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