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).
The mode of binding of sperm and somatic H1 histones to DNA has been investigated by analyzing the effect of their addition on the electrophoretic mobility of linear and circular plasmid molecules. Low concentrations of sperm histones do not appear to alter the electrophoretic mobility of DNA, whereas at increasing concentrations, an additional DNA band is observed near the migration origin. This band then becomes the only component at higher values. In contrast, somatic histones cause a gradual retardation in the mobility of the DNA band at low concentrations and aggregated structures are observed only at higher values. Experiments on the H1 globular domain obtained by limited proteolysis indicate that the mode of binding to DNA depends on the H1 globular domain. The arginine residues appear to be relevant for the different effects as indicated by experiments on sperm histone and on protamine with arginines deguanidinated to ornithines. The modified molecules influence DNA mobility like somatic H1s, indicating that the positive guanidino groups of arginines cannot be substituted by the positive amino groups of ornithines. Modifications of the amino groups of lysines show that these residues are necessary for the binding of H1 histones to DNA but they have no influence on the binding mode.
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