Chemical Modification of Acidic Residues of Cytochrome c with Safranin: pH Effect on Structure and Function of the Modified Protein

Utilizing cytochrome c (Cyt c), we studied the covalent attachment of safranin O to a protein could affect the protein properties. The effect of covalent attachment of safranin to Cyt c was explored using spectroscopic techniques, which revealed changes in the secondary and tertiary structure of the protein. Using Trp 59 fluorescence emission (lambda(em)=355 nm) and safranin fluorescence profile (lambda(em)=587 nm), we investigated the changes in the structure of the heme moiety and the binding of safranin molecules to the Cyt c protein. Far-UV CD spectroscopy results showed that modification significantly reduced the alpha-helix content. Studies showed that the modification causes the prevention of thermal aggregation of the protein at 65 degrees C and decreases in the peroxidase activity of the protein. Investigating of the pH effect revealed that although the Cyt c modification reduced peroxidase activity, the pH shift to pH 6.0 could increase the peroxidase activity.