Porcine Pancreatic ¦Á-amylase Denaturation - Renaturation Process And Some Of Its Folding Intermediates

The unfolding and refolding procedure of porcine pancreaticα-amylase and the existence of intermediate stages has been studied by "phase diagram" method of fluorescence. Porcine pancreaticα-amylase 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 strongly helpful to understand the mechanism of the protein refolding.The phase diagram of fluorescence representing the unfolding of porcine pancreaticα-amylase shows that, when porcine pancreaticα-amylase was denatured by urea, in the absence and presence of the reducing agent 2-mercaptoethanol, there exist only one partially folded intermediate during the unfolding procedure from native state to unfolded state, which indicates that the unfolding procedure ofα-amylase follows a three-state model at this time. And in the presence of reducing agent 2-mercaptoethanol, the denaturation procedure of porcine pancreaticα-amylase induced by guanidine hydrochloride obeys a typical three-state model; while in the absence of 2-mercaptoethanol, the denaturation procedure of porcine pancreaticα-amylase induced by guanidine hydrochloride follows a four-state model, and two partially folded intermediates can be detected.The phase diagram of fluorescence representing the refolding of porcine pancreaticα-amylase shows that, when porcine pancreaticα-amylase was denatured by urea, in the absence and presence of the reducing agent 2-mercaptoethanol, there exist only one partially folded intermediate during the refolding procedure from unfolded state to native state, which indicates that the refolding procedure ofα-amylase follows a three-state model at this time. In the absence of 2-mercaptoethanol, the refolding procedure of porcine pancreaticα-amylase induced by guanidine hydrochloride follows a two-state model; while in the presence of reducing agent 2-mercaptoethanol, there exist one partially folded intermediate during the refolding procedure of porcine pancreaticα-amylase induced by guanidine hydrochloride.The reason of aggregation during the dilute refolding of denatured porcine pancreaticα-amylase in the presence of different urea concentration has been analyzed by SDS-PAGE. It was founded that the unfolded porcine pancreaticα-amylase induced by urea in the presence of reduced agent formed soluble aggregates through intermolecular disulfide bonds. There are some aggregates formed too during the refolding of the unfolded porcine pancreaticα-amylase induced by urea in the absence of reduced agent. The reason was the intermolecule mismatch of the dissociative cysteine.