Abstract
When cytochrome c purified from bovine heart muscle was acetylated with acetic anhydride under specific conditions, the acetylated cytochrome c preparations were obtained, in which 1, 2 and 3 acetyl groups had incorporated to the ε-amino groups of lysine residues. They were purified chromatographically to homogeneous states. With these preparations it was found that the enzymatic activity of cytochrome c in the cytochrome oxidase [EC 1. 9. 3. 1] reaction decreased gradually and linearly with the decrease in its basicity. The acetylation of 6 ε-amino groups in cytochrome c molecule led the protein to complete loss of the activity. From the amino acid analysis of these preparations of the acetylated cytochrome c, it was found that the 22 nd lysine residue from the amino terminus of the protein was most reactive toward acetic anhydride.
When cytochrome c was treated with a large excess amount of acetic anhydride in the absence of sodium acetate, not only its ε-amino group of lysine residue but also the hydroxyl group of tyrosine residue was acetylated completely. With this preparation, a drastic change was observed in the visible and ultraviolet absorption spectra of the oxidized form, while that of the reduced form remained unchanged. The heme of this preparation combined with CO and oxygen. It was suggested that the heme moiety of this preparation had less interaction with protein moiety than that of the native one.
