1536 PHOTOCHEMISTRY OF CHLOROPHYLL CHAP. 35 



The chloroplast preparations which showed the capacity to fix C*02 in 

 PGA and PA were found to be inactive as sensitizers of the Hill reaction 

 with quinone as oxidant; while chloroplast preparations from the same 

 plant material, made in the usual way, reduced quinone, but fixed no C*02 

 in light. This striking difference could be demonstrated with two batches 

 prepared from the same leaf material by the same procedure, except that 

 one was frozen at — 190° C. and ground while frozen, while the other was 

 cooled on ice and ground at 0° C. 



In a second paper, Fager (1952 2) described attempts to separate the 

 protein responsible for the above-described effect of light on C*02 — fixation 

 by cell free spinach juice. The material, prepared by powdering leaves at 

 — 190° C, was subjected to fractional precipitation with acetone. The 

 abundant precipitation obtained by adding up to 35% acetone fixed no 

 C*02; the precipitate brought down by 35-45% acetone took up C*02 

 mostly in ether-extractable acids, but very little in PGA or PA; Fager 

 suggested that this fraction contained the so-called "malic enzyme," 

 and was responsible for the observations of Tolmach, Ochoa and Vishniac, 

 and Arnon to be described in section 4(f) below. Over one-half of the C*()2 

 fixation in the fractions precipitated by 45-60% acetone was in PGA and 

 PA. The 0*02 fixation in this fraction (in darkness and in light) could be 

 increased 3-5 fold by 2 X 10'^ M cysteine M'ithout changing the C* dis- 

 tribution significantly. 



The enhancing effect of glyoxal on C* fixation, reported above for crude 

 juice was not found with the separated protein fraction; instead, there 

 was an inhibition (both in light and in the dark). 



TPN (1.3 X 10-^ M), or DPN (5 X 10-=* M), had no effect on light 

 induced C*02 uptake by chloroplast preparations, but increased the fixation 

 in the dark. "High energy phosphate" (ATP) caused inhibition instead of 

 the anticipated enhancement. The strongest effect of ATP was on fixation 

 in "neutral" products; this may indicate draining-off of PA (and, by 

 equilibration, also of PGA) through accelerated conversion into malic acid, 

 causing a decline in the reduction of PGA to triose. In agreement with 

 this picture, arsenate (which is known to interfere with the utilization of 

 high energy phosphate) had an effect on the C*02 fixation which was op- 

 posite to that of ATP: it enhanced slightly the total fixation, and enhanced 

 strongly the fixation of C* in neutral compounds (sugars?). 



The above-mentioned experiments by Tolmach, Vishniac and Ochoa 

 and Arnon, in which carbon dioxide was drawn into the Hill reaction by 

 supplying DPN or TPN as intermediate oxidants, will be dealt with in sec- 

 tion 2(f) below. 



