Local stabilities of horse cytochrome c metalloderivatives as probed by tryptic digestion and electrospray mass spectrometry

The porphyrin, Co-III, Mn-III and Zn-II derivatives of horse heart cytochrome c (cyt c) were characterized by electrospray mass spectrometry. The results reveal that the primary structure of the polypeptide is unaltered following the procedures used to prepare the derivatives. Together with the native Fen and Fem forms and the cyanide adduct of the latter, the derivatives were subjected to time-dependent tryptic digestion, and reversed phase HPLC analysis of the digests was carried out. Since protease susceptibility requires local conformational instability, the results indicate that the local stabilities of the proteins follow the order: Fe-II-cyt c > Co-III-cyt c > Fe-III-cyt c > Mn-III-cyt c > Zn-II-cyt c > CN-Fe-III-cyt c > por-cyt c. Large, partially digested fragments were observed by LC-MS in the digests of Co-III-cyt c (residues 1-53/55 + Co-porphyrin and 56-104) and Fe-III-cyt c (residues 8-86 + heme). No such fragments were observed in the derivatives with lower local stabilities. It is proposed that the strength of the Met80 ligand is important in controlling the initial site(s) of tryptic attack and the local stability of the horse cyt c polypeptide.