27 

 Separation of pathway enzymes by chromatography 

 Column chromatography of N. silvestris extracts 



A DEAE- cellulose column for separation of enzymes of 

 aromatic biosynthesis was prepared similarly as described 

 under purification. A 100 mg amount of protein was applied 

 to a bed volume of 102 ml of a DEAE column equilibrated in 

 50 mAT KPO4 at pH 7.5. After washing the column with about 3 

 column volumes of buffer, a 600 ml gradient from to 0.4 M 

 KCl (KPO4 buffer) was applied. 

 Column chromatography of C. sorokiniana extracts 



A DEAE-cellulose column (20 ml) was prepared, 

 equilibrated in Pipes buffer (above) , and about 29 mg of 

 protein from Chlorella were applied. The column was washed 

 with about 40 ml of the same buffer before a 35 mM to 500 mM 

 KCl gradient was added. Finally, 1 . AT KCl/Pipes buffer was 

 added to remove residual protein from the column. 

 Appropriate fractions with DQT and SDH activities in the 

 gradient were pooled and dialyzed against 5 mAf KPO4 buffer, 

 20% glycerol, and 1 mM DTT at pH 7 . 2 . An HA column (10 ml) 

 was prepared with the 5 mM KPO4 buffer, pH 7 . 2 . A 15 -ml 

 sample from above was applied to the HA column and 

 activities for DQT and SDH eluted in the wash fractions. 

 Celite 545 (shallow-gradient application of Fig. 5-1) 



A 200-g amount of ground, frozen 7-day suspension cells 

 was suspended in 200 ml of KPO4 buffer, 7.2 pH, containing 

 protectants and centrifuged as described above. A 3 -ml 



