Microchip-ESI-MS determination of dissociation constant of the lysozyme–NAG3 complex

✍ Scribed by Jaroslava Svobodová; Sonal Mathur; Alexander Muck; Thomas Letzel; Aleš Svatoš

• Book ID: 102189465
• Publisher: John Wiley and Sons
• Year: 2010
• Tongue: English
• Weight: 246 KB
• Volume: 31
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.201000041

✦ Synopsis

Abstract

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.