DISSOCIATION OF THE ENZYME-INHIBITOR COMPLEX 611 



much dilution before the inhibition is markedly reduced. If the initial in- 

 hibition is above 95%, it requires at least a hundred fold dilution to bring 

 inhibition down to 33%. Dilution of inhibited systems in zones B and C in 

 mutual depletion systems has been discussed in Chapter 3. This may be 

 summarized by stating that the greater the degree of mutual depletion 

 (i.e., the more the system approaches zone C in behavior), the less effect 

 will dilution have on the inhibition. 



Straus and Goldstein (1943) discussed quantitatively the effects of di- 

 lution on equilibrium inhibitions in the system serum cholinesterase-physo- 

 stigmine and Goldstein (1944) extended this to the kinetics of dilution. 

 The experimental data on inhibition reversal were only moderately well 

 described by an equation derived on the basis of simple dissociation. The 

 equation used is equivalent to Eq. 13-8 although it is expressed in quite 

 a different form. However, more than simple dissociation of the EI com- 

 plex may be involved here, inasmuch as physostigmine is slowly inacti- 

 vated by the enzyme (Easson and Stedman, 1936). Also the' system was 

 not always completely in zone A. Thus some deviation from the monomo- 

 lecular dissociation kinetics might be expected. It may also be noted that 

 experimental points are not available for the early phases of the reversal, 

 the first readings having been made at 40 min, at which time the inhibitions 

 had been reduced almost to their final equilibrium levels. 



Inactivation of the Enzyme During the Experimental Period 



Quantitative determinations of the reverisibility of inhibition should 

 include control tests for possible changes in the activity of the enzyme in 

 the absence of the inhibitor. Complete reversal would not, of course, be 

 anticipated in those cases where the enzyme is unstable and spontaneously 

 inactivated. Unfortunately this procedure does not reveal secondary inacti- 

 vation of the enzyme by the inhibitor. For this purpose, it is necessary 

 to determine in some way (e.g., by chemical or radioactivity analysis) 

 if the inhibitor has actually been completely dissociated from the enzyme 

 during the reversal incubation or dialysis. If it can be shown that no inhi- 

 bitor remains and yet the activity has not returned to the initial value, 

 it is strong evidence for inactivation in addition to the direct effects of the 

 inhibitor. The kinetics of a system in which the enzyme is being simulta- 

 neously inactivated as it is recovering from inhibition are complex, but the 

 behavior may be readily predicted. In general, the activity at any time 

 will be the result of the two independent processes and will depend on their 

 relative rates. 



