Weisser K, Schloos J
Zentrum der Pharmakologie, Klinikum der J.W. Goethe-Universität, Frankfurt/Main, Federal Republic of Germany.
Biochem Pharmacol. 1991 Oct 9;42(9):1729-37. doi: 10.1016/0006-2952(91)90509-4.
The relationship between serum angiotensin converting enzyme (ACE) activity and concentration of the ACE inhibitor enalaprilat was determined in vitro in the presence of different concentrations (S = 4-200 mM) of the substrate Hip-Gly-Gly. From Henderson plots, a competitive tight-binding relationship between enalaprilat and serum ACE was found yielding a value of approximately 5 nM for serum ACE concentration (Et) and an inhibition constant (Ki) for enalaprilat of approximately 0.1 nM. A plot of reaction velocity (Vi) versus total inhibitor concentration (It) exhibited a non-parallel shift of the inhibition curve to the right with increasing S. This was reflected by apparent Hill coefficients greater than 1 when the commonly used inhibitory sigmoid concentration-effect model (Emax model) was applied to the data. Slopes greater than 1 were obviously due to discrepancies between the free inhibitor concentration (If) present in the assay and It plotted on the abscissa and could, therefore, be indicators of tight-binding conditions. Thus, the sigmoid Emax model leads to an overestimation of Ki. Therefore, a modification of the inhibitory sigmoid Emax model (called "Emax tight model") was applied, which accounts for the depletion of If by binding, refers to It and allows estimation of the parameters Et and IC50f (free concentration of inhibitor when 50% inhibition occurs) using non-linear regression analysis. This model could describe the non-symmetrical shape of the inhibition curves and the results for Ki and Et correlated very well with those derived from the Henderson plots. The latter findings confirm that the degree of ACE inhibition measured in vitro is, in fact, dependent on the concentration of substrate and enzyme present in the assay. This is of importance not only for the correct evaluation of Ki but also for the interpretation of the time course of serum ACE inhibition measured ex vivo. The non-linear model has some advantages over the linear Henderson equation: it is directly applicable without conversion of the data and avoids the stochastic dependency of the variables, allowing non-linear regression of all data points contributing with the same weight.
在不同浓度（S = 4 - 200 mM）的底物Hip - Gly - Gly存在下，于体外测定了血清血管紧张素转换酶（ACE）活性与ACE抑制剂依那普利拉浓度之间的关系。根据亨德森图，发现依那普利拉与血清ACE之间存在竞争性紧密结合关系，血清ACE浓度（Et）约为5 nM，依那普利拉的抑制常数（Ki）约为0.1 nM。反应速度（Vi）对总抑制剂浓度（It）作图显示，随着S增加，抑制曲线向右发生非平行移动。当将常用的抑制性S形浓度 - 效应模型（Emax模型）应用于数据时，这表现为表观希尔系数大于1。大于1的斜率显然是由于测定中存在的游离抑制剂浓度（If）与横坐标上绘制的It之间的差异所致，因此可能是紧密结合条件的指标。因此，S形Emax模型会导致Ki的高估。所以，应用了抑制性S形Emax模型的一种修正模型（称为“Emax紧密模型”），该模型考虑了因结合导致的If消耗，以It为参照，并允许使用非线性回归分析来估计参数Et和IC50f（发生50%抑制时抑制剂的游离浓度）。该模型可以描述抑制曲线的非对称形状，并且Ki和Et的结果与从亨德森图得出的结果非常吻合。后一发现证实，体外测定的ACE抑制程度实际上取决于测定中存在的底物和酶的浓度。这不仅对于正确评估Ki很重要，而且对于解释离体测定的血清ACE抑制的时间进程也很重要。非线性模型相对于线性亨德森方程具有一些优势：它无需对数据进行转换即可直接应用，并且避免了变量的随机依赖性，允许对所有具有相同权重的数据点进行非线性回归。
