880 17. PLANNING AND REPORTING INHIBITION STUDIES 



addition of the substrates, inasmuch as the rates may change during the 

 course of the reactions due to a variety of factors. If the inhibition is 

 reported over a period of 30 min, for example, one cannot be certain 

 how much of the depression is due to the direct action of the inhibitor and 

 how much to secondary alterations in the enzyme. Unless the effect on 

 the initial rate is determined, the results may not be of quantitative sig- 

 nificance and any calculated inhibitor constants may be incorrect. The 

 problem of secondary inactivation of enzymes was discussed in Chapter 12. 



(2) Work in the range where the rate is linear with the enzyme concentration. 

 The usual expressions for inhibition kinetics are derived on the basis of a 

 linear dependence of enzyme rate on the enzyme concentration. There are 

 several reasons for nonlinearity under certain conditions, as pointed out 

 by Dixon and Webb (1958, p. 64), so that it is necessary to test the enzyme 

 being investigated under the conditions in which the inhibition is exerted. 

 Nonlinearity may imply limitation by diffusion, relative depletion of coen- 

 zymes or cofactors, impurities in the reagents used, and other situations 

 which can easily alter inhibition behavior. 



(3) Use as little enzyme as possible. There is some tendency these days 

 with the greater availability of purified enzymes to use quite high enzyme 

 concentrations in the reaction vessels. There are several reasons why these 

 high concentrations may be undesirable. In the first place, there is a greater 

 likelihood for the depletion of free substrate or inhibitor; that is, the 

 system may be in zones B or C, and more complex kinetics must be ap- 

 plied to interpret the data. In the second place, there is more opportunity 

 for impurities or other enzymes to be introduced with the tested enzyme. 

 In the third place, one often wishes to study an enzyme under approxi- 

 mately physiological conditions and within the cell the enzyme may not 

 be so concentrated. 



(4) Establish competitive inhibition by quantitative procedures. To demon- 

 strate competitive inhibition, it is not sufficient to show an increased rate 

 of the inhibited enzyme upon increase in the substrate concentration; this 

 would be expected to occur with essentially any type of inhibition. Nor is 

 it enough to show that the inhibition is reduced by increasing substrate 

 concentration, because this may be brought about by other mechanisms, 

 the reaction of the substrate with the inhibitor for example. It is necessary 

 to show by adequate plotting procedures that competitive kinetics are 

 obeyed and to cover as wide a range of substrate and inhibitor concentra- 

 tions as possible. 



(5) Utilize several analytical plots to determine the inhibition mechanism,. 

 It was pointed out in Chapter 5 that the various types of plotting procedure 

 are not equivalent and that some are more applicable to certain inhibitions 



