The ABRF-MIRG'02 study: assembly state, thermodynamic, and kinetic analysis of an enzyme/inhibitor interaction.
Fully characterizing the interactions involving biomolecules requires information on the assembly state, affinity, kinetics, and thermodynamics associated with complex formation. The analytical technologies often used to measure biomolecular interactions include analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). In order to evaluate the capabilities of core facilities to implement these technologies, the Association of Biomolecular Resource Facilities (ABRF) Molecular Interactions Research Group (MIRG) developed a standardized model system and distributed it to a panel of AUC, ITC, and SPR operators. The model system was composed of a well-characterized enzyme-inhibitor pair, namely bovine carbonic anhydrase II (CA II) and 4-carboxybenzenesulfonamide (CBS). Study participants were asked to measure one or more of the following: (1) the molecular mass, homogeneity, and assembly state of CA II by AUC; (2) the affinity and thermodynamics for complex formation by ITC; and (3) the affinity and kinetics of complex formation by SPR. The results from this study provide a benchmark for comparing the capabilities of individual laboratories and for defining the utility of the different instrumentation.
Myszka
D G
DG
University of Utah, Salt Lake City, Utah 84132, USA. david.myszka@cores.utah.edu
Abdiche
Y N
YN
Arisaka
F
F
Byron
O
O
Eisenstein
E
E
Hensley
P
P
Thomson
J A
JA
Lombardo
C R
CR
Schwarz
F
F
Stafford
W
W
Doyle
M L
ML
D000818:Animals; D002152:Calorimetry, Differential Scanning; D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000187:drug effects; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D013449:Sulfonamides / Q000737:chemistry* / Q000494:pharmacology; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D014461:Ultracentrifugation
D004791:Enzyme Inhibitors; D013449:Sulfonamides; D024402:Carbonic Anhydrase II