884 17. PLANNING AND REPORTING INHIBITION STUDIES 



by providing a physiologically correct ionic balance and pH. Intracellular 

 enzymes should be tested at intracellular pH's and cells should be tested 

 at the pH's of their usual bathing media if correlations are to be made 

 between the actions of the inhibitors on the experimental preparations and 

 the enzymes or cells under normal conditions. Of course, in many cases the 

 physiological conditions must be varied to obtain pertinent information 

 but the initial and control conditions should be as i3hysiological as possible. 

 It is actually not very important to work under conditions in which the 

 rates are maximal, as is often done. Needless to say, it is never possible to 

 achieve exact physiological conditions when studying intracellular enzymes 

 in isolated preparations, but one should come as close as is feasible. 



(2) Control the pH carefully. All solutions to be used in the enzyme test 

 or in cellular inhibition should be adjusted to the same pH so that mixing 

 will not result in any unknown shift in pH. If this is impossible for some 

 reason, the pH should be determined in the final medium. It is also well 

 not to rely completely on buffers to maintain the pH constant nor is it 

 generally good practice to use high buffer concentrations. 



(3) Use a wide range of inhihitor concentrations. It is only by using inhi- 

 bitor concentrations that give the total range of possible inhibition that 

 an enzyme may be characterized adequately and useful constants obtained. 

 Inhibition data on newly isolated enzymes today are often restricted to the 

 per cent inhibition of each inhibitor at a single concentration but often 

 such results are not very valuable. To say that an inhibitor produces a 

 95-100% inhibition at 50 mM means very little; concentrations of 1-5 vaM 

 might very well inhibit quite potently. Because of the configurations of 

 most inhibition-concentration curves, it is more valuable to give the con- 

 centration of an inhibitor that produces around 50% inhibition, if only 

 one concentration is to be reiiorted. It is particularly important in deter- 

 minations of specificity for a fairly complete inhibition range to be presented. 



(4) Use several similarly acting inhibitors for better correlations. If the 

 identification of a particular enzyme grouj) is desired, or if the relationship 

 of some metabolic process to a cell function is being studied, it is occasionally 

 useful to use several inhibitors having common actions. For example, in 

 characterizing the nature of sulfhydryl groups at or near the active cen- 

 ter, it is well to use several types of sulfhydryl reagent, such as an ar- 

 senical, a mercurial, iodoacetate or iodoacetamide, iodosobenzoate, iV-ethyl- 

 maleimide, a quinone, and other inhibitors. If inhibition is obtained with 

 some but not others, this might indicate that the mechanism of the inhi- 

 bition is not through reaction with sulfhydryl groups, or it could mean 

 that the particular sulfhydryl group is situated at the enzyme surface in 

 such a manner that only certain substances can obtain access to it. in both 

 cases interesting information being obtained. 



