872 16. SPECIFICITY OF INHIBITION 



more rapidly than others, particularly when the inhibitor chemically reacts 

 with the enzymes. Thus, during the initial stages an inhibitor such as 

 j)-chloromercuribenzoate may possible act specifically on a certain enzyme, 

 whereas later a progressively increasing number of enzymes may be inac- 

 tivated. In the second place, an initially selective inhibition may give rise 

 to many types of secondary changes that may be confused with the primary 

 inhibition. Specificity for practical purposes may be considered as refer- 

 ring to any secondary or subsidiary effects of an inhibitor, and not merely 

 to the differential actions on enzymes. 



Bearing in mind these difficulties inherent in studies on cellular pre- 

 parations, it is now necessary to say a word about the basic problem 

 that confronts one in attempting to achieve specificity — what range of 

 inhibitor concentrations is optimal? The following suggestions may be of- 

 fered in the approach to a solution of this problem. 



(A) The first thing that must be done is to determine as accurately 

 as jpossible the sensitivities of all pertinent enzymes. Often much can be 

 obtained from the literature but in most cases the enzymes tested are from 

 a variety of tissues and species so that the data do not necessarily apply 

 to the problem at hand. Therefore, the enzyme or the metabolic system to 

 be attacked should be studied in as pure a form as possible, and a concen- 

 tration-action curve constructed. All other enzymes or related reactions 

 should be tested in a similar manner as far as is practicable. If the reac- 

 tions and inhibitions can be studied directly in the cells, this will be ideal, 

 but is seldom possible. If such studies can be done, at least one knows the 

 effects to be expected at any concentration. Of course, it is always impossible 

 to test all the enzymes or metabolic pathways, so there is inevitably an 

 element of uncertainty, but without such preliminary investigation there 

 is almost complete uncertainty. It is worth empliasizing that these deter- 

 minations should be made on enzymes and systems from the same tissue 

 and species in which the specific inhibition is desired, because there are many 

 examples of enzymes whose susceptibilities to inhibitors vary greatly with 

 the source. 



(B) One should either know or make a reasonable estimate of the intra- 

 cellular pH so that the in vitro studies can be as comparable to the proto- 

 plasmic conditions as iDossible. Many enzymes are tested at their optimal 

 pH's rather than at physiological pH's; here we are not interested in the 

 behavior of the enzymes when they are most active but in their relative 

 behavior under constant conditions. 



(C) Consideration should be given to the properties of the inhibitor 

 that determine its distribution between the cells and the medium. It is oc- 

 casionally possible to make an approximate prediction of the ratio of the 

 intracellular to the extracellular inhibitor concentrations, based on the 



