SUGGESTIONS OF A GENERAL NATURE 887 



poorly understood. However, in general one should not use a particular 

 inhibitor unless consideration of the situation leads to the conclusion that 

 the results, whatever they are, will be meaningful in some way. 



(3) Use inhibitors that are as pure as possible. It is important to be able 

 to attribute with certainty the effects observed to the inhibitor used if 

 reliable conclusions are to be drawn and thus it is advisable to have either 

 assurance of the purity of an inhibitor that is purchased or to purify it 

 before use. In many cases the commercially available inhibitors are not 

 pure. It must never be forgotten that enzymes and living cells are often 

 highly sensitive to trace impurities, such as the heavy metals, and only 

 very small amounts are required to produce significant effects. Some inhi- 

 bitors are unstable and alter chemically with time to products that may or 

 may not be active; one is seldom certain of the interval between the prepa- 

 ration of the inhibitor and its purchase, so that in many instances repuri- 

 fication is mandatory. Particular attention must be paid to certain inhi- 

 bitors such as fluoroacetate, iodoacetate, the quinones, dimercaprol, allox- 

 an, and others, because experience has shown that they are occasionally 

 quite impure as obtained and are unstable. One must, also be cautions 

 in using solutions of inhibitors over several days since many inhibitors 

 are unstable in solution even when stored in the cold. In cases of doubt, 

 it is always better to prepare the inhibitor solutions fresh for each expei'- 

 iment. 



(4) Interpret inhibition studies in as quantitative a manner as possible. 

 The attempt has been made throughout this volume to put the study of 

 inhibition on a quantitative basis because it is believed that a greater 

 amount of information may be obtained by using such an approach. In 

 the case of isolated enzymes it is best to determine the inhibitor constants, 

 Ki, when possible, and to record changes in these or any other pertinent 

 constants with variations in the experimental conditions. The presentation 

 of some type of rate constants for the development of inhibition and its 

 reversal is also advisable, rather than merely stating that the inhibition 

 proceeds rapidly or slowly, or that the inhibition is readily or difficultly 

 reversible. Whenever possible the thermodynamic characteristics of the 

 inhibition should be determined, especially the binding energies and the 

 entropy terms. Even where absolute binding energies cannot be calculated, 

 the relative binding energies of the inhibitors within a series are often val- 

 uable in demonstrating the interaction energies contributed by certain 

 groups. If the aim is to characterize an enzyme active site, a quantitative 

 approach based on physical and chemical principles is apt to be rewarding. 

 When one turns to the application of inhibitors to cell or tissue preparations, 

 or to whole animals, and especially in the correlation of functional and 

 metabolic changes, it is of the utmost importance to obtain and present 



