Unfolding Kinetics of Bovine Trypsinogen

The unfolding kinetics of bovine trypsinogen were studied by a fluorescence‐detected stopped‐flow technique at pH 5.8. Trypsinogen unfolding appeared to be a rather complex reaction. Two phases, fast (with a time constant in the millisecond range) and slow, were detected in the range 2–7 M guanidium chloride (GdmC1). The natural logarithm of the rate constant of the slow phase exhibited strong dependence on [GdmCl], changing from hundreds of seconds at low denaturant concentration to about 20 ms at 7 M GdmC1. The curvature of this dependence further suggests a complex mechanism of unfolding. Generally, similar kinetics were observed for the trypsinogen · Ca complex. Small differences could be noticed, however, for the fast phase. In agreement, Ca2+ influenced only this stage of the reaction. Analysis of the dependence of the time constant of the fast phase on [CaCl2] indicates that at 4 M GdmC1, trypsinogen · Ca unfolds about sixfold slower than free zymogen, and that native trypsinogen at 4 M GdmCl still exhibits high affinity for Ca2+. Limited data on trypsin unfolding show virtually an identical dependence of the slow phase on [GdmC1]; the fast phase, however, was not observed.