From small-molecule reactions to protein folding: Studying biochemical kinetics by stopped-flow electrospray mass spectrometry

This work introduces stopped-how electrospray ionization (ESI) mass spectro metry (MS) as a method for studying fast biochemical reaction kinetics. Aft er initiating a reaction by rapid mixing of two solutions, the mixture is t ransferred to a reaction vessel and a steady liquid how to the ESI source o f the mass spectrometer is established. The kinetics are studied in real ti me by monitoring selected ion intensities as a function of time. In order t o characterize the performance of this setup the acid-induced demetallation of chlorophyll a was studied. It was found that the reaction is second ord er in acid concentration and that pseudo-first-order rate constants of up t o roughly 7 s(-1) can be measured reliably. Stopped-flow ESI MS was also ap plied to study the acid-induced denaturation of myoglobin. The data present ed here confirm the occurrence of a short-lived unfolding intermediate duri ng this reaction. Stopped-flow ESI MS can provide information that is not a ccessible by optical rapid-mixing experiments. Therefore it appears that th is novel technique has the potential to become a standard tool for kinetic studies in a number of different fields. (C) 2001 Academic Press.