PREPARATION, PRESERVATION AND ACTIVATION OF CHLOROPLASTS 1555 



also includes a large amount — of the order of one third of the total — of 

 lipoidic materials, including carotenoids and phospholipides (c/. Vol. I, 

 chapter 14). It is likely — although it cannot be proved at present — that 

 at least some of these materials are essential and that any fractionation 

 which leaves them behind will lead to a loss of photocatalytic activity. 

 The Hill reaction, although it must be simpler than complete photosynthe- 

 sis, still is a complex process, partly photochemical and partly enzymatic. 

 Its occurrence may be bound to the preservation of a definite pattern of 

 pigments, prosthetic groups of one or several enzymes, proteins and lipoids. 



The "photocatalytic activity" of chloroplastic material usually is de- 

 fined as the (initial) rate of the Hill reaction with a given oxidant, related 

 to unit amount of chlorophyll. This activity can only be increased if : (a) 

 the activity of the preparation as measured before fractionation was limited 

 by an enzymatic component (i. e., if the measurement was made in satu- 

 rating light, and with saturating concentration of the oxidant) ; and (b) 

 if a fraction can be obtained in which this limiting enzyme is enriched in 

 relation to chlorophyll. 



If chlorophyll and the limiting enzyme are enriched together, activity 

 tests related to unit chlorophyll content will show no change at all. It is, 

 therefore, desirable to check the results of fractionation also by measuring 

 the photochemical activity per unit mass (or per unit amount of nitrogen, if 

 only protein fractionation is considered). 



Following is a brief review of the chloroplast fractionation experiments. 



French, Holt, Powell and Anson (1946) made a preliminary study of 

 the effects of various treatments (freezing, lyophylizing, disintegration by 

 supersonic waves, and centrifugation) on the photochemical activity of a 

 chloroplast suspension. They considered the results as indicating the 

 possibility of successful fractionation of the chloroplast material and con- 

 centration of active ingredients. 



Holt, Smith and French (1950) (c/. Holt and French 1949) attempted 

 fractionation by high-speed centrifugation, acid coagulation, salting-out, 

 and adsorption and elution. They used a spinach chloroplast suspension 

 in 10% propylene glycol; it was disintegrated by supersonic vibrations. 

 When the resulting colloidal dispersion was fractionated by centrifugation, 

 the first precipitate, obtained after 5 min. at 7600 g. and presumably com- 

 posed of the largest particles, was found to contain one half of the total N 

 present; its activity (per unit weight of N) was lower than the average, 

 while that of the supernatant was twice as high. Centrifuging the super- 

 natant for 10 min. at 8600 g. precipitated one half of the remaining nitrogen, 

 the activity per unit N remaining the same in the precipitate and in the 

 supernatant. The latter was centrifuged 30 min. at 3200 g., again pre- 

 cipitating about one half of the remaining N content. In this case, the 



