856 15, EFFECTS OF VARIOUS FACTORS ON INHIBITION 



types of sites may also vary. This is why it is imijortant, as pointed out 

 by Potter, in the titration of tissue enzymes with potent inhibitors that 

 the specificity of the inhibitor for the enzyme be very high. This is the 

 specificity not only with respect to other enzymes but to all binding sites 

 or substances. Thus antimycin may block very specifically one step in 

 electron transjiort and interfere with no other enzyme systems, but this 

 does not mean that it cannot be bound significantly by other substances 

 in the cells. If the affinity of an extraneous binding site for the inhibitor is 

 great, it may not require a very high concentration of this site to reduce 

 the inhibition appreciably. 



This treatment applies to zone C behavior. In zone A one would not 

 expect any effect of extraneous binding sites on the inhibition while in 

 zone B the situation would be intermediate between zones A and C. This is 

 one way of looking at it. Another is to assume that the boundary conditions 

 are defined not only by the binding to the enzyme but to all the sites, 

 which is somewhat more reasonable because experimentally the behavior 

 of the inhibition will correspond to a zone determined by the total binding. 

 In other words, the i^resence of sufficient extraneous binding sites to re- 

 duce the free inhibitor concentration will transfer a system from zone A 

 into zone B, or from zone B into zone C. 



Detection of extraneous binding is usually not easy. If the inhibition is 

 truly in zone C, a plot of the inhibition against the total concentration 

 of the inhibitor will no longer give a straight line passing through the 

 origin (Eq. 15-121). However, in an impure enzyme preparation, if it 

 is found that the inhibition at constant inhibitor concentration varies 

 with the amount of the enzyme present, it is not immediately evident 

 whether the depletion of free inhibitor is due to the enzyme itself or to 

 extraneous binding substances. One can determine the inhibition in prep- 

 arations of varying i3urity; if the inhibition becomes greater as the en- 

 zyme is purified, it is possible that other substances are binding the enzyme, 

 but on the other hand the enzyme may be made more sensitive by the pu- 

 rification or the extraneous binding sites may be located on the enzyme. 

 In the latter case it is very difficult to determine the extent of such extra- 

 neous binding but it is usually not so important to do so as in situations 

 where other substances are interfering with the inhibition. In certain 

 instances, one can directly determine the degree of inhibitor binding in a 

 preparation by equilibrium dialysis or by other techniques and, if the ap- 

 proximate concentration of the enzyme is known, some estimate may be 

 made of the extent of extraneous binding. The nature of the inhibitor is 

 often the best clue because some inhibitors are obviously rather strongly 

 bound to a variety of cell components; for example, the heavy metal ions 

 or the mercurials are bound to many proteins and nucleic acids, so that 

 evidence of free inhibitor depletion would usually be interpreted in terms 

 of extraneous binding. 



