SOLUBILIZATION AND PROPERTIES OF THE DPNH DEHYDROGENASE IO5 



reasonably certain to represent the flavoprotein responsible for DPNH 

 oxidation in mitochondria. 



Considerable effort was expended on elaborating a reliable assay 

 method for the enzyme. Early in this work it became apparent that, 

 contrary to general impression, the assay of DPNH dehydrogenase in 

 mitochondria or in particles such as ETP is a relatively difficult task. 

 Among electron acceptors to be employed phenazine methosulphate was 

 eliminated because of its rapid non-enzymic reaction with DPNH, 

 quinones, such as menadione, because of their failure to react with the 

 isolated dehydrogenase at significant rates, and 2,6-dichlorophenol- 

 indophenol because of relatively high blanks and the great dependence of 

 the measured activity on dye concentration characteristic of this oxidant. 



2 4 6 8 



l/ml, FelCN)^**" 



Fig. I. Ferricyanide assay of particulate DPNH dehydrogenase in the presence 

 of 6 X lo"^ M DPXH. The assays were performed in the presence of 120 /^/moles 

 phosphate, pH 7-4, i-8 /xmoles DPXH, DPXH oxidase, and quantities of o-oi 

 M ferricyanide as indicated, in a total volume of 3 ml. The determinations were 

 made at 30 using a recording spectrophotometer and a 30 to 60 sec. total reaction 

 time. The reduction of ferricyanide was followed at various wave lengths and 

 corrected to £400- X — X, assay without further additions, O— o, in the presence 

 of 2 X io~^ M antimj'cin A, • — •, in the presence of io~^ m cyanide. 



A suitable method was eventually elaborated which is based on the 

 spectrophotometric measurement of the initial rate (15 or 3c sec.) of re- 

 duction of ferricyanide [12]. The application of this method to heart mito- 

 chondria or to particles deri^■ed therefrom, such as ETP, entails several 

 problems, some of which are illustrated in Fig. i. This figure is a Line- 

 weaver-Burk plot of the variation of measured activity with ferricyanide 

 concentration in an ETP preparation at moderately high (6 x iq-^ m) 

 initial substrate concentration. In the absence of inhibitors (crosses) a 

 definite break is seen in the curve relating reciprocal activity to reciprocal 

 ferricyanide concentration. The reason for this is that ferricyanide has two 



