Regio-specific adsorption of cytochrome c on negatively charged surfaces.

Studies are reported on the identification of the chromatographic contact domain of equine cytochrome c during its interaction with negatively charged sorbents. A negatively charged resin was designed that would simultaneously adsorb the protein electrostatically and covalently bind it through amide bond formation to succinate groups coupled to the support in an ester linkage. Protein immobilization occurred through lysine residues participating in electrostatic adsorbed cytochrome c to the resin surface. After covalent bond formation in the interface between the protein and the sorbent, ester linkages coupling succinate groups to the support were hydrolyzed, and the protein was released. Lysine residues on the protein that had participated in covalent capture were labeled with succinate residues. The tagged protein was then tryptic-mapped and the peptides were examined by matrix-assisted laser desorption ionization mass spectrometry to determine the position of the amino acids that had been tagged. Comparing the tagged sites with the X-ray crystallographic structure of cytochrome c, it was concluded that a single face of the protein dominated the adsorption process and the 3-D structure of the protein remained largely undisturbed during adsorption.