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Microchip‐ESI‐MS determination of dissociation constant of the lysozyme–NAG 3 complex
Author(s) -
Svobodová Jaroslava,
Mathur Sonal,
Muck Alexander,
Letzel Thomas,
Svatoš Aleš
Publication year - 2010
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201000041
Subject(s) - lysozyme , chemistry , dissociation constant , chromatography , dissociation (chemistry) , microfluidics , analytical chemistry (journal) , reagent , miniaturization , substrate (aquarium) , nanotechnology , materials science , biochemistry , receptor , oceanography , geology
An on‐line microfluidic system for determination of dissociation constants of enzyme/substrate weak interactions by nanoESI‐MS is introduced. The microchip was designed to permit the enzyme and the substrate to mix by molecular diffusion in a pressure‐driven laminar flow. Introduction of reagent solutions into the chip was an optimized combination of a micropump and an autosampler to enable automation of the measurements. The system performance was tested for monitoring of non‐covalent interactions of hen egg white lysozyme with N ‐acetyl glucosamine oligomers. Dissociation constant ( K D ) of the hen egg white lysozyme‐ N ‐acetyl glucosamine oligomer complex was determined by non‐linear regression analysis, and the range of K D values (39±6×10 −6 and 19.6±8×10 −6 M for manual and automated infusions, respectively) confirms the previously reported values. Such miniaturization of a continuous‐flow enzyme assay system to a microfluidic format can maximize the capabilities of mass spectrometric detection, reduce the sample size and analysis time required, as well as the associated costs for on‐line enzymatic kinetic studies.