Purification and Characterization of Myofibril-Bound Serine Proteinase Inhibitor from Larimichthys crocea

To investigate the structural characteristics of a myofibril-bound serine proteinase inhibitor (MBSPI) from Larimichthys crocea and to elucidate its inhibitory mechanism against MBSP.

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.

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.

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.