Comparison of Myoglobins from Harbor Seal, Porpoise, and Sperm Whale

Abstract

Abstract The pH rate profile for uncatalyzed formation of a complex between cyanide and sperm whale ferrimyoglobin at 25° and total ionic strength 0.11 has been established over the pH range 5 to 11 utilizing an integrated reversible second order rate equation of general applicability. The profile may be interpreted as reflecting three important routes for complex formation: (a) addition of cyanide ion to an unprotonated myoglobin, (b) addition of hydrocyanic acid to the unprotonated myoglobin (or the kinetically indistinguishable addition of cyanide ion to a protonated myoglobin), and (c) addition of hydrocyanic acid to the protonated myoglobin. Complex formation is strongly subject to catalysis by certain buffers: catalysis is most evident at intermediate values of pH and becomes negligible below pH 6 and above pH 10. Effective buffers include phosphate, glycine, and cyanide itself. Other buffers with forms capable of simultaneously accepting 2 protons through hydrogen bonding are also effective. Addition of cyanide to ferrimyoglobins from pilot whale, porpoise, and harbor seal is also subject to buffer catalysis. Guanidination of sperm whale ferrimyoglobin and carboxymethylation in the crystalline state have only small effects on the reactivity of the protein toward cyanide or on the sensitivity of this reaction to buffer catalysis. Carboxymethylation in solution, in contrast, markedly reduces reactivity toward cyanide. The hypothesis is developed that the buffer-binding site may lie between arginine-45 and histidine-64. A reasonable mechanism for the buffer catalysis is proposed.