PHOTOSYNTHETIC MACROMOLECULES OF CHLOROBIUM THIOSULFATOPHILUM 



313 



excepting the inclusions, could apply to typical non-photosynthetic 

 bacteria [9, 11, 12, 13, 28, 32] such as Escherichia coli (Fig. 4). 



CRUDE EXTRACTS 



It is rather difficult to rupture this organism but two methods have 

 been used successfully: breaking frozen cells in the Hughes pressor 

 exposing a suspension of 2 g. of cells (wet weight) and i g. of very fine 

 synthetic sapphire abrasive (Linde B) in 40 ml. of o-i m tris (hydroxy- 

 methylamino methane) buffer at pH 7-8 to sonic oscillation for 2 min. at 

 0-5° in a 10 K.C. Ra\i;heon oscillator. 



0.6 



0.5 



-| — [ — 1 — I — 1 1 — I — \ — I — I r 



IN VIVO 



CLEARED EXTRACT 



"T 1 1 1 1 ! 1 T" 



400 



500 



700 



800 



900 



600 

 X m ^ 



Fig. 5. Illustration of the correspondence between the absorption spectrum 

 of the photosynthetic pigments in vivo and in the cell-free extracts. The divergence 

 at lower wavelengths is attributable to differences in light scattering which were 

 not completely compensated by the use of opal glass. 



Cells and debris are remo\ed from the crude extract by two successive 

 centrifugations for 30 min. with refrigeration (5 ) at 26000 g. The 

 "cleared" extract has an absorption spectrum which corresponds with the 

 in vivo spectrum (Fig. 5). The small differences which are observed are 

 attributable to differences in light scattering. This agreement provides 

 some assurance that the phvsicochemical characteristics of the pigment 

 bearer have not been disturbed greatly during the process of cell 

 disintegration. 



A pigmented fraction, free from other macromolecular constituents, 

 can be prepared by repetitive centrifugation for 2 hr. at 144 000 g under 

 refrigeration {^ ). The progress of the fractionation is indicated by changes 

 in the components observed in the analytical ultracentrifuge and by 



