title: Enzyme kinetics above denaturation temperature: a temperature-jump/stopped-flow apparatus.
abstract: 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 degrees 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.
authors.affiliation: Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary.
authors.forename: Bálint
authors.initials: B
authors.lastname: Kintses
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Simon
authors.forename: Máté
authors.initials: M
authors.lastname: Gyimesi
authors.forename: Júlia
authors.initials: J
authors.lastname: Tóth
authors.forename: Balázs
authors.initials: B
authors.lastname: Jelinek
authors.forename: Csaba
authors.initials: C
authors.lastname: Niedetzky
authors.forename: Mihály
authors.initials: M
authors.lastname: Kovács
authors.forename: András
authors.initials: A
authors.lastname: Málnási-Csizmadia
mesh_terms: D000818:Animals; D004023:Dictyostelium / Q000201:enzymology; D007700:Kinetics; D009154:Mutation; D024462:Myosin Type II / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D024462:Myosin Type II