Disruption of secondary structure by oxidative stress alters the cross-linking pattern of myosin by microbial transglutaminase

Porcine myofibrillar protein (MP) was oxidatively stressed in an iron-H₂O₂ radical-producing system then subjected to microbial transglutaminase (TGase, E:S=1:20) at 4°C. Changes in the MP secondary structure and cross-linking site on myosin (subfragments S1, S2, rod, light meromyosin, and heavy meromyosin) after TGase treatment were investigated. Circular dichroism and FTIR recorded unraveling of helixes caused by both oxidation and TGase. The loss of α-helix due to TGase treatment was oxidation-dependent, namely, mild oxidation (0.1-1mM H₂O₂)>non-oxidation>moderate oxidation (5-20mM H₂O₂). Moreover, oxidation altered the myosin cross-linking pattern: TGase-initiated S1 cross-linking (which dominated non-oxidized MP) partially shifted to the rod under 0.1-0.5mM H₂O₂ and extensively to the S2 site with 20mM H₂O₂. Unraveling of the helical structure and formation of disulfide bonds due to oxidation were implicated in the altered myosin cross-linking pattern during subsequent TGase reactions.