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

  1. Author:
    Cannon, M. J.
    Papalia, G. A.
    Navratilova, I.
    Fisher, R. J.
    Roberts, L. R.
    Worthy, K. M.
    Stephen, A. G.
    Marchesini, G. R.
    Collins, E. J.
    Casper, D.
    Qiu, H. W.
    Satpaev, D.
    Liparoto, S. F.
    Rice, D. A.
    Gorshkova, I. I.
    Darling, R. J.
    Bennett, D. B.
    Sekar, M.
    Hommema, E.
    Liang, A. M.
    Day, E. S.
    Inman, J.
    Karlicek, S. M.
    Ullrich, S. J.
    Hodges, D.
    Chu, T.
    Sullivan, E.
    Simpson, J.
    Rafique, A.
    Luginbuhl, B.
    Westin, S. N.
    Bynum, M.
    Cachia, P.
    Li, Y. J.
    Kao, D.
    Neurauter, A.
    Wong, M.
    Swanson, M.
    Myszka, D. G.

  2. Author Address

    Myszka, DG, Univ Utah, Ctr Biomol Interact Anal, Salt Lake City, UT 84132 USA Univ Utah, Ctr Biomol Interact Anal, Salt Lake City, UT 84132 USA. SAIC, Prot Chem Lab, Ft Detrick, MD 21702 USA. Rikilt Inst, Wageningen, Netherlands. Univ N Carolina, Dept Microbiol & Immunol, Chapel Hill, NC 27510 USA. Locus Pharmaceut, St Petersburg 19422, Russia. Genzyme Corp, Framingham, MA 01701 USA. Agensys Inc, Santa Monica, CA 90404 USA. Myriad Pharmaceut, Salt Lake City, UT 84108 USA. NICHD, LMG, NIH, Bethesda, MD 20892 USA. Eli Lilly & Co, Indianapolis, IN 46285 USA. Appl Biosyst Inc, Bedford, MA 01730 USA. Biacore RCS, Durham, NC 27713 USA. Berlex Biosci, Richmond, CA 94804 USA. Biogen Inc, Cambridge, MA 02142 USA. Pfizer, Dept Biochem, San Diego, CA 92121 USA. Human Genome Sci Inc, Rockville, MD 20850 USA. Allergan, Irvine, CA 92612 USA. NCI, Frederick, MD 21702 USA. Regeneron Pharmaceut Inc, Tarrytown, NY 10591 USA. Univ Zurich, Dept Biochem, Zurich, Switzerland. Biacore AB, Uppsala, Sweden. Agilent Technol, Palo Alto, CA 94303 USA. Univ Colorado, Hlth Sci Ctr, Dept Biochem & Mol Genet, Boulder, CO 80309 USA. Monsanto Co, St Louis, MO 63198 USA. Zyomyx Inc, Hayward, CA USA. EOS Biotechnol, San Francisco, CA USA. Pharmacie, Kalamazoo, MI USA.
    1. Year: 2004
  2. Journal: Analytical Biochemistry
    1. 330
    2. 1
    3. Pages: 98-113
  4. Type of Article: Article
  1. 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 MOW). 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 x 10(6) M-1 s(-1) and 6.7 +/- 2.5 x 10(-2) s(-1). respectively, which corresponded to an average equilibrium dissociation constant (K-D) of 2.6 +/- 1.4 x 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. (C) 2004 Elsevier Inc. All rights reserved

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