Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology.

摘要: Abstract To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30,000 Da) was chosen as model system. Both reagents were stable and their posed challenge measure because of low molecular weight analyte fast association rate constant. Each investigator created three density surfaces CAII an identical dilution series acetazolamide (ranging from 4.1 1000 nM). The greatest in results observed during enzyme immobilization step since each provided own surface activating reagents. Variability quality responses likely product how well investigators’ instruments had been maintained. determine reaction kinetics, fit globally 1:1 that included term for mass transport. averaged dissociation constants 3.1 ± 1.6 × 10 6  M −1  s 6.7 ± 2.5 × 10 −2 , respectively, which corresponded average equilibrium constant ( K D ) 2.6 ± 1.4 × 10 −8  M. provide benchmark interpreting biosensor highlight keys areas should be considered when analyzing molecule interactions.