Potentiometric titration curves of oxidized and reduced horse heart cytochrome c

✍ Scribed by M. A. Marini; G. E. Marti; R. L. Berger; C. J. Martin

• Publisher: Wiley (John Wiley & Sons)
• Year: 1980
• Tongue: English
• Weight: 729 KB
• Volume: 19
• Category: Article
• ISSN: 0006-3525
• DOI: 10.1002/bip.1980.360190412

✦ Synopsis

Abstract

Potentiometric titration curves of oxidized and reduced horse heart cytochrome c in 0.15__M__ KCl at 20°C have been obtained by timed titration (0.125–0.500 μmol/sec) from the isoionic points (pH 10.2–10.4) to pH 3 and back to the isoionic point. Computer‐assisted (PROPHET) data acquisition and blank corrections give curves with good precision with a maximum standard deviation of 0.3 groups for an average error of 1%. The potentiometric titration curve of reduced cytochrome c is reversible within the precision of the method and for the pH range studied. The potentiometric curves for oxidized cytochrome c titrated upscale (pH 3–10) and downscale (pH 10–3) are not reversible. However, they show the same ionization behavior after the initial downscale titration. This is probably the result of a conformational change. Comparison of the data herein reported with the titration curves of oxidized cytochrome c already published by others indicates good agreement on the basis of a normalization of the concentration of protein or on the basis of 25 titrable groups between the acid end point and the isoionic pH.

Titration of the 2 μmol imidazole in the upscale or downscale direction gives the correct analytical concentration and p__K__′ after correction for the solvent titration. Titration of reduced cytochrome c in the presence and absence of an additional equivalent of imidazole gave a difference titration curve, which indicates that a group on the protein shifts from p__K__′ 5.8 to p__K__′ 5.3 in the presence of imidazole. The p__K__′ of imidazole, in the presence of the protein, remains at a nearly normal value of 7.34.