374 R. LUMRY AND J. D. SPIKES 



EXPERIMENTAL DETAILS 



Both tlic nuinometric and potentiometric techniques were used 

 for determining Hill reaction velocities as previously described (1). 

 The potentiometric technique was preferred because of its greater pre- 

 cision, speed of measurement, and applicability at low electron 

 acceptor (oxidant) concentration, as will be discussed later. Most of 

 the experiments were carried out with washed sugar beet, Swiss 

 chard, or rhubarb chard chloroplast fragments prepared and stored as 

 previously described (2). Chloroplast fragments stored in 0.5 M su- 

 crose at -85°C. did not show any loss of activity or change in other 

 properties for periods of at least one year. If stored samples of the 

 same batch of material were removed at intervals and thawed accord- 

 ing to a precise time schedule under conditions of controlled tempera- 

 ture, it was found over extended periods that Hill reaction velocities 

 were obtained which agreed within two or three standard deviations 

 of the method (1.5% to 3%) used for rate measurements. At the 

 present time it is of importance to do all the experiments in a series 

 with samples from the same batch of chloroplasts, since it has not 

 been found possible to prepare different batches of material with iden- 

 tical properties. One or more preparative variables are not yet under 

 control. Plant growth conditions also have a marked effect on chloro- 

 plast properties. For example, it has been suggested by preliminary 

 experiments in this laboratory that chloroplasts from sugar-beet 

 plants grown under short photoperiods show a large decrease in the 

 Hill reaction rate per chlorophyll molecule at light saturation. It may 

 be that natural inhibitors for one or more of the subprocesses of the 

 Hill reaction are formed under certain environmental conditions and 

 that these inhibitors cannot be removed by the usual washing tech- 

 niques. Alternatively, and more probable, necessary cellular partici- 

 pants in the Hill reaction may be partially remo\'ed or chemically 

 altered (as by oxidation) during the preparatory process, and they 

 may be reduced in concentration as a result of particular combina- 

 tions of growth conditions. 



Chloroplasts prepared under nitrogen from plants (especially sugar 

 beet) grown under mid-summer conditions exhibit up to a 50% 

 increase in Hill reaction activity after being incubated for a short time 

 at 20° to 25°C. (3). Yahics of QoT of over 3000 ha\e been obtained 

 with sugar-beet chloroplasts prepared in this manner with ferricyanide 

 as the oxidant. This activation phenomenon is confined to the rates 



