Microchip-ESI-MS determination of dissociation constant of the lysozyme-NAG(3) complex.
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 x 10(-6) and 19.6+/-8 x 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.
Svobodová
Jaroslava
J
Mass Spectrometry Research Group, Max Planck Institute for Chemical Ecology, Jena, Germany.
Mathur
Sonal
S
Muck
Alexander
A
Letzel
Thomas
T
Svatos
Ales
A
D000117:Acetylglucosamine / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D009113:Muramidase / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
C421864:hen egg lysozyme; D009113:Muramidase; D000117:Acetylglucosamine