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Originally published as Biophys J. BioFAST on September 29, 2006.
doi:10.1529/biophysj.106.092833
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Biophysical Journal 91:4605-4610 (2006)
© 2006 The Biophysical Society

Enzyme Kinetics above Denaturation Temperature: A Temperature-Jump/Stopped-Flow Apparatus

Bálint Kintses *, Zoltán Simon *, Máté Gyimesi *, Júlia Tóth *, Balázs Jelinek *, Csaba Niedetzky {dagger}, Mihály Kovács * and András Málnási-Csizmadia *

* Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary; and {dagger} Supertech Ltd., Pécs, Hungary

Correspondence: Address reprint requests to Andras Malnasi-Csizmadia, Tel.: 36-1-209-0555-8780; Fax: 36-1-381-2172; E-mail: malna{at}elte.hu.

We constructed a "temperature-jump/stopped-flow" apparatus that allows us to study fast enzyme reactions at extremely high temperatures. This apparatus is a redesigned stopped-flow which is capable of mixing the reactants on a submillisecond timescale concomitant with a temperature-jump even as large as 60°C. We show that enzyme reactions that are faster than the denaturation process can be investigated above denaturation temperatures. In addition, the temperature-jump/stopped-flow enables us to investigate at physiological temperature the mechanisms of many human enzymes, which was impossible until now because of their heat instability. Furthermore, this technique is extremely useful in studying the progress of heat-induced protein unfolding. The temperature-jump/stopped-flow method combined with the application of structure-specific fluorescence signals provides novel opportunities to study the stability of certain regions of enzymes and identify the unfolding-initiating regions of proteins. The temperature-jump/stopped-flow technique may become a breakthrough in exploring new features of enzymes and the mechanism of unfolding processes.




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[Abstract] [Full Text] [PDF]


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Copyright © 2006 by the Biophysical Society.