PREPARATION, PRESERVATION AND ACTIVATION OF CHLOROPLASTS 1543 



chlorophyll). Holt and French (1946) used ultrasonic waves to disinte- 

 grate spinach chloroplasts, while French and co-workers (1950) pressed 

 chloroplast suspensions through a needle valve under high pressure. In 

 view of the granular structure of leaf chloroplasts (c/. chapter 14 and 37 A) 

 suspensions containing relatively small chloroplast fragments have been 

 sometimes described as "grana preparations" (Warburg and Liittgens 1944, 

 1946; Aronoff 1946). There is, however, no positive evidence that these 

 preparations consisted of pure grana, without admixture of the "stroma." 



Furthermore, we now know (chapter 37A) that many chloroplasts, par- 

 ticularly in algae, contain no grana, but lamellae, running through their 

 whole bodies. 



According to Arnon, and Punnett (section 1), whole chloroplasts differ 

 from fragments in affinity for certain oxidants, pH maximum, and other 

 photochemical characteristics. 



In dealing with unicellular algae (instead of leaves) short-cuts can be 

 used. For example, disintegration and dispersion of unicellular algae can 

 be carried out in one operation by pressing an algal suspension through a 

 needle valve. Arnold and Oppenheimer (1950) found that certain blue- 

 green algae (Chroococcus) can be disintegrated simply by squeezing their 

 suspensions between the cylinder and the barrel of a hypodermic syringe. 

 Some red algae disintegrate spontaneously when placed in distilled water. 

 Punnett and Fabiyi's (1953) method of smashing Chlorella and other uni- 

 cellular algae was mentioned in the preceding section. 



French (1950) pointed out that any procedure adopted for the prepara- 

 tion of chloroplast suspensions, by necessity, must be a compromise be- 

 tween the desire to obtain a material of high activity, and the requirement 

 of purity and uniformity. He recommended disintegrating 100 g. of leaves 

 in 150 cc. water in a Waring Blendor for 0.5-1 min., filtering through cloth, 

 and centrifuging 15 min. at 0° C. at 12,000 g. This throws down a white 

 layer of starch and cell nuclei, and above it a green layer of chloroplastic 

 material. The supernatant is brownish and contains very little chloro- 

 phyll. The green precipitate can be removed by a spatula and resuspended. 



In section (b) we will describe stabilization of photochemical activity 

 of chloroplasts by the addition of methanol. To minimize losses of activity 

 during preparations, it has been suggested that leaf grinding and disper- 

 sion be carried out, from the beginning, in 15% methanol. 



(b) Loss of Activity. Stabilization 



Most troublesome in the quantitative study of the Hill reaction is the 

 rapid deterioration of the active material in storage and in use. 



The rate of deterioration depends on the nature of the preparation, and 

 the results may vary with the type of oxidant used to measure it. Crude 

 suspensions usually deteriorate faster than separated chloroplasts (Arnon 



