Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.
Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.
复制标题
使用 HPLC 检测进行平衡透析，测量非血红素铁卤化酶的底物结合亲和力。
DOI：
10.1101/2024.04.03.588023
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Damodaran,AnoopRama
中科院分区：
文献类型：
--
作者：
Smithwick,ElizabethR;Bhagi-Damodaran,Ambika;Damodaran,AnoopRama
Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis with subsequent detection with High Performance Liquid Chromatography (HPLC). This method can be performed in anaerobic glove bag settings, requires readily available HPLC instrumentation for subsequent detection, and is adaptable to meet the needs of a variety of substrate affinity measurements.
