364 S. Paleus and H. Tuppy 



MATERIAL AND METHODS 

 Preparation of Rhodospirillum Rubrum Cytochrome c 



Lyophilized bacterial cells were extracted with H2SO4 at pH 4. From the 

 extract, the cytochrome c was obtamed by fractionation with ammonium 

 sulphate (KeiUn and Hartree, 1937), dialysis and lyophihzation. A sample 

 thus prepared was kindly put at our disposal by Professor M. D. Kamen. 

 It was further purified by chromatography on a column of CM-W cellulose 

 (1-6 X 10 cm) prepared according to Peterson and Sober (1956) and buffered 

 with 0-015 M ammonium acetate of pH 6-5. The preparation of cytochrome 

 c (470 mg) was dissolved in 2 ml of this buffer, reduced by the addition of 

 solid Na2S204 and transferred to the column. A dark brown, fast movmg 

 band separated from the red cytochrome band which moved slowly, if at all. 

 When the buffer of pH 6-5 was replaced by 0-05 m ammonium acetate of 

 pH 7-5, the red material started moving down the column and divided into 

 three bands. The main fraction (R = 0-4, E270/E551 = 0-96) yielded 71-5 mg 

 of dry, salt-free cytochrome c which was used for all subsequent experiments. 



Preparation of the Haemopeptide and Removal of the Haem 



After hydrolysis of the bacterial cytochrome c with trypsin, the haemo- 

 peptide formed could be precipitated from the digest with ammonium 

 sulphate and purified by adsorption on talc and by column partition chro- 

 matography as described previously for other haemopeptides (Tuppy and 

 Bodo, 1954; Tuppy and Paleus, 1955). The haemin part of the haemo- 

 peptide was split from the peptide moiety using silver sulphate in acetic acid 

 solution accordmg to the method of Paul (1949; 1950). Performic acid was 

 used to oxidize the thiol groups of cysteine residues to sulphonic acid residues. 

 The haemin-free oxidized peptide obtained was essentially homogeneous as 

 determined by electrophoresis on paper in a formic acid-acetic acid buffer 

 of pH 2 (Werner and Westphal, 1955). 



Enzymic Hydrolyses 



The crystalline chymotrypsin used was purchased from the Worthington 

 Biochemical Corporation. The subtilisin was a gift from Ing. M. Ottesen, 

 Carlsberg Laboratory, Copenhagen. Digestions were carried out at pH 7-5 

 to 7-8 for 20 hr at 25°C (with chymotrypsin) or for 24 hr at 37°C (with 

 subtilisin), the ratio between enzyme and substrate (w/w) being about 1: 15. 



End Group Determinations 



For the characterization of N-terminal amino-acid residues the DNP 

 method (Sanger, 1945; see also Sanger and Thompson, 1953) was employed. 

 C-terminal residues were determined using the hydrazinolysis procedure of 

 Akabori, Ohno and Narita (1952). 



