91 



was no difference in activity levels from that seen with Bis 

 Trispropane buffer. 



A temperature curve was followed for each activity of 

 the two bifunctional proteins (Fig. 5-7) using optimal assay 

 conditions for activity. Optimal activity occurred at about 

 40°C for the SDH functional domain and at about 32''C for the 

 DQT functional domain of either protein. Extract of each 

 bifunctional protein was heated to 30°C, 40°C or 50°C for 5 

 to 25 min, then assayed at room temperature (24°C) as shown 

 in Fig. 5-8. Activity decreased rapidly at all three 

 temperatures, by 10 min of incubation at 50°C no activity 

 was detected for either protein. Even at 3 0°C the enzymes 

 were subject to inactivation. Substrate was shown to 

 protect the functional SDH domain of both enzymes from 

 thermal inactivation (Fig. 5-9) . With the addition of NADP* 

 prior to incubation at 50°C for 5 min, SP-I retained 67% of 

 its SDH activity and SP-II retained about 61% of the SDH 

 activity compared to 8% and 15% SDH activity retained, 

 respectively, when NADP* was not added. SHK was 

 considerably less protective against thermal inactivation 

 with only 18% and 21% retention of SDH activity for SP-I and 

 SP-II, respectively. Without substrate present, SP-I was 

 thermally inactivated to 50% and SP-II to 60% of activity 

 after 2 min at 50°C. 



