Biosensor-Based Estimation of Kinetic and Equilibrium Constants

✍ Scribed by John G. Quinn; Richard O'Kennedy

• Book ID: 102565864
• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 139 KB
• Volume: 290
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4919

✦ Synopsis

The steady-state affinity constant for the interaction of glutathione S-transferase (GST) with anti-GST immunoglobulin (IgG) was determined by solution-phase equilibrium analysis. A Biacore concentration assay for the determination of free anti-GST IgG was employed giving a Kd value of 6.83 x 10(-10) M. A simple 1:1 solution-phase, equilibrium model approximated the data well. Furthermore, saturation studies showed a maximum occupation of approximately 50%. The choice of affinity-capture ligand, used to anchor anti-GST IgG to the hydrogel, influenced the interaction curves, as evidenced by contact-time-dependent dissociation-phase curves. This was apparent when performing the analysis on anti-mouse Fc-coated surfaces. When the interaction was conducted on a protein A-coated CM5 sensor chip, the interaction conformed well to ideal behavior and was selected for kinetic analysis of the GST interaction.