The Reactivity of the Lysyl Residues of Cytochrome b5

Abstract

Abstract The modification of α- and e-amino groups in cytochrome b5 with acetic anhydride, succinic anhydride, and 2,4,6-trinitrobenzene 1-sulfonic acid (trinitrobenzene sulfonate) was described. Acylation of these residues increases the rate of heme dissociation from the protein in the presence of urea, without altering the spectrum or enzymatic reduction of the cytochrome. Trinitrophenylation of all of the α- and e-amino groups results in a marked alteration of the protein structure. Even more extensive changes in the protein structure occurred when trinitrophenylation was carried out in the absence of the heme. The kinetics of trinitrophenylation showed that 3 of the lysyl residues in cytochrome b5 are particularly reactive. These residues are largely confined to the heme peptide core rather than to the lysyl residues near the amino- and carboxyl-terminal ends of the protein. Because the completely acetylated apocytochrome b5 recombines with heme to yield a cytochrome b5 with unaltered spectral properties, the α- and the e-amino groups are not involved directly in either heme binding or the catalytic activity of cytochrome b5.