Abstract: Objective: To investigate the structural characteristics of a myofibril-bound serine proteinase inhibitor (MBSPI) from Larimichthys crocea and to elucidate its inhibitory mechanism against MBSP. Methods: MBSPI was purified from the skeletal muscle of L. crocea using ammonium sulfate fractionation, DEAE-Sepharose, and SP-Sepharose ion-exchange chromatography. Its structural characteristics were analyzed by circular dichroism (CD) spectroscopy and intrinsic fluorescence spectroscopy. Inhibition kinetics was employed to determine the inhibitory mechanism. Results: MBSPI was identified as a monomeric protein with a molecular mass of 55 kDa and exhibited specific immunoreactivity with rat polyclonal antibody against white croaker-derived MBSPI. Its primary structure exhibited a high identity to that of glucose-6-phosphate isomerase (GPI). MBSPI was predominantly composed of α-helices and had a thermal denaturation temperature of 53 ℃. At temperatures above 53 ℃, its secondary structure underwent significant changes, Specifically, the α-helix content decreased and the random coil and antiparallel β-sheet contents increased. Kinetic analysis revealed that MBSPI exhibited a competitive inhibitory effect on MBSP with an IC50 of 0.03 µmol/L. MBSPI effectively inhibited MBSP-induced degradation of myosin heavy chain (MHC), actin, and tropomyosin in myofibrillar proteins. Conclusion: The endogenous inhibitor MBSPI from L. crocea skeletal muscle can significantly suppress MBSP-induced autolysis of myofibrillar proteins, providing a new strategy for the utilization of water-soluble proteins in surimi-based product processing.

Key words: Larimichthys crocea; myofibril-bound serine proteinase; inhibitor; glucose-6-phosphate isomerase; inhibitory mechanism