The Study About Porcine Pepsin Unfolding-Refolding Process And Partially Folded Intermediate

The unfolding and refolding procedure of porcine pepsin and the existence of intermediate stages has been studied by"phase diagram"method of fluorescence. Porcine pepsin aggregates and how they formed during the refolding procedure were studied by lauryl sodium sulfate-polyacrylamide gel electrophoreses (SDS-PAGE). The conclusion of this study will be helpful to understand the mechanism of the protein refolding.The fiuorescence spectrum of the denatured pepsin indicates that atλ_ex=280nm, when porcine pepsin was denatured by urea, theλemof Typ do not shift,but when it was denatured by guanidine hydrochloride , theλemof Typ shift red,and the intensity of it become weak,at the same time the apex of Tyr appears.The phase diagram of fluorescence representing the unfolding of porcine pepsin shows that, when porcine pepsin was denatured by urea, in the absence and presence of the reducing agent 2-mercaptoethanol, there have no partially folded intermediate during the unfolding procedure from native state to unfolded state, which indicates that the unfolding procedure of pepsin obeys a two-state model at this time. And the denaturation procedure of porcine pepsin induced by guanidine hydrochloride in the absence and presence of reducing agent 2-mercaptoethanol, obeys a typical three-state model, one partially folded intermediate can be detected.The phase diagram of fluorescence representing the refolding of porcine pepsin shows that, when porcine pepsin was denatured by urea, in the absence and presence of the reducing agent 2-mercaptoethanol, the denatured pepsin changes from unfolded state to native state, which indicates that the refolding procedure of pepsin follows a two-state model at this time. In the refolding procedure of porcine pepsin induced by guanidine hydrochloride follows a three-state model,that is to say there exists only one partially folded intermediate during the refolding procedure of porcine pepsi induced by guanidine hydrochloride.The phase diagram of fluorescence representing the unfolding of porcine pepsin shows that, when the pH of the buffer changed from 2 to 9,the porcine pepsin changed from native state,partially folded intermediate 1,partially folded intermediate2,to unfolded state.It follows a four-state model. The existent of aggregation during the dilute refolding of denatured porcine pepsin in the presence of different urea concentration has been detected by SDS-PAGE. The result shows that there no aggregation form in that process.