From small-molecule reactions to protein folding: Studying biochemical kinetics by stopped-flow electrospray mass spectrometry
From small-molecule reactions to protein folding: Studying biochemical kinetics by stopped-flow electrospray mass spectrometry
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DOI：
10.1006/abio.2001.5062
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发表时间：
2001-05-01
影响因子：
2.9
通讯作者：
Konermann, L
中科院分区：
文献类型：
--
作者：
Kolakowski, BM;Konermann, L
本文介绍了一种研究快速生化反应动力学的电喷雾质谱方法。通过快速混合两种溶液引发反应后，将混合物转移到反应容器中，并建立稳定的液体流到质谱仪的ESI源。通过监测作为时间的函数的所选离子强度，在真实的时间中研究动力学。为了表征该装置的性能，研究了叶绿素a的酸诱导脱金属。结果发现，该反应是二级酸浓度和伪一级速率常数高达约7 s（-1）可以可靠地测量。停流电喷雾质谱也被用于研究肌红蛋白的酸诱导变性。这里提供的数据证实了在该反应过程中存在短暂的解折叠中间体。停流ESI MS可以提供光学快速混合实验无法获得的信息。因此，看来这种新的技术有可能成为一个标准的工具，在许多不同的领域的动力学研究。(C)北京：科学出版社.
This work introduces stopped-how electrospray ionization (ESI) mass spectrometry (MS) as a method for studying fast biochemical reaction kinetics. After initiating a reaction by rapid mixing of two solutions, the mixture is transferred to a reaction vessel and a steady liquid how to the ESI source of the mass spectrometer is established. The kinetics are studied in real time by monitoring selected ion intensities as a function of time. In order to characterize the performance of this setup the acid-induced demetallation of chlorophyll a was studied. It was found that the reaction is second order in acid concentration and that pseudo-first-order rate constants of up to roughly 7 s(-1) can be measured reliably. Stopped-flow ESI MS was also applied to study the acid-induced denaturation of myoglobin. The data presented here confirm the occurrence of a short-lived unfolding intermediate during this reaction. Stopped-flow ESI MS can provide information that is not accessible by optical rapid-mixing experiments. Therefore it appears that this novel technique has the potential to become a standard tool for kinetic studies in a number of different fields. (C) 2001 Academic Press.