greater than 1000 MuM). The concentration procedure following optical or EPR characterization often causes changes in the sample's redox state. Ambiguity in the redox state of the suceptibility sample, uncertainties in the level of the sample's impurity paramagnetism, and the intrinsic lack of resolution of the static susceptibility method when compared with resonance techniques such as EPR and Mossbauer spectroscopy have led to unreliable interpretation of susceptibility experiments. Moreover, a fundamental problem for high molecular weight proteins has been the impossibility of achieving the concentration necessary for a susceptibility experiment. We have developed new techniques to measure the magnetic properties of low concentration (greater than 50 MuM) metalloprotein samples using a commercial superconducting SQUID susceptometer. We have combined this new capability with EPR and Mossbauer spectroscopy by developing a multi-instrument sample holder. We can now study the same frozen metalloprotein sample (without thawing the sample or changing its concentration or holder) with both EPR and Mossbauer prior to its study with the susceptometer. With this combination of techniques the redox state of the susceptibility sample can be fully defined and its level of impurity paramagnetism measured making reliable interpretation of the susceptibility data possible. In addition, new experiments combining photolysis at low temperatures with susceptibility measurements are now feasible. We will study bacterial and mammalian cytochrome oxidase, a bacterial Rieske protein and other representative [2Fe-2S] proteins, E. coli sulfite reductase, and D. gigas ferredoxin II. One of our goals is to lay the ground work for susceptibility studies of biochemical kinetics at room temperature.

金属蛋白的磁化率研究具有潜在的巨大意义 价值。 很少有其他技术可以测量整数的磁性 自旋态和其他顺磁态的 EPR 沉默 弛豫展宽等原因。 金属蛋白敏感性研究的困难在于 敏感度计缺乏灵敏度，导致需要高度 浓缩样品（大于 1000 MuM）。 浓度 光学或 EPR 表征后的程序通常会导致变化 在样品的氧化还原状态下。 氧化还原态的模糊性 敏感性样品，样品杂质水平的不确定性 顺磁性，以及静态分辨率的内在缺乏 磁化率方法与 EPR 等共振技术的比较 和穆斯堡尔光谱导致了不可靠的解释 敏感性实验。 此外，高的一个根本问题 蛋白质的分子量一直是不可能实现的 药敏实验所需的浓度。 我们开发了测量低磁性材料的新技术 使用浓度（大于 50 MuM）金属蛋白样品 商用超导 SQUID 感受器。 我们结合了这个新的 通过开发 EPR 和穆斯堡尔光谱能力 多仪器样品架。 我们现在可以研究同样的冷冻 金属蛋白样品（无需解冻样品或改变其 在研究之前与 EPR 和 Mossbauer 进行联系 与感受计。 通过这种技术组合，氧化还原 可以完全定义敏感性样品的状态及其水平 测量的杂质顺磁性可以做出可靠的解释 可能的敏感性数据。 此外，新的实验结合了 现在通过磁化率测量进行低温光解 可行的。 我们将研究细菌和哺乳动物细胞色素氧化酶，一种细菌 Rieske蛋白和其他代表性[2Fe-2S]蛋白，大肠杆菌亚硫酸盐 还原酶和 D. gigas 铁氧还蛋白 II。 我们的目标之一是为敏感性研究奠定基础 室温下的生化动力学。