Different methodologies for determining the dissociation equilibrium constant (K_i) of protein tight binding inhibitors are frequently found in the scientific literature. Taking into account that the K_i value is the main parameter characterizing the inhibition strength, its determination often represents the first step during the characterization of a potential drug. The purpose of this review is to summarize the current information related to tight binding inhibitors K_i values determination and discuss about the importance of different factors as the enzyme concentration, the inhibitor concentration dilution series, the enzyme-inhibitor incubation time and the dose-response data mathematical fitting. For this aim, the bi-functional SmCI protease inhibitor is used as a tool for exemplifying the experimental and mathematical steps performed during tight binding inhibitors K_i values determination. In addition, the natural and the different recombinant forms of SmCI were used to go deeply into the comparison of some mathematic approaches that are frequently used in the literature. Finally, other biochemical techniques that could be potentially used for tight binding inhibitors K_i values determination are also commented.

在科学文献中经常发现用于确定蛋白紧密结合抑制剂的解离平衡常数（K _i）的不同方法。考虑到K _i值是表征抑制强度的主要参数，其确定通常代表潜在药物表征过程中的第一步。这篇综述的目的是总结与紧密结合抑制剂K _i有关的最新信息。值测定，并讨论酶浓度，抑制剂浓度稀释系列，酶抑制剂温育时间和剂量反应数据数学拟合等不同因素的重要性。为了这个目的，双功能SmCI蛋白酶抑制剂被用作例证在紧密结合抑制剂K _i值测定期间进行的实验和数学步骤的工具。另外，天然和不同重组形式的SmCI用于深入比较文献中常用的一些数学方法。最后，还评论了可能用于紧密结合抑制剂K _i值测定的其他生化技术。