Figure 4. Determination of stoichiometry of alkylation by quinacrine mustard.
A sample of NADPH-reduced TryR (1 μM) was inactivated by 10 μM QM. Aliquots were removed at intervals, quenched with 2-mercaptoethanol and residual activities were determined as described in Figure 2. Residual activity is fitted to the equation for a single exponential decay (open circles). The remainder of each quenched sample was denatured with trichloroacetic acid to remove bound FAD, washed and re-dissolved by proteolytic digestion. Residual covalently bound QM was determined spectrophotometrically as described in Experimental Procedures (closed circles).