Figure 1.

Unfolding kinetics by pulse proteolysis. After protein unfolding is initiated by adding urea, aliquots are dispended into tubes. At each time point, pulse proteolysis is performed by adding thermolysin into an aliquot and incubating for a minute. After proteolysis is quenched by EDTA, remaining intact proteins are quantified by SDS PAGE. Unfolding kinetic constants are determined by curve-fitting of the change of band intensities to an appropriate rate equation.