Attwood, P.V. and Geeves, M.A. (2004) Kinetics of an enzyme-catalyzed reaction measured by electrospray ionization mass spectrometry using a simple rapid mixing attachment. Anal Biochem, 334 (2). pp. 382-9. ISSN 0003-2697 .
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Mass spectrometry offers a potential means of measuring virtually all enzyme-catalyzed reactions by simultaneously measuring the concentrations of substrates, products, and intermediates where there are differences in mass between them. To perform these measurements the reaction mixture must be aged for different times and then ionized. Electrospray ionization mass spectrometry provides the most direct means of measuring these reactions. Here we describe a simple reaction mixing and ageing attachment for an electrospray ionization mass spectrometer, built from commercially available components. We have employed this device to measure the kinetics of a model reaction, namely the hydrolysis of N2-(carbobenzyloxy)-L-lysine-p-nitrophenyl ester-catalyzed by trypsin. In this way we were able to measure the kinetics of substrate depletion, product formation, and changes in both free enzyme and acyl-enzyme intermediate concentration in the approach to steady state. With this device we were able to measure reaction times down to about 640 ms.
|Additional information:||0003-2697 (Print) Journal Article|
|Uncontrolled keywords:||Catalysis Hydrolysis Kinetics Lysine/*analogs & derivatives/chemistry/metabolism Research Support, Non-U.S. Gov't Spectrometry, Mass, Electrospray Ionization/*instrumentation/*methods Time Factors Trypsin/*metabolism enzyme kinetics; rapid reaction; electrospray ionization mass spectrometry; trypsin|
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences|
|Depositing User:||Michael Geeves|
|Date Deposited:||29 May 2009 05:55|
|Last Modified:||29 May 2009 05:55|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/13199 (The current URI for this page, for reference purposes)|
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