MUTUAL DEPLETION SYSTEMS 71 



50% inhibition for various acceptable errors in the calculated inhibition. 

 If one is quite critical regarding the acceptable error, zone B kinetics must 

 be used over a much wider range than when only low accuracy is required. 



Let us consider two examples applying these results. If an inhibitor with 

 Ki = 10~' M is being used (many inhibitors are known with K-?, as small 

 as this or smaller) and the acceptable error is set at 5%, zone A equations 

 are no longer permissible (assuming inhibitions around 50%) when (E/) is 

 greater than 0.21 or (E^) is greater than 2.1 X 10"^ M. If the molecular weight 

 of the enzyme is 50,000 per active center, this concentration of enzyme 

 corresponds to 0.0001%. Enzyme concentrations of this magnitude may be 

 relatively common. In fact, in an homogenate, which is usually around 0.3% 

 tissue, this enzyme concentration will occur if the concentration in the 

 original tissue is 0.03%. If one has a reasonable estimate of the enzyme 

 concentration, it is possible to decide which inhibitors will yield zone B 

 or C behavior on the basis of their ^/s. Thus the concentration of cholin- 

 esterase in human serum has been found to be 2.3 X 10~^ M (Myers, 1952 a) 

 and inhibitors with K-b less than 1.1x10^^ M would require zone B or 

 if less than 5.7 X 10"^° M would require zone C kinetics. Several inhibitors 

 indeed possess K-b in this range with respect to cholinesterase. The diffi- 

 culty at the present time in applying such reasoning is the lack of knowledge 

 of enzyme concentrations in either tissues or isolated preparations. A meth- 

 od for determining enzyme concentration in certain cases will be presented 

 in the following section. 



Finally, one may inquire as to what boundaries can be set for an inves- 

 tigation of enzyme inhibition where the range between 10% and 90% in- 

 hibition will be encountered. Approaching zone B from zone A, from Fig. 3-11 

 it may be seen that zone B kinetics should be used when (E/) becomes grea- 

 ter than 0.12; approaching zone B from zone C, the system enters zone B 

 when (E/) becomes less than 100. Thus when one includes a wide range of 

 possible degrees of inhibition, the width of zone B increases if the specified 

 accuracy throughout is desired. 



Determination of Enzyme Concentration with Potent Noncompetitive 



Inhibitors 



It was pointed out by Easson and Stedman (1936) that if Eq. 3-32 is re- 

 written as: 



-^ = ,-^ + (E,) (3-42) 



a plot of (I^)/^ against 1/(1— ■?') would show a slope of K^ and an intercept 

 with the {lt)li axis at (E^). Thus (E^) and (E/) can be determined and this 

 was done for serum cholinesterase using miotine as the inhibitor. Another 

 method, probably more accurate, was used by Myers (1952 a) for serum 



