Rapid unfolding of pig pancreas α-amylase: Kinetics, activity and structure evolution

• Kinetics of rapid unfolding of α-amylase was disclosed on the microsecond scale. • Detailed structural changes of α-amylase during the unfolding was described. • Unfolding of α-amylase from pig pancreas by guanidine hydrochloride was studied. • Contribution of H-bond structure of water to protein unfolding was disclosed. • One intermediate existed in the unfolding of pig pancreas α-amylase. α-Amylase plays an important role in food processing and in-vivo digestion. Many biological functions of α-amylase are affected by unfolding. The pre-steady-state rapid unfolding kinetics of α-amylase remains unknown. In this study, the rapid unfolding kinetics of porcine pancreatic α-amylase (PPA) with guanidine hydrochloride (GdmHCl) were investigated by stopped-flow spectroscopy. Structural characterization of PPA by fluorescence spectroscopy, and molecular dynamics simulation showed that the unfolding process of PPA might start from the internal active center, where the β-sheet structure was destroyed, followed by the exposure of hydrophobic amino acid residues. Further research revealed that GdmHCl denaturized PPA not by complexing with PPA. The surrounding H-bond network of water was changed by GdmHCl. This research improves our understanding of the unfolding kinetics of the PPA on the microsecond scale. It also provides the evidence experimentally of the surrounding water contribution to protein denaturization.