THE CHLOROPLAST REACTION 27 1 



is an essential element of his scheme. Kamen referred to Hill's early papers, while 

 I am referring to his later work. 



Whittingham : I don't think he considered oxygen as essential. 



Amen: This is a good point to have cleared up. The second question is this: 

 It is rather important, insofar as photosynthesis by chloroplasts is concerned, to 

 make sure where the cji^ochromes are localized in the green leaf. Have you any 

 comment on that? Is there any evidence which would compel one to the conclusion 

 that they are localized in the chloroplasts? 



Whittingham : If you ask me for quantitative data, I have none. But I am quite 

 certain that they are located, at least in part, in the chloroplasts. 



Amon : Are the cytochromes obtained from the chloroplasts after separating the 

 latter from the cell? 



Whittingham : Yes. 



Chance : I want to make a short comment on Dr. Kamen's stimulating specu- 

 lation concerning direct excitation of cytochrome in Rhodospirillum ruhrum. 

 It seems that the spectra of the forms involved in photosynthesis are very nearly 

 identical with those involved in respiration. Therefore, if an excited state does 

 exist, it seems to have no characteristic absorption spectrum — because the cyto- 

 chrome molecules affected by light have the same bands as those affected by oxygen. 

 Perhaps, excitation does not cause a measurable band shift: in the respiratory 

 oxidative phosphorj-lation system, some of the enzymes probably exist in "high- 

 energy" forms (at least, they can form high-energy phosphate bonds); yet, in this 

 case, we do not detect any unusual spectral bands. 



Kamen : This work emphasizes the need for reexamination of the spectra of the 

 purified pigments. Up to now, I believe, nobody has ever had such pure forms to 

 play with. In the particular case of bacterial cytochromes, the region where you 

 would expect significant absorption bands would be in the near infrared. It is a 

 matter of some difficulty to get cytochrome preparations so pure that you have 

 the right to believe, when you measure the absorption spectrum, that a certain 

 characteristic band can be assigned to this compound. In the case of cytochrome/, 

 it has never been obtained in pure enough form so you could look for the band at 

 750 m;u, which one would expect to exist. 



Amon : I would like to offer a comment on Ohmura's experiments discussed by 

 Dr. Whittingham. This also pertains to the question raised by Dr. Vishniac the 

 other day. I think it is a simple yet important matter which greatly complicates 

 discussions of chloroplasts and oxidative phosphorylation. Chloroplasts and mito- 

 chondria are both present in green cells, chloroplasts being much larger in size. 

 Any technique used so far for breaking leaves will inevitably break some chloro- 

 plasts. Differential centrifugation will separate two fractions from the leaf. At 

 least two. One would consist of whole chloroplasts and the other of broken chloro- 

 plasts plus mitochondria. The latter fraction is often referred to as "chloroplast 

 fragments." This combined fraction is thus a mixture of broken chloroplasts and 

 mitochondria. From this mixture the two kinds of particles are no longer sepa- 

 rable on the basis of size. 



I will now state the point which is documented elsewhere (Biochimica et Bio- 

 physica Acta (13)). If the discussion allows enough time we may bring the 

 evidence in later. We find that photosynthetic phosphorylation, independent of 



