Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods.
The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface- and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are performed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase II bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content, and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors.
Day
Yasmina S N
YS
Center for Biomolecular Interaction Analysis, University of Utah, School of Medicine, 50 N Medical Drive, Salt Lake City, UT 84132, USA.
Baird
Cheryl L
CL
Rich
Rebecca L
RL
Myszka
David G
DG
D000818:Animals; D024402:Carbonic Anhydrase II / Q000737:chemistry*; D002417:Cattle; D003619:Dansyl Compounds / Q000737:chemistry*; D007700:Kinetics; D011485:Protein Binding; D013379:Substrate Specificity; D013449:Sulfonamides / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
C118985:4-carbobenzoxybenzene sulfonamide; D003619:Dansyl Compounds; D013449:Sulfonamides; C001026:5-dimethylaminonaphthalene-1-sulfonamide; D024402:Carbonic Anhydrase II