title: Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology.
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; 30000 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 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.
authors.affiliation: Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132, USA.
authors.forename: Michelle J
authors.initials: MJ
authors.lastname: Cannon
authors.forename: Giuseppe A
authors.initials: GA
authors.lastname: Papalia
authors.forename: Iva
authors.initials: I
authors.lastname: Navratilova
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fisher
authors.forename: Lindsey R
authors.initials: LR
authors.lastname: Roberts
authors.forename: Karen M
authors.initials: KM
authors.lastname: Worthy
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Stephen
authors.forename: Gerardo R
authors.initials: GR
authors.lastname: Marchesini
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Collins
authors.forename: Dave
authors.initials: D
authors.lastname: Casper
authors.forename: Huawei
authors.initials: H
authors.lastname: Qiu
authors.forename: Daulet
authors.initials: D
authors.lastname: Satpaev
authors.forename: Stefano F
authors.initials: SF
authors.lastname: Liparoto
authors.forename: Dax A
authors.initials: DA
authors.lastname: Rice
authors.forename: Inna I
authors.initials: II
authors.lastname: Gorshkova
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Darling
authors.forename: Donald B
authors.initials: DB
authors.lastname: Bennett
authors.forename: Michael
authors.initials: M
authors.lastname: Sekar
authors.forename: Eric
authors.initials: E
authors.lastname: Hommema
authors.forename: Amy M
authors.initials: AM
authors.lastname: Liang
authors.forename: Eric S
authors.initials: ES
authors.lastname: Day
authors.forename: Jean
authors.initials: J
authors.lastname: Inman
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Karlicek
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Ullrich
authors.forename: Dianne
authors.initials: D
authors.lastname: Hodges
authors.forename: Teresa
authors.initials: T
authors.lastname: Chu
authors.forename: Eric
authors.initials: E
authors.lastname: Sullivan
authors.forename: Jack
authors.initials: J
authors.lastname: Simpson
authors.forename: Ashique
authors.initials: A
authors.lastname: Rafique
authors.forename: Béatrice
authors.initials: B
authors.lastname: Luginbühl
authors.forename: Susanne Nyholm
authors.initials: SN
authors.lastname: Westin
authors.forename: Magdalena
authors.initials: M
authors.lastname: Bynum
authors.forename: Paul
authors.initials: P
authors.lastname: Cachia
authors.forename: Yue-Jin
authors.initials: YJ
authors.lastname: Li
authors.forename: Daniel
authors.initials: D
authors.lastname: Kao
authors.forename: Amy
authors.initials: A
authors.lastname: Neurauter
authors.forename: Melanie
authors.initials: M
authors.lastname: Wong
authors.forename: Michael
authors.initials: M
authors.lastname: Swanson
authors.forename: David G
authors.initials: DG
authors.lastname: Myszka
mesh_terms: D000086:Acetazolamide / Q000737:chemistry* / Q000378:metabolism; D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D015588:Observer Variation; D011485:Protein Binding; D012108:Research Personnel; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000592:standards
chemical_list: D024402:Carbonic Anhydrase II; D000086:Acetazolamide