Kinetic determination of tight-binding impurities in enzyme inhibitors

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 microM). The resulting material was treated as "unknown." Our kinetic equation predicts that such adulterated material should show I(50)=0.40 microM, 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.