Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology
Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology
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DOI：
10.1016/j.ab.2004.02.027
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发表时间：
2004-07-01
影响因子：
2.9
通讯作者：
Myszka, DG
中科院分区：
文献类型：
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作者：
Cannon, MJ;Papalia, GA;Myszka, DG
为了衡量与Biacore分析相关的实验变异性，36名不同的研究人员分析了类似条件下的小分子/酶相互作用。选择乙酰唑胺(222g/mol)与碳酸氢酶II(CAII；30000 Da)结合作为模型体系。这两种试剂都很稳定，由于分析物的低分子量和快速的结合速率常数，它们的相互作用对测量构成了挑战。每个研究人员创建了三种不同密度的CAII表面，并分析了相同的乙酰唑胺稀释系列(从4.1到MoW)。由于每个研究人员都提供了自己的表面活化剂，因此在酶固定化步骤中观察到的结果差异最大。乙酰唑胺结合反应质量的变化很可能是研究人员仪器维护得很好的结果。为了确定反应动力学，来自不同密度表面的响应被全局地拟合为1：1相互作用模型，该模型包括质量传输项。平均缔合和解离速率常数分别为3.1+/-1.6×10(6)M-1 S(-1)和6.7+/-2.5×10(-2)S(-1)。分别对应于2.6+/-1.4×10(-8)M的平均平衡解离常数(K-D)。这些结果为解释生物传感器的结合常数提供了一个可变性的基准，并强调了在分析小分子相互作用时应该考虑的关键区域。(C)2004 Elsevier Inc.保留所有权利。
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.