65 

 almost completely removed from the column by the end of a 

 45% of ammonium sulfate saturation step. The major activity 

 peak fractions were completely separated from fractions 

 containing the minor peak of activity and eluted at about 

 25% of ammonium sulfate saturation. Fractions with DQT 

 activity were located coincidently with the two peak of SDH 

 activity. There were no column fractions containing only 

 one of these two activities. Fractions from each activity 

 peak were pooled and labeled SP-I for the major peak of 

 activity and SP-I I for the minor peak of activity. Proteins 

 (SP-I and SP-II) were further purified similarly by 

 chromatography steps. It was calculated from this 

 chromatographic step that the major peak, SP-I, accounts for 

 90% or greater of the total activity in the crude extract. 

 The fold purifications calculated for the SP-I and SP-II 

 were based on this assumption. The proteins eluted in the 

 wash fractions of the CM columns in the second 

 chromatographic step of purification. Figure 4-2 shows 

 profiles of SP-I and SP-II elutions from the third step of 

 purification on DEAE-cellulose columns in Epps buffer at pH 

 8.6. Each protein peak eluted at about . 3M KCl in the 

 gradient and had coincident DQT and SDH activities. The 

 fourth chromatographic step was an HA column which eluted 

 each protein in the wash fractions. This step was followed 

 by another DEAE column, equilibrated at a lower pH of 7.2. 

 Each bifunctional protein eluted in the gradient of an NADP* 



