The greening reaction of skipjack tuna (Katsuwonus pelamis) metmyoglobin promoted by free cysteine during thermal treatment

Background: Tuna muscle greening is a problem that occurs after heating. A hypothesis has been postulated to address this problem, involving a conserved Cys residue at position 10 (Cys-10) present on tuna myoglobin (Mb) that is exposed during the thermic treatment, forming a disulfide bond with free cysteine (Cys) in the presence of trimethylamine oxide (TMAO), resulting in the greening of the tuna Mb. Methods: We present a study using skipjack tuna (Katsuwonus pelamis) metmyoglobin (MbFe(III)-H₂O) where the effect of free Cys (1–6 mM), TMAO (1.33 mM), and catalase on the greening reaction (GR) was monitored by UV-vis spectrometry during thermal treatment at 60 ℃ for 30 min. Moreover, the participation of Cys-10 on the GR was evaluated after its blocking with N-ethymaleimide. Results: The GR occurred in tuna MbFe(III)-H₂O after heat treatment with free Cys, forming sulfmyoglobin (MbFe(II)-S) as the responsible pigment for the tuna greening. However, the rate constants of MbFe(II)-S production depended on Cys concentration (up to 4 mM) and occurred regardless of the TMAO presence. We postulate that two consecutive reactions involve an intermediate ferrylmyoglobin (promoted by H₂O₂) species with a subsequent MbFe(II)-S formation since the presence of catalase fosters the reduction of the rate reaction. Moreover, GR occurred even with blocked Cys-10 residues in tuna Mb and horse Mb (without Cys in its sequence). Discussion: We found that GR is not exclusive to tuna Mb´s, and it can be promoted in other muscle systems. Moreover, Cys and thermal treatment are indispensable for promoting this pigmentation anomaly.