1556 PHOTOCHEMISTRY OF CHLOROPHYLL CHAP. 35 



sediment was 3.4 times more active per unit N weight than the original ma- 

 terial, while the supernatant was only one half as active. The highest 

 ratio of active material to total protein was thus found in the precipitate 

 from the third centrifugation. However, an increase of specific activity 

 by a factor of 3 or 4 is disappointing compared to the results obtained in 

 experiments with water-soluble enzymes. Fractional coagulation by acid 

 of the same (supersonically dispersed) chloroplast material produced, in 

 the range pH 5.3-6.0, a series of precipitates which, when resuspended at 

 pH 6.5, exhibited no marked difference in photochemical activity. Total 

 precipitation at pH 4.6 led to complete loss of activity. Salting-out, e. g., 

 with 30-40% Na2S04, also produced no marked enrichment. Detergents, 

 such as dupanol and vetanol, which are of help in solubilization and frac- 

 tionation of some proteins, destroyed photochemical activity. French 

 and Milner (1951) reported that addition of 1 volume saturated (NH4)2S04 

 to 4 volumes of a dispersion of chloroplast material precipitated nearly all 

 green material, leaving in solution some colorless proteins^not more than 

 Vs of the total protein content. The resuspended precipitate showed only a 

 slight photochemical activity, whether related to chlorophyll or to nitrogen 

 content. A green precipitate could also be obtained by acidification, but 

 its activity was similarly poor. Precipitates obtained by addition of salts 

 to methanol-stabilized dispersions are more active than the starting ma- 

 terial, but this appears to be due to activation by coagulation rather than to 

 enrichment (cf. below). 



Attempts to coagulate dispersed chloroplast material with alcohols 

 were unsuccessful; even in 95% ethanol, precipitation occurred only after 

 several hours; the alcoholic solution was photochemically inactive. Simi- 

 lar experiments with methanol later led French and co-workers to the dis- 

 covery of the stabilizing influence of methanol on chloroplastic matter, 

 which was described in section (c). 



Adsorption of the chloroplast dispersion on a column (Supercel) at pH 

 5.5 gave, according to Holt, Smith and French (1950), a green band, which 

 could be partially eluted in neutral buffer or distilled water. However, the 

 easily eluted fraction had the same activity as the noneluted residue. Ac- 

 cording to French and Milner (1951), fractional adsorption on Fuller's 

 earth or charcoal leaves, as the last, unadsorbed residue (a few % of the 

 total) a dispersion that may be 2-3 times more active than the original ma- 

 terial. Aronoff (1946) found that when "grana" from spinach chloroplasts 

 were precipitated at 6700 g., the remaining supernatant, which was clear but 

 showed a Tyndall cone, had a remarkably high photochemical activity with 

 quinone (about ten times that of the precipitate, related to equal chloro- 

 phyll amounts) ; its activity remained almost constant for about 2 hrs. at 19 

 klux. Dried in a lyophylizer and redissolved, the product kept its activity; 



