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The colour of buffalo (Bubalus bubalis L.) meat is darker than bovine meat. Since meat colour depends on the concentration of myoglobin (Mb) and its oxidation state, we have determined the main structural and functional properties of buffalo Mb. Buffalo Mb was purified from longissimus dorsi muscles and its molecular mass determined by ESI Q-TOF mass spectrometry. The molecular mass 17,034.50 was 86.20 Da higher than the bovine Mb. This was confirmed by analysing its primary structure, using a combined approach based on Edman degradation and MALDI-TOF mass spectrometry. Comparing the amino acid sequences of both Mbs, we found three amino acid differences out of 153 amino acid residues. One is a conservative substitution (D(bov)141E(buf)), and the other two (A(bov)19T(buf) and A(bov)117D(buf)) are nonconservative. These amino acid substitutions are unlikely to cause structural changes because they are located far from the heme binding pocket, as revealed by the 3D structure of buffalo Mb elaborated by homology modelling. Stability analyses show no difference with the bovine Mb for helix E and only minor differences in the stability values for helices A and G. Moreover, autoxidation rates of purified buffalo and bovine myoglobins at 37 degrees C, pH 7.2, were almost identical, 0.052+/-0.001 h(-1) and 0.054+/-0.002 h(-1), respectively, as were their oxygen-binding Kd values, 3.7+/-0.1 microM and 3.5+/-0.1 microM, respectively. The percent of MetMb values were almost identical. The results presented here suggest that the darker buffalo meat depends on factors other than the oxidation rate of its Mb, as, for example, the Mb content (0.393+/-0.005 g/100 g of tissue) and consequently MetMb, which are almost twice as high as bovine meat (Mb: 0.209+/-0.003 g/100 g of tissue).

ABSTRACT:It is generally accepted that dry-aged buffalo (B. bubalis) meat becomes dark faster than bovine (B. taurus) meat, discouraging consumer purchase. We have investigated whether this faster darkening process might depend on structural and/or kinetic differences between buffalo and bovine myoglobins (Mbs). To this end, we have purified to homogeneity buffalo Mb from Longissimus dorsi muscle and obtained both its Mr (17,034.5) and the complete amino acid sequence, which, compared with the bovine one, showed three amino acid substitutions: D bo 141E bu, A bo 19T bu and A bo 117D bu. As revealed by the 3D structure, they were located on the surface of the protein, far from the heme binding pocket, and did not cause appreciable structural changes. Autoxidation rates of purified buffalo and bovine myoglobins at 37 °C, pH 7.2, were almost identical (0.052±0.001 h −1 and 0.054±0.002 h −1 , respectively), as were their oxygen-binding Kd values (3.7±0.1 μM and 3.5± 0.1 μM, respectively). These data indicate that the structure of the heme pockets in the two proteins is similar, with similar functional properties. Moreover, the percent of MetMb values in the purified buffalo and bovine samples, after the same time from slaughtering, were almost identical (57% and 47%, respectively). The results presented here suggest that the faster darkening of buffalo meat depends on factors other than the oxidation rate of its Mb, as, for example, the Mb content (0.393±0.005 g/100 g) and consequently its MetMb content, which is almost twice as high as bovine meat (0.209±0.003 g/100 g), and likely other factors.

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In protein deposition disorders, a normally soluble protein is deposited as insoluble aggregates, referred to as amyloid. The intrinsic effects of specific mutations on the rates of protein aggregation and amyloid formation of unfolded polypeptide chains can be correlated with changes in hydrophobicity, propensity to convert a-helical to b sheet conformation and charge. In this paper, we report the aggregation rates of buffalo, horse and bovine apomyoglobins. The experimental values were compared with the theoretical ones evaluated considering the amino acid differences among the sequences. Our results show that the mutations which play critical roles in the rate-determining step of apomyoglobin aggregation are those located within the N-terminal region of the molecule.