SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Enzyme kinetics above denaturation temperature: a temperature-jump/stopped-flow apparatus
Balint Kintses 1, Zoltan Simon 1, Mate Gyimesi 1, Julia Toth 1, Balazs Jelinek 1, Csaba Niedetzky 2, Mihaly Kovacs 1 and Andras Malnasi-Csizmadia 1*
1 Eotvos Lorand University
2 Supertech Ltd.
* To whom correspondence should be addressed. E-mail: amc26{at}le.ac.uk.
Submitted on July 6, 2006
Revised on August 3, 2006
Accepted on 23 August 2006
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Abstract |
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We constructed a temperature-jump/stopped-flow apparatus which allows studying fast enzyme reactions at extremely high temperatures. This apparatus is a redesigned stopped-flow which is capable of mixing the reactants on a submillisecond time scale 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.
Key Words:
human enzyme, myosin, temperature-induced denaturation, temperature-jump, transient kinetics, tryptophan fluorescence
