Unfolding Kinetics of Bovine Trypsinogen

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 (GdmCI). 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, CaZ+ influenced only this stage of the reaction. Analysis of the dependence of the time constant of the fast phase on [CaClJ indicates that at 4 M GdmCI, trypsinogen . Ca unfolds about sixfold slower than free zymogen, and that native trypsinogen at 4 M GdmCl still exhibits high affinity for Ca''. Limited data on trypsin unfolding show virtually an identical dependence of the slow phase on [GdmCI] ; 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.Keywords: trypsinogen; unfolding kinetics; transition state; denaturation; guanidinium chloride.The mechanism by which an unfolded polypeptide chain acquires a specific, densely packed and cooperative three-dimensional structure still remains one of the major unsolved questions of molecular biology (Anfinsen, 1973 ;Creighton, 1990;Fersht, 1993 ;Miranker and Dobson, 1996). The process of protein folding in vivo and in viti'o is often on a milisecond-second timescale. Thus, fast-reaction techniques are required to characterize the process in terms of intermediate(s) and transition state(s).Usually, studies focus on small (below 25-20 kDa) proteins, including myoglobin (Chiba et al., 1994), lysozyme (Denton et al., 1994;Dobson et al., 1994;Itzhaki et al., 1994), lactalbumin (Kuwajima et al., 1989), basic pancreatic trypsin inhibitor (Darby et al., 1995;Weissman and Kim, 1995), Q subunit of tryptophan synthase (Chen and Matthews, 1994;Ogasahara and Yutani, 1994) and cytochrome c (Elove et al., 1992). Particularly coherent and detail 'folding pictures' have been reached for two proteins, barnase and chymotrypsin inhibitor 2 from barley, due to application of a large number of carefully designed mutant proteins (Fersht, 1993(Fersht, , 1994Otzen et al., 1994). Two general conclusions can be drawn from these studies.(a), the folding pathway is more complicated for larger proteins than for smaller ones. Two-s...

Partially folded intermediates during trypsinogen denaturation

“…The fast phase is not a single-phase event under mildly denaturing conditions and in the slow phase the authors showed evidence of intermediates accumulating under strongly folding conditions. A similar kinetics was observed for the trypsinogen-Ca 2+ complex and limited data on trypsin unfolding show a virtually identical behavior (13). More recently Ruan et al (14) studied the effect of hydrostatic pressure on the unfolding of bovine trypsin.…”

“…The authors showed that the stability of trypsinogen is increased by calcium ion, by the dipeptide Ile-Val and by basic pancreatic trypsin inhibitor (BPTI) and proposed a two-state transition in equilibrium. The unfolding kinetics of trypsinogen was studied by Otlewski et al (13) using a fluorescencedetected stopped-flow manifesting two independent phases. The fast phase is not a single-phase event under mildly denaturing conditions and in the slow phase the authors showed evidence of intermediates accumulating under strongly folding conditions.…”

Effects of Essential Oil Extracted from <i>Nigella sativa</i> (L.) Seeds and Its Main Components on Human Neutrophil Elastase Activity

“…Moreover, no research has been carried out to investigate the eŠects of bioactive molecules present in the extract of this medicinal plant on human neutrophil elastase (HNE) activity. This enzyme has been reported to play a crucial role in extracellular proteolytic processes at sites of in‰ammation [11][12][13] and capable of cleaving many proteins with important biologic functions, especially elastin, an important extracellular matrix protein that plays a mechanical function in the lungs. Thus it became evident that HNE is involved in the pathogenesis of diŠerent in‰ammatory diseases such as emphysema, cysticˆbro-sis (CF), and COPD.…”

Mutations in the N- and C-terminal Tails of Potato Carboxypeptidase Inhibitor Influence Its Oxidative Refolding Process at the Reshuffling Stage

“…The question of whether these proteins need the tails for correct refolding, as does PCI, is an interesting issue. The trypsin squash inhibitors have a very similar structural topology to PCI and the same disulfide bridge pattern (28). The sequence alignment of a large number of the squash inhibitors shows that the cysteine-reinforced core contains 25 amino acids (27 in PCI); there are zero to five amino acids at the N terminus and, in most cases, only one glycine residue at the C terminus, after the last cysteine of the T-knot core.…”

“…If the fourth residue of the C terminus, Pro 36 , is also deleted, no formation of the native-like species is detected. Both in the NMR and x-ray structure of PCI, a hydrogen bond between the carbonyl oxygen of Pro 36 (in the tail) and the amide nitrogen of Trp 28 (in the core) is observed. Our results seem to indicate that this hydrogen bond, which could be formed by any residue in position 36, could participate significantly in directing the native disulfide pairing of PCI.…”