Publisher Summary This chapter describes purification method and physicochemical data for hexadecaheme cytochrome c or high molecular weight cytochrome c (HMC) from Desulfovibrio vulgaris ( D. vulgaris ) Miyazaki (DvM) and D. vulgaris Hildenborough (DvH). For purification of hexadecaheme cytochrome c from DvH, wet cells are suspended in 2–2.5 vol of the 25 m M buffer solution, and disrupted with an ultrasonic disintegrator for 10 minutes. The cells should be stirred and cooled by ethanol-dry ice during sonication. The cytochrome is eluted by a linear gradient of phosphate from 25 to 300 m M with a total volume of 1000 ml. The HMC is eluted at about 100 m M phosphate. The molecular weight of HMC from DvH is determined by amino-acid sequence analysis, whereas that of HMC from DvM is estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the DvH protein calculated from the elution volume on gel filtration is slightly larger. The total iron content of these cytochromes c is analyzed by atomic-absorption spectroscopy using a standard curve obtained from properly diluted iron ammonium sulfate solutions.