SUGGESTIONS FOR PLOTTING AND DETERMINATION 189 



racy could certainly be achieved by using mean values from several exper- 

 iments. 



The following suggestions are meant to be only a guide to the quanti- 

 tative study of enzyme inhibition and are of a quite general nature. It is 

 likely that many investigators in the past have not fully realized the com- 

 plexity of most enzyme systems and for this reason their interpretations 

 and constants are suspect. It is better to anticipate a degree of complexity 

 and then experimentally reduce the system to its simplest terms than to 

 assume simplicity at the beginning. 



(A) Experimental Procedure 



(1) Attempt to measure initial reaction rates or determine them by 

 extrapolation in order to avoid effects of reduction in substrate concen- 

 tration, rise in product concentration, or spontaneous loss of activity of 

 the enzyme. 



(2) Use the proper controls so that the rates measured can be attributed 

 specifically to the enzyme studied. The occurrence of a nonenzymic reac- 

 tion should be investigated as well as the possibility of a nonsubstrate 

 (endogenous) reaction. 



(3) Vary the concentrations of all the known components of the enzyme 

 system, including (H+), independently. If a system contains a coenzyme 

 or activator, its effect upon the rate in the presence and absence of the in- 

 hibitor may be just as important as the effect of substrate. 



(4) Utilize as wide a range of concentrations as possible so that accuracy 

 in plotting may be increased and deviations from linearity may be more 

 readily detected. Care must be taken in using high concentrations because 

 the rate may be reduced by secondary or nonspecific mechanisms, such 

 as ionic strength effects. For example, it is probable that all experimental 

 curves on a plot of 1/u against 1/(S) will turn up sharply as the 1/u axis 

 is approached due to these mechanisms. 



(5) Give consideration to the time factors in the inhibition, i.e., the 

 rates at which inhibition is achieved or disappears. A system that is truly 

 competitive may be interpreted as noncompetitive if the displacement of 

 the inhibitor from the enzyme by substrate is low, i.e., if equilibrium is 

 not reached. The equations that have been presented in previous chapters 

 are derived on the basis of equilibrium states. 



(B) Plotting Procedure 



(1) Plot the data in several different ways, e.g., by the six different 

 methods discussed above or other methods applicable to special situations. 

 This is important for two reasons. In the first place, errors in interpreta- 



