PREPARATION, PRESERVATION AND ACTIVATION OF CHLOROPLASTS 1553 



Fig. 35.13 shows that, according to Milner, Koenig et al. chloride is 

 not the best "reactivator" of chloroplast dispersions; it is exceeded by 

 sulfates (found inactive by Warburg and Liittgens, and Arnon and What- 

 ley). However, the activating effect of (NH4)2S04, for example, was ex- 

 tremely short-lived, and was soon replaced by inactivation. 



Milner, French et al. also investigated the effect of some cations, vnih. 

 and without addition of "versene" (ethylene diamine tetra-acetic acid, a 

 chelating agent). In these experiments, versene, which is an acid, was 

 neutralized by KOH to 7>H 6.5. The potassium salt of versene alone had a 

 marked reactivating effect — probably similar to that of other potassium 

 salts. Addition of versene to a divalent salt solution, which in itself had 

 an activating effect, produced no important changes; but its addition to 

 a 5 X 10 ~^ M CUSO4 solution (which, by itself, was strongly poisonous) 

 leads to effective reactivation. (No reactivation was possible in 3 X 10 ~^ 

 M CUSO4.) Versene also reactivated dispersions inactivated by HgCb. 

 Ferrous sulfate had no activating effect. 



Gerretsen (195O0 described a strong enhancing effect of Mn + + ions on 

 the change of redox potential, which occurs in crude chloroplast suspensions 

 from Avena upon exposure to light (without added oxidants) under aerobic 

 conditions. He suggested an interpretation in terms of manganous ions 

 playing a role in the enzymatic mechanism of the liberation of oxygen. 



Clendenning and Gorham (1950^) found that the initial rate of the Hill 

 reaction with quinone was two or three times higher with crude suspensions 

 than with separated chloroplasts, and that repeated washing reduced it 

 still further; only about }4> of the loss caused by washing could be restored 

 by the addition of potassium chloride. 



Gorham and Clendenning (1952) investigated the effect of anions on 

 chloroplast activity in more detail, and arrived at conclusions which dif- 

 fered from those of both Warburg and Arnon. They grew spinach, Swiss 

 chard, beet and millet in ordinary soil, and in chloride-containing and 

 chloride-free nutrient solutions. Chloroplasts prepared from these plants 

 were analyzed for chloride content, and their photochemical activity studied 

 with various oxidants. The activity of crude suspensions was about the 

 same in the three kinds of plants. Separation from cell sap and cytoplasm 

 affected strongly the activity of preparations from plants grown in nutrient 

 solutions — either with or without chloride — but material from soil grown 

 plants could be washed extensively without inactivation. The activity 

 lost by washing could be restored by anions, the order of effectiveness being 

 Cl~> Br~ > CN~ > Hoagland's salt mixture (nitrate -{- sulfate -f phos- 

 phate) > NO3- > I- > F- > CIO3- > BrOs- > IO3- > SO4— (no effect). 

 Ascorbate and thiocyanate caused inhibition. Maximum restoration 

 could be achieved with [Cl~] > 3.3 X 10~^ mole/1. Pre-illumination ex- 



