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Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology |
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| Authors: | Cannon Michelle J Papalia Giuseppe A Navratilova Iva Fisher Robert J Roberts Lindsey R Worthy Karen M Stephen Andrew G Marchesini Gerardo R Collins Edward J Casper Dave Qiu Huawei Satpaev Daulet Liparoto Stefano F Rice Dax A Gorshkova Inna I Darling Ryan J Bennett Donald B Sekar Michael Hommema Eric Liang Amy M Day Eric S Inman Jean Karlicek Shannon M Ullrich Stephen J Hodges Dianne Chu Teresa Sullivan Eric Simpson Jack Rafique Ashique Luginbühl Béatrice Westin Susanne Nyholm Bynum Magdalena Cachia Paul Li Yue-Jin Kao Daniel Neurauter Amy Wong Melanie Swanson Michael Myszka David G |
| Affiliation: | a Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132, USA b Protein Chemistry Laboratory, SAIC, Frederick, MD 21702, USA c Biomolecular Detection, Rikilt Institute, Wageningen, Netherlands d Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27510, USA e Locus Pharmaceuticals, Blue Bell, PA 19422, USA f Genzyme Corp., Framingham, MA 01701, USA g Agensys, Inc., Santa Monica, CA 90404, USA h IDEC Pharmaceuticals Corp., San Diego, CA 92191, USA i Myriad Pharmaceuticals, Salt Lake City, UT 84108, USA j LMG NICHD NIH, Bethesda, MD 20892, USA k Eli Lilly and Co., Indianapolis, IN 46285, USA l Applied Biosystems, Bedford, MA 01730, USA m Biacore RCS, Durham, NC 27713, USA n Berlex Biosciences, Richmond, CA 94804, USA o Biogen Inc., Cambridge, MA 02142, USA p Biochemistry Department, Pfizer, San Diego, CA 92121, USA q Human Genome Sci., Inc., Rockville, MD 20850, USA r Allergan, Irvine, CA 92612, USA s NCI, Frederick, MD 21702, USA t Regeneron Pharmaceutical, Tarrytown, NY 10591, USA u Department of Biochemistry, Universitaet Zürich, Zürich, Switzerland v Biacore AB, Uppsala, Sweden w Agilent Technologies, Palo Alto, CA 94303, USA x Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, USA y Monsanto, Chesterfield, MO 63198, USA z Zyomyx, Inc., Hayward, CA, USA aa EOS Biotechnology, South San Francisco, CA, USA ab Pharmacie, Kalamazoo, MI, USA |
| 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 a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators’ instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1 ± 1.6 × 106 M−1 s−1 and 6.7 ± 2.5 × 10−2 s−1, respectively, which corresponded to an average equilibrium dissociation constant (KD) of 2.6 ± 1.4 × 10−8 M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions. |
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