The Conformation of Horse Heart Apocytochrome c

The covalently bound heme moiety in horse heart cytochrome c was cleaved from the polypeptide chain by reaction with silver sulfate in acidic solution. The apoprotein was extracted with acid acetone, treated with 2-mercaptoethanol, and subjected to differential sedimentation to remove the heme moiety, protein bound silver, and polymerized apoprotein, respectively. Monomeric apocytochrome c has a sedimentation coefficient of 1.2 S, an intrinsic viscosity of 15.5 ml per g, and a large negative maximum in its circular dichroic spectrum at 199 nm. All 3 histidyl residues can be carboxymethylated, the phenolic ionization of all 4 tyrosyl residues is normal giving an apparent pK of 10.2, and the ultraviolet absorption spectrum of the single tryptophanyl residue can be perturbed by addition of 20% ethylene glycol. These results are identical with those of unfolded cytochrome c and are characteristic of a polypeptide in a randomly coiled conformation. Therefore the heme-polypeptide interactions in cytochrome c must be essential to stabilization of the globular conformation of the native protein.