Determination of the dead time of a stopped-flow fluorometer

Analytical Biochemistry
P BrissetteV Massey

Abstract

This investigation was carried out to develop a convenient alternative method for examining the performance and determining the dead time of a stopped-flow fluorometer. We examined the kinetics for the formation of the fluorescent Mg2+-8-hydroxyquinoline chelate in aqueous solutions. The reversible association of the Mg2+ ion with 8-hydroxyquinoline is a second-order process whose on and off rate constants are dependent on pH. We estimated that the Mg2+ ion chelate has a fluorescence quantum yield of 0.02 in aqueous solutions. Using this reaction we measured the dead time of a stopped-flow fluorometer at different pH values. Measurements of the dead time were found to be reproducible and accurate. The Mg2+-8-hydroxyquinoline reaction fulfills the requirements for a convenient test reaction for dead time measurement of stopped-flow fluorometers. Although the usefulness of the reaction is primarily to determine the dead times of stopped-flow instruments operating in the fluorescence mode, the reaction can also be used for testing an instrument operating in the absorbance mode.

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Citations

Sep 23, 2003·Journal of Molecular Biology·Kiyoto KamagataKunihiro Kuwajima
Jan 27, 2004·Journal of Molecular Biology·Richard C Stewart, Ricaele Van Bruggen
Apr 16, 1998·Biochimica Et Biophysica Acta·L A BassoA R Walmsley
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Apr 17, 2013·Proceedings of the National Academy of Sciences of the United States of America·Joseph M MurettaDavid D Thomas
May 23, 2015·The Journal of Physical Chemistry. B·Yuuka TakanoJun-ya Kohno
Dec 3, 2014·Analytical Biochemistry·Sandra MiticSimon de Vries
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Dec 1, 2012·Expert Review of Proteomics·Maxim V Berezovski, Gleb G Mironov
Dec 7, 2018·The Journal of Biological Chemistry·Laura K GuntherChristopher M Yengo
Feb 14, 2006·The Journal of Biological Chemistry·François J M ChartierManon Couture
Jun 23, 2010·Biochemistry·Anna K Eaton, Richard C Stewart
Feb 26, 2011·Analytical Chemistry·Gleb G MironovMaxim V Berezovski

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