THOMAS P. SINGER 



batch of venom under the conditions of Table I, 90^0 or more of the 

 activity may be obtained in solution. 



Conditions: 80 min. incubation with i mg. A^aja iiaja per 25 mg. protein at 

 30'^. Solubilization varied from 62 to 78%. Second incubation yields 22% more 

 enzyme in solution. 



The fact that the enzyme is in true solution has been shown by the 

 usual criteria : it does not sediment in i hr. at 144 000 x g even after 12 hr. 

 dialysis or repeated freezing and thawing and it may be readily fractionated 

 with (NH4)2S04 in a manner characteristic of soluble proteins. 



The enzyme has been purified by two cycles of (NH4)2S04 fractiona- 

 tion at pH 8-0 and the resulting preparation has a specific activity of about 

 200 /xmoles of DPNH oxidized /min. /mg. protein (biuret basis, coefficient 

 = 0-095) ^^ 3° ' pH 7 •4. Fractionation on calcium phosphate gel or 

 hydroxylapatite has failed to increase the purity further. The enzyme is 

 not held on carboxymethylcellulose at pH 6-8 and it is excluded on 

 Sephadex G75. Fractionation on DEAE cellulose is not feasible, since the 

 enzyme is extremely strongly adsorbed on this ion exchanger. The turn- 

 over number per mole of flavin is at least 350 000 at 30°, pH 7*4 in the 

 ferricyanide assay. 



Present knowledge of the properties of the enzyme may be summed 

 up as follows. The enzyme is gratifyingly stable and may be preserved for 

 prolonged periods in the frozen state with little or no loss of activity. 

 Even after 96 hr. at room temperature (21 ) at the pH of optimum stability 

 (pH 7-5) 8o")o of the activity remained. 



The dehydrogenase does not act on TPNH, nor is TPNH an inhibitor. 

 DPN, however, is a powerful competitive inhibitor; the competition is 

 again with respect to the electron acceptor (Fig. 7). This inhibitory efl^ect 

 of the oxidation product is another reason why accurate assays must be 

 based on the measurement of initial rates at infinite ferricyanide con- 

 centration. 



The apparent Y^j^j for DPNH, based on assays in which the DPNH 

 concentration was varied at infinite ferricyanide concentration, is i x io~^M 



