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 (GdmCl). 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 GdmCl. 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 GdmCl, 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 [GdmCl]; the fast phase, however was not observed. Moreover, in the 3-4.5 M GdmCl range, a separate phase was detected. It is postulated that this phase is a manifestation of the activation-domain unfolding. The Eyring plots for the fast phase of . trypsinogen and trypsinogen.Ca unfolding are linear, indicating little change in heat capacity for this stage of reaction. The slow step of unfolding, however, shows significant curvature which indicates a substantial increase in heat capacity.
Otlewski
J
J
Institute of Biochemistry and Molecular Biology, University of Wroclaw, Poland.
Sywula
A
A
Kolasinski
M
M
Krowarsch
D
D
D000818:Animals; D002118:Calcium / Q000737:chemistry; D002417:Cattle; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013696:Temperature; D014357:Trypsin / Q000737:chemistry; D014362:Trypsinogen / Q000737:chemistry*
D006146:Guanidines; D014362:Trypsinogen; D014357:Trypsin; D019791:Guanidine; D002118:Calcium