PREPARATION, PRESERVATION AND ACTIVATION OF CHLOROPLASTS 1559 



mentation experiments of particles falling into discrete groups; rather 

 the dispersion was completely heterogeneous, with random distribution of 

 particles. About 25% sedimented in 15 min. at 20,000 g.; the rest in 10 

 min. at 60,000 g. About 90% had a molecular weight of 6-7 X 10«, 10% 

 seemed to be smaller. Under the electron microscope, most particles ap- 

 peared to be <2 m/x in size; however, a number of particles 8 m^ in size 

 were observed, and some agglomerates of the 8 mju particles, about 25 m/x 

 in size, also were noted. Such groups had not been noticeable in sedimenta- 

 tion experiments, which were, however, made with a different batch of 

 chloroplasts. 



Since the chlorophyll-bearing material of chloroplasts contains about 

 25% lipides, the effect of lipide-dissolving agents was studied by Milner 

 et al. (1950), They shook a chloroplast dispersion with petroleum ether, 

 extracting 2-3% of the total lipides. This caused the loss of up to one 

 half of photochemical activity, which could not be restored by KCl. How- 

 ever, when the lipide extract was evaporated and the residue, dissolved 

 in a little ether, returned to the depleted dispersion, the activity was re- 

 stored, and the material could be still further activated by chloride. The 

 same effect could be obtained more simply by adding a little ether to the 

 aqueous phase after the petroleum ether extraction, and removing the ether 

 again by evaporation in vacuum. It thus seems that the effect of petroleum 

 ether (as well as that of salts) is associated with changes in the size and 

 colloidal structure of the active particles rather than with the removal (or 

 addition) of specific chemical constituents. After the larger and more ac- 

 tive particles had been broken into smaller less active fragments, addition 

 of salts causes them to coagulate again and thus restores activity. Pe- 

 troleum ether, by removing some of the hpoid "glue," may cause the par- 

 ticle to fall apart into fragments. Addition and subsequent evaporation 

 of ether may cause redistribution of the remaining lipides, bringing them 

 from the interior to the surface, and thus cause renewed coagulation to 

 larger units. 



French and Milner (1951) mention that some detergents were found to 

 split chloroplast particles into smaller fragments without destroying their 

 photochemical activity, but this method has not proved useful for prepara- 

 tory purposes. 



All these experiments, however inconclusive, add to the impression that 

 the photocatalytic activity of chloroplast preparations, as determined by 

 the Hill reaction, is associated, not with one or two specific enzymatic 

 components, but with a more or less complex structure which cannot be 

 broken into small units without losing its activity. Perhaps this could be 

 explained by the assumption — derived from kinetic experiments on photo- 

 synthesis and the Hill reaction in whole cells — that one of the enzymes 



