Infrared spectroscopic analysis of salt bridge formation between cytochrome b5 and cytochrome c

The infrared spectrum of a solution of a protein contains bands due to both the peptide backbone and the amino acid side chains. Generally, the bands due to the peptide backbone, between 1700 and 1600 cm-1, are analyzed to determine the secondary structure of the protein; the bands due to the amino acid side chains, between 1600 and 1500 cm-1, are largely ignored. When cytochrome b5 is mixed with cytochrome c, under conditions that favor ionic complex formation, changes are seen in protein secondary structure and also in a band at 1562 cm-1. The band at 1562 cm-1 is due to the side-chain carboxyl of Glu residues, rather than those of Asp residues that show a band at 1585 cm-1, and the changes in the band at 1562 cm-1 indicate that when the two proteins interact, three ionized carboxyl groups of Glu become involved in salt bridge formation. This result is identical with that obtained by previous theoretical studies and suggests that infrared spectroscopy may be a rapid and quantitative method for the study of ionic interactions between proteins.