Tryptic Hydrolysate o/Rhodospirillum Rubrum Cytochrome c 365 



RESULTS 



In the hydrolysate of the haemin-free oxidized polypeptide (prepared from 

 the haemopeptide as described in the foregoing section) ten different ami no- 

 acids were identified by paper chromatography — alanine, aspartic acid, 

 cysteic acid, glutamic acid, glycine, histidine, leucine, lysine, phenylalanine 

 and threonine. The lysine present in the polypeptide may be expected to be 

 the C-terminal residue of the amino-acid sequence, since the polypeptide 

 has been formed by the action of trypsin which is known to split bonds 

 involving the carboxyl groups of lysine and arginine residues. The N- 

 terminal residue was shown to be cysteic acid. After dinitrophenylation of 

 the polypeptide and subsequent hydrolysis, however, the hydrolysate was 

 found to contain, besides DNP-cysteic acid, free cysteic acid as well. This 

 finding is accounted for by the presence in the polypeptide of two cysteic acid 

 residues, one of which is terminal and the other is not. 



Chymotryptic hydrolysis of the polypeptide (Table 1) gave rise to three 

 main split products which were separated from each other by high-voltage 

 electrophoresis at pH 2 and by paper chromatography in butanol-acetic acid : 

 (I) CyS03H-[Leu, Ala, CySOgH, His], (II) Thr-Phe, and (III) Asp-Glu- 

 [Gly, Ala, Asp]-Lys. Split product (I) with N-terminal cysteic acid, must 

 be derived from the N-terminal side of the polypeptide chain and the lysine- 

 containing peptide (III) from the C-terminal side. Thr-Phe, then, must be 

 located between these two. After dinitrophenylation of peptide III and 

 partial hydrolysis (with cone. HCl at 37°C, for 8 days) two ether-soluble 

 products were separated and characterized, DNP-Asp and DNP-Asp-Glu, 

 which demonstrates that glutamic acid must adjoin the N-terminal aspartic 

 acid residue. 



In the subtilisin hydrolysate of the polypeptide (Table 1) five products 

 were present in relatively high concentration : CySOgli-Leu-Ala, CySOgH- 

 His, Thr-Phe, [Asp, Glu, Gly, Ala], and Asp(NH2)-Lys. Among the split 

 products obtained in small amounts there were two tripeptides containing 

 liistidine: Ala-[CyS03H, His] and CyS03H-[His, Thr]. Other minor 

 components of the hydrolysate were shown to be [Gly, Ala], [Asp, Glu, Gly], 

 [Thr, Phe, Asp, Glu, Gly] and free alanine. 



From the findings presented above it may be concluded that the amino acid 

 sequence in the haemin-free oxidized polypeptide is 



1 2 3 4 5 6 7 8 9 10 11 12 13 



CySOsH— Leu— Ala— CySOsH— His— Thr— Phe— Asp— Glu— Gly— Ala— AspCNHg)— Lys 



The results obtained leave it undecided whether residues 8 and 9 are free 

 aspartic acid and free glutamic acid or whether these amino acids are in the 

 amide form, as asparagine and glutamine residues. 



The two cysteic acid residues contained in the sequence have arisen from 

 and taken the place of two haem-bound cysteine residues present in the 



