Hydrogen ion titration of horse heart ferricytochrome c.
Continuous hydrogen ion titration curves of deionized solutions of horse heart ferricytochrome c have been obtained at 25 degrees C. at a constant ionic strength of 0.10 from pH 3.0 to 11.0. Titration of the oxidized protein in KCl required 28.4 equiv over that pH range, and a small hysteresis between the forward and reverse limbs was displayed. The Linderstrom-Lang approximation, which takes into account electrostatic interactions between charged groups on the protein surface, was used in a computer simulation program to analyze the forward and reverse limbs of the titration curve separately. The results indicated 1 alpha-, 12 beta- and gamma-, and 1 heme propionic carboxylic, 1 imidazole, 1 phenolic, and 18 epsilon-amino residues appear to titrate normally. Variations in the electrostatic interaction factor omega suggest conformational changes in the protein at the extremes of pH, although the relationship of the variations in omega to the magnitude of the conformational changes does not appear to be strictly quantitative for cytochrome c. These results show the acid-base behavior of cytochrome c to be complex in nature, and suggest that the Lindenstrom-Lang model may not be adequate for cytochrome c.
Hydrogen ion titration of horse heart ferricytochrome c. Continuous hydrogen ion titration curves of deionized solutions of horse heart ferricytochrome c have been obtained at 25 degrees C. at a constant ionic strength of 0.10 from pH 3.0 to 11.0. Titration of the oxidized protein in KCl required 28.4 equiv over that pH range, and a small hysteresis between the forward and reverse limbs was displayed. The Linderstrom-Lang approximation, which takes into account electrostatic interactions between charged groups on the protein surface, was used in a computer simulation program to analyze the forward and reverse limbs of the titration curve separately. The results indicated 1 alpha-, 12 beta- and gamma-, and 1 heme propionic carboxylic, 1 imidazole, 1 phenolic, and 18 epsilon-amino residues appear to titrate normally. Variations in the electrostatic interaction factor omega suggest conformational changes in the protein at the extremes of pH, although the relationship of the variations in omega to the magnitude of the conformational changes does not appear to be strictly quantitative for cytochrome c. These results show the acid-base behavior of cytochrome c to be complex in nature, and suggest that the Lindenstrom-Lang model may not be adequate for cytochrome c.