THE CHLOROPLAST 101 



This method of obtaining a pigment-complex is similar to that 

 used for the visual complex, rhodopsin, from retinal rods. From the 

 chloroplasts of Etiglcna and spinach we have extracted in 1.8 per 

 cent recrystallized digitonin a pigment-protein complex containing 

 both chlorophyll and carotenoids (Wolken, 1956b; Eversole and 

 Wolken, 1958). Other such chloroplastins have been extracted 

 previously by Smith (1941a, 1941b, 1941c). We have been able 

 to determine the physical and chemical properties of chloroplastin 

 and to compare them with those of the chloroplast in vivo. The 

 absorption spectrum of chloroplastin closely approximates the ab- 

 sorption spectra of the pigments in the in vivo chloroplasts ( Fig. 3 ) . 

 Chloroplastin ^ appears to be a homogeneous complex, judging from 

 its sedimentation in the analytical ultracentrifuge and its electro- 

 phoretic pattern (Wolken, 1956b, 1958). 



Temperature and Wave Length. The bleaching of chlorophyll 

 in Euglena can be induced by growing the organisms in darkness or, 

 in light, by raising the temperature above 32° C. Chloroplastin 

 (pH 7.2) can be bleached by light or by heat, analogous to the 

 organisms in vivo. Chloroplastin bleaches at a rate proportional to 

 the amount of light or heat energy absorbed, causing a steady de- 

 crease in optical density with the disappearance of the maximum 

 absorption peak at 675 m^u,; it is bleached to pheophytin and unknown 

 products. Below 560 m/x, bleaching is accomplished by light energy 

 alone; above 560 m/x, the combined effects of light and heat are 

 required. The total activation energy for bleaching is 48.3 kcal/mole 

 (Wolken and Mellon, 1957). 



Photochemistry. Photochemical reactions analogous to photo- 

 synthesis were carried out with Euglena chloroplastin, e.g., the rate 

 of photoreduction of the dye, 2,6-dichlorobenzenoneindophenol, the 

 evolution of oxygen, and the conversion of inorganic phosphate to 

 labile phosphate (ATP). 



The rate of photoreduction of the dye, 2,6-dichlorobenzenone- 

 indophenol (3 X 10~'' M), was measured at 600 myu, after illumina- 

 tion for 1-4 minutes with 300 foot-candles. In 20 per cent of the 

 preparations, photoreduction of dye was complete within 2 minutes 

 with no accompanying reduction in darkness. The molecular turn- 

 overt with a typical preparation indicated that 7 X 10^ molecules of 



'' Chloroplastin is not a purified macromolecule as it contains a cytochrome c type 

 haem protein as well as other impurities. 



