Equilibrium titration of protein-ligand binding affinity in the presence of volatile reagents--a semiautomated approach

Abstract

An existing method of equilibrium titration was significantly improved for investigating the effects of volatile anesthetics on Ca(2+) binding characteristics of human recombinant cardiac troponin C in in vitro conditions. The modified method increases stability of volatile compound concentrations in solution and allows for faster and more accurate data acquisition. The time to complete a titration series could be reduced from 28.3 +/- 6.2 min to 9.3 +/- 2.1 min, whereas the dispersion for pK(d) was decreased from 2.16 +/- 0.27 to 0.63 +/- 0.27. The method utilizes a semiautomatic approach to continuously monitor stability of fluorescence signals in a sealed chamber with greatly reduced air space.