Unfolding kinetics of bovine trypsinogen

Eur J Biochem. 1996 Dec 15;242(3):601-7. doi: 10.1111/j.1432-1033.1996.0601r.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / chemistry
  • Cattle
  • Guanidine
  • Guanidines / chemistry
  • Kinetics
  • Models, Molecular
  • Protein Denaturation
  • Protein Structure, Tertiary
  • Temperature
  • Trypsin / chemistry
  • Trypsinogen / chemistry*

Substances

  • Guanidines
  • Trypsinogen
  • Trypsin
  • Guanidine
  • Calcium