A Continuous-Flow System for High-Precision Kinetics Using Small Volumes

• Published in: Analytical chemistry (Washington) Vol. 75; no. 15; pp. 3681 - 3687
• Main Authors: Zhou, Xianzhi, Medhekar, Rohit, Toney, Michael D
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.08.2003
• Subjects: Analytical chemistry; Chemistry; Diffusion; Dithionitrobenzoic Acid - pharmacology; Dithiothreitol - pharmacology; Exact sciences and technology; Flow Injection Analysis - instrumentation; Flow Injection Analysis - methods; Hydrolysis; Kinetics; Nitrophenols - metabolism; Organophosphorus Compounds - metabolism; Protein Tyrosine Phosphatases - metabolism; Protons; Spectrometric and optical methods; Yersinia pestis - enzymology; Catalytic reaction; Molecular diffusion; Kinetic method; Yersinia pestis; Hydrolysis; Kinetic isotope effect; Absorption spectrometry; Laminar flow; Bacteria; Diode array; Continuous flow method; Enterobacteriaceae; Enzymatic reaction
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac034068j

A generally applicable continuous-flow kinetic analysis system that gives data of a precision high enough to measure small kinetic isotope effects for enzymatic and nonenzymatic reactions is described. It employs commercially available components that are readily assembled into an apparatus that is easy to use. It operates under laminar flow conditions, which requires that the time between the initiation of the reaction in the mixer and the observation be long enough that molecular diffusion can effect a symmetrization of the concentration profile that results from a thin plug of reagents introduced at the mixer. The analysis of a second-order irreversible reaction under pseudo-first-order conditions is presented. The Yersinia pestis protein tyrosine phosphatase catalyzed hydrolysis of p-nitrophenyl phosphate is characterized with the system, and a proton inventory on k cat is presented.