MAGNETIC SUSCEPTIBILITY STUDIES OF METALLOPROTEINS

金属蛋白的磁化率研究具有潜在的重要意义 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，这些顺磁态对于 如弛豫展宽等原因。 金属蛋白易感性研究的困难在于 真空计的灵敏度不足，导致需要高度 浓缩样品（大于1000 μ M）。 浓度 程序 在光学或EPR表征之后， 在样品的氧化还原状态下。 氧化还原状态的模糊性 样品的可接受性，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 磁化率法与EPR等共振技术相比 和穆斯堡尔谱导致对 敏感性实验 此外，一个根本问题是， 分子量的蛋白质已经不可能实现 敏感性实验所需的浓度。 我们已经开发了新的技术来测量低的磁性， 浓度（大于50 μ M）的金属蛋白样品， 商用超导SQUID温度计。 我们结合了这个新的 通过开发一种新的电子顺磁共振和穆斯堡尔谱仪， 多仪器样品保持器。 我们现在可以研究同样的冰冻 金属蛋白质样本（无需解冻样本或改变其 浓度或保持器）与EPR和穆斯堡尔在其研究之前 用磁力计 通过这种技术组合， 可以完全定义敏感性样品的状态， 杂质顺磁性测量作出可靠的解释， 可能的敏感性数据。 此外，新的实验结合 在低温下的光解与磁化率测量， 可行 我们将研究细菌和哺乳动物的细胞色素氧化酶， Rieske蛋白和其他代表性的[2Fe-2S]蛋白，E.大肠杆菌亚硫酸盐 还原酶和D.巨铁氧还蛋白II. 我们的目标之一是奠定基础工作的易感性研究， 室温下的生化动力学。

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.