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Plot of E◦ vs. pH produced a straight line with a slope of −46.0mV pH−1 (the correlative coefficient is 0.994). This value was smaller than the theoretical value of −58.0mV pH−1 at 20 ◦C for a single proton coupled, reversible one electron ransfer. According to the literature , it may be that the protonation of trans-ligands influences the heme iron and amino acids around Hb or that the protonation of the water molecules coordinates to the central iron, both of which affect the protons involved in electrochemical reaction of Hb.
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