Interaction of Astaxanthin Stereoisomers with Porcine Pancreatic α-Amylase

In this study, the inhibitory effects (3S, 3′S) and (3R, 3′R) astaxanthin (AST), derived from Haematococcus pluvialis and Xanthophyllomyces dendrorhous, respectively, were examined on porcine pancreatic α-amylase (PPAA), and the interaction mechanism between AST and PPAA was elucidated using circular dichroism (CD) spectroscopy, synchronous fluorescence spectroscopy, endogenous fluorescence spectroscopy, and molecular docking. The results showed that (3S, 3’S) (> 94% pure) and (3R, 3’R) (> 96% pure) AST were successfully prepared and identified. They showed no significant differences in inhibitory effects on PPAA activity. Both isomers slightly affected the microenvironment of tryptophan residues in PPAA, but they did not alter the overall protein conformation. Moreover, molecular docking analysis revealed that the binding site of AST isomers to PPAA was located within its catalytic pocket, which was identical to that of the enzyme inhibitor acarbose. The isomers interacted with the key amino acid residues in the catalytic center of the enzyme, namely Asp197, Glu233, and Asp300. Notably, Glu233 formed hydrogen bonds with both stereoisomers, indicating that they inhibited PPAA activity through a competitive mechanism. Endogenous fluorescence spectroscopy further demonstrated that the interaction between AST stereoisomers and PPAA exhibited static quenching, suggesting the formation of nonfluorescent complexes in the ground state. Although (3R, 3’R) AST exhibited lower thermodynamic parameters, shorter hydrogen bond lengths, and interaction with more amino acid residues, its binding affinity was comparable to that of (3S, 3’S) AST, thereby indicating that its inhibitory activity was not affected by the stereo-configurational difference. These findings provide a theoretical basis for the use of AST as a natural product for preventing hyperglycemia, and offer scientific support for the development of novel PPAA inhibitors and functional foods.