292 



Fulvio Perini 



The pellet was re suspended in the same kind of buffer mixture as 

 above and kept at -20° C till needed and then thaved. The 

 supernatant liquid after centrifugation \ras saved, while the 

 sediment was resuspended in a buffered (pH 6.5) mixture of sodium 

 chloride -phosphate and subjected to the same treatment as above. 

 (Buffered salt mixtures used in this investigation are always 

 0.1 ionic strength in phosphate.) The two supernatant liquids 

 were combined and ammonivim sulfate was added to 0.^ saturation, 

 keeping the pH near neutrality. The sediment was discarded and 

 the supernatant liquid dialysed against phosphate buffer (0.02 M, 

 pH 6.8) and added to a column of diethylaminoethyl-cellulose 

 (DEAE). The portion which was not adsorbed could then be added, 

 after a brief dialysis period against the same buffer, to a 

 carboxymethyl-cellulose (ClAC) . The band from DEAE contained 

 cytochrome 552, which could be eluted with 0.25 ionic strength 

 buffered salt mixture (pH 6.8), and rechromatographed for further 

 purification. Cytochrome 556 was eluted from QAC with O.O5 M 

 phosphate buffer (pH 6.8). Each cytochrome preparation v/as 

 completely free of other haem compo\mds. 



Cytochrome 556 alone could be prepared in higher yields from 

 etiolated and plastid-less mutants by adjusting the pH of the 

 extracting mixture to 7*5 • 



A ratio of 300-400 chlorophyll molecuJ.es to 1 cytochrome 552 

 molec\ile was found even in purified preparations, indicating 

 excellent recovery. The amount of cytochrome 556 was 15-20^ 

 that of cytochrome 552. 



FPNR 



Slightly higher yields of cytochrome 552 were obtained when 

 acetone powders were made, but complications arose in the purifi- 

 cation because a brov/n-red pigment \>ra,s extracted xmder conditions 

 of high salt concentration. The highest yields were at pH 8.0. 

 An acetone powder, made from cells which had been exhaustively 

 extracted for soluble cytochromes, as described, was suspended 

 in ammoniiim sulfate {O.h sat., pH 8.0). The supernatant liquid 

 after centrifugation was then fractionated with ammonium sulfate. 



