84 



(containing all fractions of the S-protein) , the 

 bifunctional protein was determined to have only one native 

 molecular weight of about 63 kD. 

 Activity stained gels 



In order to optimize conditions for a native PAGE 

 activity stain gel, various combinations of substrate and 

 dyes were studied with concentrated crude extract, data not 

 shown. All activity stained gels were assayed at optimal 

 conditions as described in Methods (Chapter II) . Activity 

 stained bands required both substrate, SHK and NADP* for 

 visibility. Crude extract revealed 3 bands on native gels 

 after application of the activity stain (Fig. 5-4) . In Fig. 

 5-5, purified SP-I and SP-II had 2 activity stained bands 

 each. SP-II migrated at a faster rate than did SP-I. 

 pH and temperature optima of S-proteins 



Both functional domains of the two bifunctional 

 proteins were studied with respect to pH optima (Fig. 5-6) . 

 SDH activity was followed in a pH range of 6.5 to 10 in 200 

 mM Bis Trispropane buffer and showed optimal activity at pH 

 9.0 for both proteins. DQT, assayed by the coupled assay 

 method had very low activities when the same buffer was used 

 over the same pH range for both SP-I and SP-II. A pH range 

 from 6.1 to 9.5 in potassium phosphate, Epps or glycine 

 buffers, each at 100 mAf resulted in higher activities and 

 showed optimal activity at pH 7.25 for both proteins. When 

 the three buffers above were used for the SDH assay there 



