FACTORS IMPORTANT IN INHIBITION 103 



important to realize that the nature of the dehydrogenase has been defined 

 by the dye used. Unfortunately it is not known from what components most 

 dyes accept hydrogen-atoms or electrons. 



The final transfer to the dye is generally a reaction that is nonbiological 

 in the sense that it replaces another transfer reaction occurring in the intact 

 system. An inhibitor may possibly interfere with the dye reduction and 

 yet exert no action, or a different type of action, on the normal transfer 

 sequence. That the final dye reduction step is involved in the kinetics and 

 may often be limiting is indicated by the fact that the over-all rate is often 

 dependent on dye concentration and that different dyes lead to greatly 

 different over-all rates. The inhibitor may compete with the dye, in which 

 case the dissociation constant of the enzyme-dye complex is involved. It 

 is also possible that the flow of hydrogen-atoms or electrons is not exclu- 

 sively to the dye; in mechanism 3-107 the acceptor D competes with com- 

 ponent X2 and division of the flow will depend on several factors. Such a 

 possibility is often circumvented by using anaerobic conditions or inhibit- 

 ing the reaction to oxygen with cyanide. It is preferable, whenever possible, 

 to determine the reduction of some normal component but, even here, the 

 situation may not be simple; the reduction of added DPN+, for example, 

 may be inhibited differently than the reduction of tightly-bound DPN+. 



Calculation of the inhibition has often been made from an equation of 

 the type: 



. aiQ - ait,,) 



aits) 



(3-108) 



where t, is the time required for a specified amount of dye reduction in the 

 presence of the substrate and ^j, is the time when both substrate and inhibi- 

 tor are present. This equation involves two assumptions: (a) that the rate 

 of the reaction is inversely proportional to the time required to reach a 

 certain end-point and (b) that there is no endogenous activity. If the rate 

 of dye reduction is linear or if reduced dye appears exponentially as the 

 substrate is oxidized, according to the equation (DH2) = (So){l — e^^O 

 where (So) is the initial substrate concentration, Ijt is proportional to the 

 rate but this need not be so in all cases; this point should be verified under 

 the experimental conditions employed. With regard to the second assump- 

 tion, it is always necessary to introduce two other measurements, unless 

 they can be shown to be unimportant. The correct equation for the inhi- 

 bition is: 



ri ^ 1 ri in/ri ^ 1 



(3-109) 



where t^ is the time required in the absence of substrate (endogenous) and t^ 

 is the time in the presence of only the inhibitor. These two terms correct for 



