Myoglobin from Atlantic and Tinker mackerels: Purification, characterization and its possible use as a molecular marker

Here, we report the characterization (purification, autoxidation rate, pseudoperoxidase activity) and amino acid sequence determination of S. scombrus (Atlantic mackerel) and S. colias (Tinker mackerel) mioglobins (Mbs), considering the increasing consumption of fresh and canned mackerel meat and Mb implication in meat storage (e.g.: browning and lipid oxidation). We found that Atlantic mackerel Mb has major autoxidation rate (0.204 +/- 0.013 h-1) compared to Tinker mackerel Mb (0.140 +/- 0.009 h-1), while the pseudoperoxidase activity is major for Tinker mackerel (Km: 87.71 +/- 7.19 mu M; kcat: 0.32 s-1) Mb with respect to Atlantic mackerel (Km: 96.08 +/- 6.91 mu M; kcat: 0.50 s-1). These functional differences are confirmed by primary structure determination, in which six amino acid substitutions are found, with the first N-terminal amino acid residue acetylated. Furthermore, we predicted by AphaFold 3D model both fish Mbs and used them to investigate the possible structural differences. In addition, phylogenetic analysis using Mb sequences from Scombridae family confirms that Atlantic and Tinker mackerels are two distinct species. Finally, an analytic qualitative RP-HPLC method to distinguish S. scombrus and S. colias specimens was developed considering the different retention times of the two mackerel apoMbs.