From Small-Molecule Reactions to Protein Folding: Studying Biochemical Kinetics by Stopped-Flow Electrospray Mass Spectrometry
✍ Scribed by Beata M Kolakowski; Lars Konermann
- Book ID
- 102567252
- Publisher
- Elsevier Science
- Year
- 2001
- Tongue
- English
- Weight
- 91 KB
- Volume
- 292
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
This work introduces stopped-flow 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 flow 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.