Schebb Nils Helge, Heus Ferry, Saenger Thorsten, Karst Uwe, Irth Hubertus, Kool Jeroen
Westfalische Wilhelms-Universitat Munster, Institut fur Anorganische and Analytische Chemie, Corrensstrasse 30, 48149 Munster, Germany.
Anal Chem. 2008 Sep 1;80(17):6764-72. doi: 10.1021/ac801035e. Epub 2008 Aug 8.
A gradient HPLC approach in combination with a countergradient system for online biochemical detection (BCD) to screen for inhibitors of serine proteases is described. For gradient separations, this novel countergradient system was developed to produce a biocompatible constant solvent composition in the BCD. The countergradient system is based on retaining complete gradients in an additional preparative HPLC column, followed by subsequent and reversible elution to the separation column effluent. Major advantages compared with existing countergradient systems are that no additional LC pumps are needed and enhanced stability. The developed countergradient system was systematically characterized applying different gradient programs. Inhibitors eluting in a postcolumn continuous flow analysis interfere with the enzymatic release of fluorescent 7-amino-4-methylcoumarin (AMC) from an AMC-labeled peptide. The inhibitory activity of eluting substances is sensitively detected as the degree of reduced fluorescence intensity. This biochemical detection system (BCD) for proteases was validated with three known inhibitors of the benzamidine type. Their IC 50 values were in good accordance with the results of conventional plate reader assays. Finally, a small library of protease inhibitors was successfully screened with the combination of the BCD and the countergradient system.
本文描述了一种结合反梯度系统的梯度高效液相色谱法，用于在线生化检测（BCD）以筛选丝氨酸蛋白酶抑制剂。对于梯度分离，开发了这种新型反梯度系统，以便在BCD中产生生物相容性恒定的溶剂组成。该反梯度系统基于在一个额外的制备型高效液相色谱柱中保留完整的梯度，随后将其可逆洗脱至分离柱流出物中。与现有反梯度系统相比，主要优点是无需额外的液相色谱泵且稳定性增强。应用不同的梯度程序对开发的反梯度系统进行了系统表征。在柱后连续流动分析中洗脱的抑制剂会干扰荧光7-氨基-4-甲基香豆素（AMC）从AMC标记肽的酶促释放。洗脱物质的抑制活性通过荧光强度降低的程度来灵敏检测。这种用于蛋白酶的生化检测系统（BCD）用三种已知的苯甲脒类抑制剂进行了验证。它们的IC50值与传统酶标仪测定结果高度一致。最后，结合BCD和反梯度系统成功筛选了一个小型蛋白酶抑制剂文库。
