426 



LIGHT AND LIFE 



cold 80 per cent acetone, a behavior that is in marked contrast to 

 that ol the initodionchial cytochromes. The extraction of freshly pre- 

 pared chloroplasts by cold 80 per cent acetone removes chlorophyll 

 and allows quantitative spectroscopic and spectrophotometric ob- 

 servation of the residue. Such an extraction of chloroplasts changes 

 the oxidation state of the cytochrome components, and cytochrome / 

 becomes fiUly reduced while the highly autoxidizable /^c remains 

 oxidized. 



A spectrum showing the difference in absorption between 80 per 

 cent acetone-extracted chloroplasts and similar chloroplasts reduced 

 with hydrosidfite is shown in Fig. 2. This spectrum clearly shows a 

 single cytochrome component, a component whose low-temperature 

 absorption maxima do not correspond to any of the plant mitochon- 

 drial or microsomal cytochromes (cf. Bonner, 2) . The spectrum 

 shown in Fig. 2 is that of cytochrome h^^. No spectral changes have 

 been observed with hydrosulfite-reduced b^ preparations in the pres- 

 ence of cyanide, azide, carbon monoxide, or antimycin A. The lack 

 of any effect of carbon monoxide on the spectrum indicates a mini- 

 mum of denaturation during the acetone treatment. Frequently, 

 however, the a-band of cytochrome ^^ in the acetone-extracted prepara- 



Acetone extracted Spinach Chloroplasts 



557 



( — ) untreated -S2Q1 



( ) oxidized- S2Q1 



I 



AOD = OIO 



i 



I 



A0D=0.02 



i 



— I — I — 1 — I — I — 1 — 

 400 450 



550 



A(nn>i) 



Fig. 2. Low-teinperatiire difTcrencc spectra of 80 per cent acetone-extracted 

 spinacli cliloroplasls. 1 lie solid line, untreated - — liydrosuKite-rednced chloroplast 

 residue, shows the spectrum of cytochrome fco only. The dotted line, oxidized 

 (chlorox) — hytlrosiillite-rcdiued < liloroplast residue, shows, in addition to /'„. the 

 peak, at 551 m^u, of cytochrome /. 



