112 THOMAS P. SINGER 



The determination of the "pH optimum" of this enzyme is a particu- 

 larly difficult task. The ferricyanide assay, as described, functions very 

 satisfactorily in the pH range of about 5-5 to 7-8. In this range double 

 reciprocal plots of activity versus ferricyanide concentration show a definite, 



6 



Fig. 



l/ml. Fe(CN)j"" 

 9. Ferricyanide assay of soluble, purified enzyme at different pH values. 



zlE/min 



pH 



Fig. 10. Effect of pH on the activity of purified DPNH dehydrogenase. 

 Solid line, at fixed (i -66 x io~^ m) ferricyanide concentration; dashed line, V^ax 

 values. The pH values given are those of the reaction mixture at 30°. Buffers: 

 0-04 M phosphate (pH 5-5 to 8-5) or tris (above pH 8-5). 



moderate slope and the reaction kinetics are of zero order, while at pH 

 5-0 the slope is negligible (Fig. 9) and the reaction assumes first order 

 characteristics with respect to the substrate. As the pH is increased above 

 8-0, the slope approaches infinity and at pH 8-5 to 9 it intersects the 

 abscissa and thus no satisfactory measure of F^ax can be obtained. At 

 present no satisfactory explanation of this complex behaviour is evident. 



