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),...

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Published inJournal of biomolecular techniques Vol. 14; no. 4; pp. 247 - 269
Main Authors Myszka, D G, Abdiche, Y N, Arisaka, F, Byron, O, Eisenstein, E, Hensley, P, Thomson, J A, Lombardo, C R, Schwarz, F, Stafford, W, Doyle, M L
Format Journal Article
LanguageEnglish
Published United States The Association of Biomolecular Resource Facilities 01.12.2003
Subjects
Animals
Calorimetry, Differential Scanning
Carbonic Anhydrase II - chemistry
Carbonic Anhydrase II - drug effects
Cattle
Enzyme Inhibitors - pharmacology
Kinetics
Molecular Weight
Sulfonamides - chemistry
Sulfonamides - pharmacology
Surface Plasmon Resonance
Thermodynamics
Ultracentrifugation
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Summary: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.
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Disclaimer: Certain commercial materials, instruments, and equipment are identified in this manuscript in order to specify the experimental procedure as completely as possible. In no case does such identification imply a recommendation or endorsement by NIST nor does it imply that the materials, instruments, or equipment identified are necessarily the best available for the purpose.
D. G. Myszka, Center for Biomolecular Interaction Analysis, University of Utah School of Medicine, Room no. 4A417, 50 N. Medical Drive, Salt Lake City, Utah 84132 (phone: 801-585-5358; fax: 801-585-3015; e-mail: david.myszka@cores.utah.edu)
The study was funded, in part, by the U.S. Department of Defense DARPA (F30602-00-2-0609).
ISSN:1524-0215