Ionization of tyrosine residues in horse-heart ferricytochrome c and its guanidinated and acetylated-guanidinated derivatives

John R. Cronin, Brenda A. Farringer, Ronald A. Nieman, Devens Gust

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Abstract

Spectrophotometric titration curves were obtained at 242 nm for native and fully gaunidinated horse-heart ferricytochrome c. The cytochrome c data were fit over the pH range 9-12 (I = 0.35) by a theoretical curve with pK′ values of 10.35 and 11.70. The slope of the experimental data increases sharply above pH 12.5 suggesting that two tyrosine residues with pK′ values greater than 12.5 are exposed by conformation change. The guanidinated cytochrome c data after correction for the alkaline spin-state transition were fit over the entire pH range 9-13.6 (I = 0.35) by a theoretical curve with pK′ values 10.37, 10.78, 11.50, and 13.60. These results along with viscosity measurements indicate that the unfolding transition occurs at higher pH in the guanidinated derivative. N-Acetylimidazole was used to acetylate specific tyrosyl groups of guanidinated cytochrome c. Assignments of acetylated tyrosine residues were confirmed by peptide mapping of 14C-labelled derivatives. Spectrophotometric titrations with rapid data acquisition of two monoacetylated derivatives allowed assignments of pK′1 (10.37) to Tyr-67 and pK′4 (13.60) to Tyr-97. The basis for the large differences in acidity and chemical reactivity of these two residues is not obvious from the crystallographic structure and may arise from differences in solvent access due to motions of the polypeptide chain.

Original languageEnglish (US)
Pages (from-to)325-335
Number of pages11
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume828
Issue number3
DOIs
StatePublished - Apr 29 1985

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Keywords

  • (Horse heart)
  • Acetylation
  • Chemical modification
  • Cytochrome c
  • Guanidination
  • Spectrophotometric titration
  • Tyrosine residue

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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