Abstract The finite excess of the non-limiting enzymes in a coupled-enzyme assay causes an initial delay that can lead to inaccuracies in the kinetic read-out of substrate assays. We derive a new correction term to the well-known analytical Lambert W based expression of the progress curve. The key parameter in comparing the activities of the various enzymes in the chain is their pseudo first order rate constant k ' = V m a x / K m . The order in which the limiting (L) and non-limiting (NL) enzymes appear in the chain has little to no effect on the final progress curves. On simulated data the model gives good recoveries of unknown substrate concentrations as well as of the k L ' of the limiting enzyme, even with excess ratio of the non-limiting over the limiting enzymes k r = k N L ' / k L ' as low as 3, and including the case of multiple non-limiting enzymes with low excess. In the presence of noise and on experimental data the new analytical expression shows superior performance in smoothing the progress curves compared to traditional non-parametric smoothing techniques. This results in reduced scatter on the dose response curves, on par or even exceeding the performance of endpoint analysis but in shorter time. The analytical expression remains very robust in the presence of low excess ratios.

中文翻译：

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偶联酶底物测定中滞后期的分析描述

摘要 偶联酶测定中非限制性酶的有限过量会导致初始延迟，从而导致底物测定动力学读数不准确。我们为众所周知的基于分析 Lambert W 的进度曲线表达式推导出一个新的校正项。比较链中各种酶活性的关键参数是它们的伪一级速率常数 k ' = V max / K m 。限制性 (L) 和非限制性 (NL) 酶在链中出现的顺序对最终进展曲线几乎没有影响。在模拟数据上，该模型给出了未知底物浓度以及限制酶 k L ' 的良好回收率，即使非限制酶与限制酶的过量比率 kr = k NL ' / k L ' 低至3、并且包括具有低过量的多种非限制性酶的情况。在存在噪声和实验数据的情况下，与传统的非参数平滑技术相比，新的分析表达式在平滑进度曲线方面表现出卓越的性能。这导致剂量反应曲线上的分散减少，达到甚至超过终点分析的性能，但时间更短。在存在低过量比率的情况下，分析表达式仍然非常稳健。与终点分析的性能相当甚至超过，但时间更短。在存在低过量比率的情况下，分析表达式仍然非常稳健。与终点分析的性能相当甚至超过，但时间更短。在存在低过量比率的情况下，分析表达式仍然非常稳健。