1544 PHOTOCHEMISTRY OF CHLOROPHYLL CHAP. 35 



and Whatley 1949), probably due to interaction with the constituents of 

 the pkisma or cell sap. On the other hand, fine dispersions of separated 

 chloroplasts often prove less staVjle than preparation containing whole 

 chloroplasts or large chloroplast fragments — perhaps because of increased 

 contact with the medium. The same reason may explain why shaking of 

 chloroplast preparations has been found to accelerate the loss of activity. 

 This was reported by Hill and Scarisbrick (1940) and confirmed by Amon 

 and Whatley (1949), who found that chard chloroplasts, if shaken, in the 

 absence of oxidants, at 15° C. (in an atmosphere of not especially purified 

 nitrogen) lose as much as 50% of their activity in 20 min. Not unrelated 

 may be the observation of Holt, Smith and French (1951) that activity is 

 lost faster in dilute than in dense chloroplast suspensions. 



The rate of "dark" deactivation is, as expected, a function of tempera- 

 ture. Thus, Warburg and Liittgens (1946) found that heating for 10 min. 

 to 40° C. reduced the rate of oxygen production by chloroplasts (with 

 quinone as oxidant) by 50% and that heating for 10 min. to 50° C. brought 

 it to a standstill. Chlorophyll appeared unchanged, and no coagulation of 

 the grana was noticeable after heating. According to French, Anson and 

 Holt (unpublished) deactivation is accompanied by a loss of fluorescence; 

 however, the decay of fluorescence intensity is much slower than the loss of 

 photochemical activity, e. g., 30% loss after 15 min. at 35° C, at which 

 time photochemical activity has declined by 80% (compare the results of 

 Zubkovich et al. below). 



The results of French, Anson and Holt indicated average temperature 

 coefficient Q^ = 6.4 — a very high value, which points to the denaturation 

 of a protein as mechanism of deactivation. French and Holt (1946) had 

 found a smaller temperature coefficient of deactivation — about 3.9 (3- 

 15° C). 



Arnon and Whatley (1949) found that heating of chloroplasts to 55° 

 for 5 min. inactivated them completely, and pointed out that the thermo- 

 lability of the "Hill enzyme" appears much greater than that of the poly- 

 phenol oxidase, whose activity in chloroplasts also was studied by Arnon 

 (c/. section 5(c) below, and chapter 37A) ; the latter activity remained un- 

 changed, after 5 min., even at 75° C. 



Although cooling of chloroplast suspensions slows do^vn the deteriora- 

 tion, Milner, French et al. (1950) found freezing (of whole or dispersed 

 chloroplasts) to reduce their activity; they also noted that frozen suspen- 

 sions were difficult to resuspend. "Lyophilized" material {i. e., material 

 dried and preserved in vacuum in the cold) retained 90% of the original 

 chloroplast activity after 48 hrs. (French, Holt, Powell and Anson, 1946). 

 Dry powder, obtained from lyophilized leaves, and stored for a week, lost 

 20% of its activity at 5° C, and 60% at 25° C. Clendenning and Gorham 



