Cytochrome Cg 409 



Once the bacteria have grown (and their cultivation has been facilitated 

 in recent years) cytochrome c^ may be prepared in a high state of purity very 

 simply. A procedure occupying three to four days is given below : 



Day 1 : Squirt a cream of living or vacuum-dried D. desulphuricans 

 (about 30 g dry wt) into 1 1. of boiling KH2PO4 (0-5%, pH 7). Allow 

 to boil for 3 min, cool and add (NH4)2S04 to 0-75 saturation. Centrifuge 

 (15 min at 2500 rev/min) and collect 'intematant' red solution by pouring 

 carefully through glass wool. Adjust to pH 2-6 with 2 N H2SO4 (check pH 

 after 60 min) and store overnight at 4°C. 



Day 2 : Centrifuge (20 min at 2500 rev/min), dissolve residue in 0-25 N 

 NH4OH, and store as fraction A. Adjust supernatant fluid to pH 2-6, stir 

 in Whatman 'Standard' powdered cellulose and pour into a chroma- 

 tography tube to form a cellulose column (about 70 ml wet cellulose). 

 Pass whole volume of effluent once through the column; do not wash 

 the column but elute red material directly with 0-25 N NH4OH. Combine 

 eluate with fraction A and dialyse overnight. 



Day 3 : Stir dialysed red solution with fine Amberhte IRC-50 (3-20 min 

 settling fraction) in the ammonium form. Pour to form a column (about 

 20 ml wet resin), wash pink resin with distilled water (20 vol ; discard 

 denatured material which is not held by the resin) and elute with 0-25 N 

 NH4OH. Dialyse and freeze dry product. Yield: 15-20 mg native 

 cytochrome c^. 



The product is at least 94% pure and is a deep red powder. It is the 

 ammonium salt of cytochrome Cg and has the following properties (Table 1). 



Table 1. Properties of cytochrome c. 



These data indicate a molecular weight approximating to that of muscle 

 cytochrome c and suggest that the molecule possesses two haematin groups 

 instead of one. The haematin groups are stable to boiling and to acid acetone 

 but are removed by reagents which split thio-ethers (acid silver sulphate, 

 mercury amalgam). The degradation products are spectroscopically closely 

 similar to those obtained from cytochrome c (Table 2). 



