Figure 5.

Unfolding kinetics by pulse proteolysis versus proteolysis kinetics. Errors in determining unfolding kinetics using proteolysis as a probe are demonstrated by simulation for the case that proteolysis also occurs through partial unfolding. In this simulation, protein unfolds with a kinetic constant of 0.01 min⁻¹ but is also digested through partial unfolding with a kinetic constant of 0.05 min⁻¹. The true unfolding kinetics is shown for comparison (solid line). When unfolding is monitored by proteolysis kinetics (dotted line), a significant error is introduced. On the contrary, pulse proteolysis does not introduce any error in the kinetic constant, even though the apparent amplitude is slightly smaller due to proteolysis of folded proteins during 1 min of pulse (dashed line).