PREPARATION, PRESERVATION AND ACTIVATION OF CHLOROPLASTS 1549 



Colloidal chloroplast dispersions containing 15% methanol can be 

 stored at —5° C. without freezing; they then retain 50% of their initial 

 activity after 10 days. Ethanol in equimoleciilar quantities had the same 

 stabihzing effect at 5° C; but a lesser one at higher temperatures. 



The more concentrated dispersions kept better in 15% methanol than 

 the dilute ones. The loss of activity is fastest in preparations the absolute 

 efficiency of which is high (fig. 35.10) ; in line with this, the effect of meth- 

 anol is much stronger in colloidal chloroplast dispersions than in crude sus- 

 pensions of chloroplast fragments. 



The most effective stabilization was achieved by macerating the leaves 

 in 15% methanol, maintaining this composition of the medium in all sub- 

 sequent operations, and keeping the temperature down to —5°. Hy- 

 drosols obtained in this way retained up to 50% of the activity of crude 

 suspensions — a much smaller loss than is commonly suffered by dispersion 

 in the absence of methanol. 



A new, promising approach has been opened by McClendon (1944) and 

 McClendon and Blinks (1952) Mith the finding that chloroplasts of red 

 algae can be prevented from losing the phycobilins, and their Hill activity 

 preserved, by preparing them in high -molecular solutions, e. g., containing 

 0.4-0.8 g./ml. of Carbowax 4000 (or 8000). 



The deterioration of chloroplast material is accelerated by illumination, 

 particularly in the absence of oxidants (Aronoff 1946^; Milner, French et 

 al. 1950; Arnon and Whatley 1949''^). To what extent photochemical 

 deactivation is prevented when appropriate oxidants (such as ferricyanide 

 or quinone) are present, so that the Hill reaction can take place, is not clear. 

 A protective effect of a sensitization substrate on the sensitizer is a well- 

 knowai phenomenon {cf. Vol. I, chapter 19). Warburg and Luttgens 

 (1946) noted it with chloroplasts and quinone, but Holt, Smith and French 

 (1950) reported that the several chloroplast fractions obtained by frac- 

 tional centrifugation of a colloidal suspension of chloroplastic matter, 

 lost their photocatalytic capacity in light, in presence as well as in the ab- 

 sence of the ferric oxalate-ferricyanide mixture. 



Spikes et al. (1950) gave a curve for the inactivation of spinach chloro- 

 plasts for the photoreduction of ferricyanide by prolonged illumination. 



(c) Activation by Anions 



Warburg and Luttgens (1944 2) first noted that after the capacity of 

 spinach or beet chloroplast suspensions to evolve oxygen with quinone 

 has been lost by dialysis, it could be restored by potassium chloride. They 

 referred to the chloride ion as a "co-enzyme" of the Hill reaction, and found 

 it to play a similar role also when ferric salts were used as oxidants instead 

 of quinone. However, this stimulation is not specific for chloride. War- 

 burg and Luttgens (1946) themselves found that bromides had a simi- 



