A combined nuclear magnetic resonance and absorbance stopped-flow apparatus for biochemical studies.
A combined nuclear magnetic resonance and absorbance stopped-flow apparatus for biochemical studies.
复制标题
用于生化研究的核磁共振和吸光度停流组合装置。
DOI：
10.1016/0003-2697(90)90119-t
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发表时间：
1990
影响因子：
2.9
通讯作者：
Parkhurst,LJ
中科院分区：
文献类型：
--
作者：
McGee,WA;Parkhurst,LJ
已开发出组合式 NMR 和吸光度停流装置，用于监测生化反应的动力学。我们证明了它在跟踪人类血红蛋白碱变性方面的有用性。该装置的制造过程中不需要吹制玻璃。尽可能使用市售阀门、注射器、管道和管道连接器。易于制造的光导用于将光传输至光学单元或从光学单元传输出光。停流装置使用 5 毫米 NMR 管，后接一个光路长度为 0.5 毫米的光学池。这样可以同时测量 NMR 和吸光度变化。在终端流速为 7.5 ml/s 时，NMR 和光学死时间分别为 60 和 260 ms。对于此处报道的研究，氧合血红蛋白用连接到 β-93 半胱氨酸的 19F 探针进行标记。 pH 7 时的天然蛋白质具有由单峰组成的 NMR 谱，而 pH 12 时完全变性的血红蛋白样品具有由三个单峰组成的谱。在变性过程中，另一个 NMR 峰迅速出现，然后随着反应时间的推移而衰减消失。 NMR 研究采用的高浓度（血红素为 2.08 mm）下的吸光度变化非常接近一级动力学。核磁共振监测的事件虽然与光学变化在同一时间范围内，但其复杂性要高得多，并且显示了多个探针在监测蛋白质展开方面的实用性。
A combined NMR and absorbance stopped-flow has been developed for monitoring the kinetics of biochemical reactions. We demonstrate its usefulness in following the alkaline denaturation of human hemoglobin. No glassblowing is required in the fabrication of the apparatus. Commercially available valves, syringes, tubing, and tubing connectors are employed whenever possible. Easily fabricated light guides are used to pipe light to and from the optical cell. The stopped-flow uses a 5-mm NMR tube followed by an optical cell with a 0.5-mm optical path length. This allows simultaneous measurements of NMR and absorbance changes. At a terminal flow velocity of 7.5 ml/s, the NMR and optical dead times were 60 and 260 ms, respectively. For the study reported here the oxyhemoglobin was labeled with a19F probe attached to the β-93 cysteine. The native protein at pH 7 has an NMR spectrum consisting of a singlet, and the fully denatured hemoglobin sample at pH 12 has a spectrum consisting of three singlets. During the denaturation process another NMR peak appears rapidly and then decays away over the time course of the reaction. The absorbance changes at the high concentration employed for the NMR study (2.08 mm in heme) follow very nearly first-order kinetics. The events monitored by NMR, though in the same time frame as the optical changes, are of much greater complexity, and show the utility of multiple probes for monitoring protein unfolding.