Microchip-ESI-MS Determination of Dissociation Constant: Lysozyme-NAG3 Complex

Jaroslava Svobodova (1) , Sonal Mathur (1) , Alexander Muck (1) , Thomas Letzel (2) , Ales Svatos (3)

1 Mass Spectrometry Group [Jena]
2 Analytical Chemistry Research Group, Department of Biopolymer Chemistry
3 Max Planck Institute for Chemical Ecology

An on-line microfluidic system for determination of dissociation constants of enzyme/substrate weak interactions by nanoelectrospray ionization mass spectrometry (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 (HEWL) with N-acetyl glucosamine oligomers (NAGn). Dissociation constant (KD) of the HEWL-NAG3 complex was determined by non-linear regression analysis, and the range of KD values (39 ± 6  10-6 M 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.

Mots clés: Life Sciences

Jaroslava Svobodova, Sonal Mathur, Alexander Muck, Thomas Letzel, Ales Svatos. Microchip-ESI-MS Determination of Dissociation Constant: Lysozyme-NAG3 Complex. Electrophoresis, 2010, 31 (15), pp.2680. ⟨10.1002/elps.201000041⟩. ⟨hal-00599461⟩