Kinetic determination of tight-binding impurities in enzyme inhibitors

• Published in: Analytical biochemistry 2003-08, Vol.319 (2), p.272-279
• Main Authors: Kuzmic, Petr, Hill, Craig, Kirtley, Matthew P, Janc, James W
• Format: Article
• Language: English
• Subjects: Binding, Competitive; Cell Line; Computer Simulation; Dose-Response Relationship, Drug; Drug Contamination; Enzyme kinetics; Enzymes; Four-parameter logistic equation; Humans; IC-50; Impurities; Inhibition constant; Inhibitor mixtures; Kinetics; Least-Squares Analysis; Mast cell tryptase; Mast Cells - cytology; Mast Cells - enzymology; Mathematics; Monte Carlo Method; Regression analysis; Serine Endopeptidases - metabolism; Serine Proteinase Inhibitors - analysis; Serine Proteinase Inhibitors - metabolism; Serine Proteinase Inhibitors - pharmacology; Statistics; Tight binding; Tryptases
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/S0003-2697(03)00248-3

A novel rate equation to characterize the dose-response behavior of a moderately potent (“classical”) enzyme inhibitor contaminated with a very potent (“tight-binding”) impurity is derived. Mathematical properties of the new rate equation show that, for such contaminated materials, experimentally observed I 50 values are ambiguous. The four-parameter logistic equation, conventionally used to determine I 50 values, cannot be used to detect the presence of tight-binding impurities in inhibitor samples. In contrast, fitting the newly derived rate equation to inhibitor dose- response curves can, in favorable cases, reveal whether the unknown material is chemically homogeneous or whether it is contaminated with a tight-binding impurity. The limitations of our method with respect to the detectable range of inhibition constants (both classical and tight-binding) were examined by using Monte-Carlo simulations. To test the new analytical procedure experimentally, we added a small amount (0.02 mole%) of a tight-binding impurity ( K i =0.065 nM) to an otherwise weak inhibitor of human mast-cell tryptase ( K i=50.4 μ M). The resulting material was treated as “unknown.” Our kinetic equation predicts that such adulterated material should show I 50=0.40 μ M, which was identical to the experimentally observed value. The best-fit value of the apparent inhibition constants for the tight-binding inhibitor was K i =(0.107±0.035) nM, close to the true value of 0.065 nM.