The absorbance coefficient of beef heart cytochrome c1

Isolated cytochrome c1 contains endogenous reducing equivalents. They can be removed by treating the protein with sodium dithionite followed by chromatography. This treatment has no effect on the reaction with cytochrome c, nor does it alter the optical spectrum, or the polypeptide or amino acid composition of the protein. Both the titration of dithionite-treated ferrocytochrome c1 with potassium ferricyanide and the anaerobic titration of dithionite-treated ferricytochrome c1 with NADH in the presence of phenazine methosulphate lead to the same value for the absorbance coefficient of cytochrome c1: 19.2 mM-1 . cm-1 at 552.4 nm for the reduced-minus-oxidised form. This value was also obtained when the haem content was determined by comparing the spectra of the reduced pyridine haemochromes of cytochrome c and cytochrome c1. Comparison of the optical spectra of cytochrome c and cytochrome c1 by integration shows equal transition moments for the transitions in the porphyrin systems of both proteins. A set of equations with which the concentration of the cytochromes aa3, b, c and c1 can be calculated from one reduced-minus-oxidised difference spectrum of a mixture of these proteins.