1542 PHOTOCHEMISTRY OF CHLOROPHYLL CHAP. 35 



The initial photochemical activity of Hill's suspensions of Chlorella 

 chloroplasts was about one-half of that of a similar suspension from pea 

 leaves; one-half of it was lost after 15 min. (at 15° C). 



Smith, French and Koski (1952) observed spectrophotometrically the development 

 of photochemical activity of washed chloroplast suspensions during the greening of 

 etiolated barley seedlings, and found that it increased proportionally with the chloro- 

 phyll content as the latter grew from 0.2 to 20 relative units. (The significance of this 

 finding depends on whether the reaction was measured in limiting or saturating light; 

 probably the second alternative prevailed and, if so, the result means that the content 

 of the rate limiting enzyme remained, during the greening, proportional to the amount 

 of chlorophyll.) 



3. Preparation, Preservation and Activation of Chloroplast Material 



(a) Preparation of Chloroplast Suspensions 



The rapid loss of photochemical activity of chloroplast preparations 

 has been noticed already by Hill and Scarisbrick (1940). This loss occurs 

 not only during illumination but also in dark storage, even at 0° C. [cf. 

 section (c)]. To obtain preparations of highest efficiency it is therefore 

 important to work fast and at as low a temperature as possible. 



It was described above that macerating at liquid air temperatures produced, in 

 Fager's experiments, chloroplast preparations capable of fixing small amounts of C *02 in 

 light, but incapable of reducing quinone. 



The usual way of preparing chloroplast suspensions is to grind leaves, 

 with or without added liquid (such as an isotonic or hypertonic sugar solu- 

 tion and a buffer to maintain the desired pH) in a Waring Blendor, tissue 

 homogenizer, or another macerator, under cooling (e. g., by mixing the 

 leaves with crushed ice). The cell wall debris, salt crystals and other 

 coarse particles can be removed by filtration through cloth, and slow centri- 

 fuging, e. g., at about 1000 g. The crude product obtained in this way 

 can be used directly (Clendenning and Gorham's "crude suspensions"), 

 or it can be freed from cell sap and soluble plasma proteins by faster and 

 longer centrifuging (<?. g., 5 min. at 20,000 g. ; or 30 min. at 2000 g.), pre- 

 cipitating whole and fragmented chloroplasts (Clendenning and Gorham's 

 "separated chloroplasts") and leaving a yellow or yellow-green supernatant. 

 All these operations should be carried out in the cold. The chloroplast ma- 

 terial can be disintegrated still further, giving colloidal dispersions of vari- 

 ous average particle size. For example, Warburg and Liittgens (1946) ob- 

 tained relatively uniform dispersions by grinding the mixture of whole and 

 broken chloroplasts by means of a glass ball hi a test tube. This product 

 was thrice precipitated in the centrifuge at 2200 g., and reground in the 

 test tube after each precipitation: its analysis showed 9% chlorophyll, 

 3% ash (1 mole P, 0.18 mole Fe, 0.02 mole Mn, 0.01 mole Zn per mole 



