title: Accurate label-free reaction kinetics determination using initial rate heat measurements.
abstract: Accurate label-free methods or assays to obtain the initial reaction rates have significant importance in fundamental studies of enzymes and in application-oriented high throughput screening of enzyme activity. Here we introduce a label-free approach for obtaining initial rates of enzyme activity from heat measurements, which we name initial rate calorimetry (IrCal). This approach is based on our new finding that the data recorded by isothermal titration calorimetry for the early stages of a reaction, which have been widely ignored, are correlated to the initial rates. Application of the IrCal approach to various enzymes led to accurate enzyme kinetics parameters as compared to spectroscopic methods and enabled enzyme kinetic studies with natural substrate, e.g. proteases with protein substrates. Because heat is a label-free property of almost all reactions, the IrCal approach holds promise in fundamental studies of various enzymes and in use of calorimetry for high throughput screening of enzyme activity.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Kourosh Honarmand
authors.initials: KH
authors.lastname: Ebrahimi
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Peter-Leon
authors.initials: PL
authors.lastname: Hagedoorn
authors.affiliation: DSM Biotechnology Center, Alexander Fleminglaan 1, 2613 AX Delft, the Netherlands.
authors.forename: Denise
authors.initials: D
authors.lastname: Jacobs
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Wilfred R
authors.initials: WR
authors.lastname: Hagen
mesh_terms: D006358:Hot Temperature; D007700:Kinetics; D008962:Models, Theoretical