Rapid determination of enzyme kinetics from fluorescence: Overcoming the inner filter effect
Rapid determination of enzyme kinetics from fluorescence: Overcoming the inner filter effect
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DOI：
10.1016/j.ab.2007.07.008
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发表时间：
2007-12-01
影响因子：
2.9
通讯作者：
Van Doren, Steven R.
中科院分区：
文献类型：
--
作者：
Palmier, Mark O.;Van Doren, Steven R.
荧光变化可方便地监测酶动力学。不幸的是，随着荧光底物的吸光度随着浓度的增加而增加，它失去了线性。当激发波长和发射波长的吸光度之和超过0.08时，这种内滤波效应(IFE)改变了表观初始速度K-m和k(Cat)。表观初始速度的IFE失真可以在不进行荧光团稀释分析的情况下得到校正。利用衬底在激发波长和发射波长的消光系数，可以在曲线拟合过程中模拟内滤波效应，以获得更准确的米氏参数。一种更快、更简单的方法是从进度曲线推导出k(Cat)和K-m。使用基质金属蛋白酶12和碱性磷酸酶说明了仅从两到三条进度曲线获得可靠和可重复性的k(CAT)和K-m估计的策略。酶活性部位浓度和特异性常数k(Cat)/k(M)的准确估计(与S的一条进展曲线)
Fluorescence change is convenient for monitoring enzyme kinetics. Unfortunately, it loses linearity as the absorbance of the fluorescent substrate increases with concentration. When the sum of absorbance at excitation and emission wavelengths exceeds 0.08, this inner filtering effect (IFE) alters apparent initial velocities, K-m and k(cat). The IFE distortion of apparent initial velocities can be corrected without doing fluorophore dilution assays. Using the substrate's extinction coefficients at excitation and emission wavelengths, the inner filter effect can be modeled during curve fitting for more accurate Michaelis-Menten parameters. A faster and simpler approach is to derive k(cat) and K-m from progress curves. Strategies to obtain reliable and reproducible estimates of k(cat) and K-m from only two or three progress curves are illustrated using matrix metalloproteinase 12 and alkaline phosphatase. Accurate estimates of concentration of enzyme-active sites and specificity constant k(cat)/k(m) (from one progress curve with [S]