A novel dual-channel enzymatic inhibition measurement (DEIM) method was developed to improve the repeatability with light/heavy isotope substrates, producing reliable relative standard deviations (< 3%) by employing acetylcholinesterase (AChE) as the model enzyme. The matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was adapted for enzyme-inhibited method due to its good salt-tolerance and high throughput; meanwhile, dual-channel enzymatic reactions were performed to improve the repeatability of each well. The acetylcholinesterase inhibition measurement was conducted by mixing the quenched enzyme reaction solution of blank group (with heavy isotope as substrate) and experimental group (with light isotope as substrate), of which the inhibition rate might be affected by isotope effects. Hence, inverse study and Km measurement were implemented to validate the method. The inverse study shows similar inhibition rate (68.9 and 70.3%) and the Km of isotope substrates are analogous (0.139 and 0.135 mM), which demonstrated that the novel method is feasible to AChE inhibition measurement. Finally, the method was applied to herb extracts, half of which exhibit inhibition to AChE. The precise dual-channel enzymatic inhibition measurement (DEIM) method could be regarded as a promising approach to potential enzyme inhibitor screening.

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开发了一种新颖的双通道酶抑制测量（DEIM）方法，以提高轻/重同位素底物的可重复性，并通过使用乙酰胆碱酯酶（AChE）作为模型酶来产生可靠的相对标准偏差（<3％）。基质辅助激光解吸/电离飞行时间质谱仪（MALDI-TOF MS）具有良好的耐盐性和高通量，适用于酶抑制法。同时，进行双通道酶促反应以提高每个孔的重复性。通过将空白组（以重同位素为底物）和实验组（以轻同位素为底物）的淬灭酶反应溶液混合进行乙酰胆碱酯酶抑制作用的测定，其抑制率可能受到同位素效应的影响。因此，Km测量被实施以验证该方法。反向研究显示相似的抑制率（68.9％和70.3％），同位素底物的Km相似（0.139和0.135 mM），这表明该新方法对于AChE抑制测量是可行的。最后，该方法应用于草药提取物，其中一半表现出对AChE的抑制作用。精确的双通道酶促抑制测量（DEIM）方法可以被视为潜在的酶抑制剂筛选的有前途的方法。

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