PREPARATION, PRESERVATION AND ACTIVATION OF CHLOROPLASTS 1557 



in contrast to the precipitated chloroplast, this material could be stored for 

 weeks in the dry state, in the cold, without losing its activity. The dialysis 

 of this supernatant into distilled water precipitated additional green ma- 

 terial, but the solution still showed the chlorophyll band. The yellow color 

 appeared to be due to a protein with absorption bands at 337 and 270 mju ; 

 the yellow "prosthetic group" of this protein was not separable by dialysis. 

 Spectrally, it appeared to be a flavone, but it did not show the oi-ange color 

 upon reduction, which is characteristic of flavones. 



The high activity of the supernatant, obtained according to Aronoff's 

 procedure, was not confirmed by Clendenning and Gorham (1950^, using 

 Hill's solution. With quinone, they noted an increase of efficiency (related 

 to unit chlorophyll amount) compared to the green precipitate by not more 

 than a factor of 2. 



French and his co-Avorkcrs (Milncr et al. 1950, and Milner 1951) macer- 

 ated leaves of Beta in a water-ice mixture in a Waring Blendor. The 

 slurry was filtered through muslin and the filtrate centrifuged 1 min. at 

 12,000 g., all at a temperature not above 2° C. The sediment, which 

 consisted of whole and broken chloroplasts, was resuspended in distilled 

 water (2 mg. Chl/cm.^). Attempts to disperse this suspension by means 

 of a colloid mill were unsuccessful. Ultrasonic wave generator (250 watt) 

 was then used, with precautions to prevent the temperature from rising 

 >5° C. The undispersed material was reprecipitated at 12,000 g. ; the 

 green supernatant was clear, but showed a strong Tyndall cone. 



In this way, 5-10% of the original chloroplast material could be con- 

 verted into a colloidal dispersion. Additional 10-15% could be dispersed 

 by grinding the sediment from the last centrifugation with sand. The 

 dispersion was about 50% less active photochemically (per unit chlorophyll 

 amount) than the original material. 



A more complete dispersion could be obtained by forcing the chloroplast 

 suspension through a small opening (a needle valve from an ammonia 

 cylinder) under high pressure. 40 cc. of suspension were placed in a pre- 

 cooled steel cylinder connected to the valve, and pressed through the valve 

 by a 60-ton hydraulic press. Despite cooling with ice, the material came 

 out at about 15° C. It was diluted to 0.5 mg. Chl/cm.^ and centrifuged at 

 12,000 g.; the dark-green supernatant now contained as much as 60-70% 

 of the total chlorophyll, but its photochemical activity was only 25% of 

 that of the original material. This loss was found to decline with the pres- 

 sure used and the duration of exposure to this pressure. At low pressure, 

 less material is dispersed, and less activity is lost (fig. 35.14). The supe- 

 riority of the dispersion obtained by the valve method over that obtained 

 by supersonics is illustrated by figure 35.15. 



Dispersions which were obtained by passage through the needle valve 



