Measurement of enzyme kinetics using microscale steady-state kinetic analysis

Langmuir : the ACS Journal of Surfaces and Colloids
Nathaniel J Gleason, Jeffrey D Carbeck

Abstract

This paper describes a new technique--microscale steady-state kinetic analysis (microSKA)--that enables the rapid and parallel analysis of enzyme kinetics. Rather than physically defining a microscopic reactor through microfabrication, we show how the relative rates of reaction and transport in a macroscopic flow chamber, where the enzyme is immobilized on one wall of the chamber, results in the confinement of an enzyme-catalyzed reaction to a microscopic reactor volume adjacent to this wall. This volume has linear dimensions that are orders of magnitude smaller than the physical dimensions of the system (i.e., micrometer vs millimeter). Conversion within this volume is monitored at steady state as a function of position, rather than time. In this way, limitations due to reactor dead time and mixing are avoided. We use microSKA to determine kinetic parameters for the alkaline phosphatase-catalyzed de-phosphorylation of nonfluorescent methylumbelliferyl phosphate (MUP) to fluorescent 7-hydroxy-4-methylcoumarin (HMC) at two different values of pH. Kinetic parameters measured with microSKA are in good agreement with values obtained using conventional methods, if one takes into account effects of immobilization on enzyme activity. ...Continue Reading

References

Mar 21, 1998·Proceedings of the National Academy of Sciences of the United States of America·U KettlingM Eigen
Nov 11, 1999·Journal of Molecular Recognition : JMR·B GoldsteinC Wofsy
Jan 14, 2000·Electrophoresis·V DolníkS Jovanovich
May 12, 2000·Science·P J EngM L Rivers
Feb 13, 2001·Current Opinion in Biotechnology·M KrishnanM A Burns
May 1, 2001·Nature Biotechnology·A HatchP Yager
Jun 29, 2001·Nature·S TakayamaG M Whitesides
Jul 12, 2001·Annual Review of Biomedical Engineering·G M WhitesidesD E Ingber
Feb 9, 2002·Journal of Chromatography. a·Julia Khandurina, András Guttman
Feb 19, 2002·Current Opinion in Biotechnology·D Wahler, J L Reymond
Feb 19, 2002·Current Opinion in Biotechnology·E T FarinasF H Arnold
Jul 27, 2002·Analytical Chemistry·James P Smith, Vicki Hinson-Smith
Aug 31, 2002·Chembiochem : a European Journal of Chemical Biology·Jean-Louis Reymond, Denis Wahler
Nov 7, 2002·Journal of the American Chemical Society·Gi Hun Seong, Richard M Crooks
Nov 16, 2002·Analytical Chemistry·James B Delehanty, Frances S Ligler
Nov 28, 2002·Nature Genetics·Gavin MacBeath
Feb 5, 2003·Current Opinion in Biotechnology·Brian M PaegelRichard A Mathies
Sep 11, 2003·Analytical Chemistry·Gi Hun SeongRichard M Crooks

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Citations

Dec 14, 2011·Analytical Chemistry·Piro SiutiMitchel J Doktycz
Jun 15, 2007·Chemical Communications : Chem Comm·Malene S ThomsenBernd Nidetzky
Jan 1, 2008·Biotechnology & Genetic Engineering Reviews·Masaya MiyazakiHideaki Maeda
Feb 5, 2008·Electrophoresis·Gloria OliverAnuj Chauhan
Aug 29, 2006·Trends in Biotechnology·Masaya Miyazaki, Hideaki Maeda
Aug 2, 2005·Biotechnology Advances·Pawel L UrbanNeil C Bruce
Jul 21, 2011·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Yuya AsanomiHideaki Maeda
Dec 17, 2008·The Journal of Physical Chemistry. a·Kenichi YamashitaHideaki Maeda
Mar 10, 2009·Analytical Chemistry·Cheng-Che LeeYuh-Shyong Yang
Feb 24, 2011·Journal of the American Chemical Society·Sergey MozharovJohn M Girkin
Sep 1, 2017·Analytical Chemistry·Hector D Neira, Amy E Herr
Dec 15, 2006·Analytical Chemistry·Matthew B KerbyAnubhav Tripathi
Mar 1, 2006·Analytical Chemistry·Barry R LutzDaniel T Schwartz
Jun 15, 2006·Analytical Chemistry·Petra S DittrichAndreas Manz

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