Partially folded intermediates during trypsinogen denaturation

Abstract

The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was DG H 2 O = 6.99 ± 1.40 kcal/ mol for guanidine hydrochloride and DG H 2 O = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

Spray‐freeze‐drying for protein powder preparation: Particle characterization and a case study with trypsinogen stability

“…This does not refold on rehydration and hence remains inactive, but not aggregated. Circular dichroism and SEC have indeed shown that urea or guanidine‐HCl induced unfolding of bovine trypsinogen in water is not a simple transition, but rather involves a molten globule intermediate 21. Our results show, therefore, that aggregation of trypsinogen is minimal during SFD/rehydration, but that a substantial fraction of the protein is partially and irreversibly unfolded to an inactive state, i.e., a molten globule.…”

Mobile phase modifier effects in multimodal cation exchange chromatography

“…It is not clear why this particular family of proteins exhibited such different behavior. It may be due to the propensity of these proteins to undergo minor structural changes in the presence of low concentrations of guanidine (Greene and Pace, 1974; Martins and Santoro, 1999). The varying responses to guanidine led to many examples of improved selectivity among the proteins in this library.…”