Determination of T Using Tight Binding Inhibitors

All these aspects of tight binding inhibition can potentially offer important advantages in terms of clinical efficacy, dosing interval, and patient safety (see Swinney, 2004, for an excellent review of some of the clinical advantages of tight binding inhibition and other nonclassical inhibition mechanisms). 

In Section 7.2 we presented one method for determining E T from the effects of apparent enzyme concentration on the measured value of IC50 for tight binding inhibitors. Another convenient way to determine [E T derives from the nature of Morrison s equation. When the ratio E T/A (PP equals or exceeds 200, the fractional velocity decreases very steeply with increasing inhibitor concentration, in an essen- 

by either titrating tight binding inhibitor concentration at a high, fixed enzyme concentration, and vice versa, one can obtain highly accurate estimates of the total enzyme concentration in a sample. These methods are commonly used to 

Copeland, R. A. (1994), Methods in Protein Analysis A Practical Guide to Laboratory Protocols, Chapman and Hall, New York. 

Copeland, R. A. (2000b), Enzymes A Practical Introduction to Structure, Mechanism and Data Analysis, 2nd ed., Wiley, New York. 

---