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多位Biacore技术用户对小分子/酶相互作用的比较分析。

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

作者信息

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

机构信息

Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132, USA.

出版信息

Anal Biochem. 2004 Jul 1;330(1):98-113. doi: 10.1016/j.ab.2004.02.027.

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.

摘要

为评估与Biacore分析相关的实验变异性,36位不同的研究人员在相似条件下分析了一种小分子/酶相互作用。选择乙酰唑胺(222 g/mol)与碳酸酐酶II(CAII;30000 Da)的结合作为模型系统。两种试剂都很稳定,由于分析物分子量低且缔合速率常数快,它们之间的相互作用对测量构成了挑战。每位研究人员创建了CAII的三种不同密度表面,并分析了相同稀释系列的乙酰唑胺(范围为4.1至1000 nM)。由于每位研究人员提供了自己的表面活化试剂,因此在酶固定步骤中观察到结果的最大变异性。乙酰唑胺结合响应质量的变异性可能是研究人员仪器维护程度的产物。为确定反应动力学,将来自不同密度表面的响应整体拟合到包含传质项的1:1相互作用模型中。平均缔合和解离速率常数分别为3.1±1.6×10⁶M⁻¹s⁻¹和6.7±2.5×10⁻²s⁻¹,这对应于平均平衡解离常数(Kₐ)为2.6±1.4×10⁻⁸M。这些结果提供了从生物传感器解释结合常数时变异性的基准,并突出了分析小分子相互作用时应考虑的关键领域。

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