190 5. DETERMINATION OF MECHANISMS AND CONSTANTS 



tion may be avoided; there is no single method of plotting which can dis- 

 tinguish between all the various possible mechanisms of inhibition. Sec- 

 ondly, the constants obtained by the different methods may be compared 

 and more accurate values reached. The best types of plots will vary with 

 the system studied so that no general statement can be made as to the 

 relative values of the various techniques. Deviations from linearity may 

 be more obvious in certain types of plots; e.g., type C plotting provides 

 somewhat more accurate interpretation at low substrate concentrations 

 and nonlinear behavior in this range is more readily detected, while type F 

 plotting often gives a more direct determination of the constants. Never- 

 theless, it is advisable always to utilize different methods as is evident 

 from inspection of the sample plots presented in this chapter. 



(2) Plot the data with respect to the concentrations of all the known 

 components of the system. For example, in type A plotting it is necessary 

 to construct graphs of l|v^ against 1/(A) and similarly for coenzymes or 

 hydrogen ions. The results may indicate noncompetitive inhibition from 

 a plot of l/Vj against 1/(S) and yet the inhibition may be due to competi- 

 tion with another component. Type F plots may be made with the concen- 

 tration of activator or coenzyme as the abscissa instead of substrate con- 

 centration. If such plots are not made, errors both in interpretation and 

 determination of constants are possible. The slopes and intercepts usually 

 have values dependent on the concentrations and dissociation constants 

 of the other components of the reaction. 



(3) It is generally better to plot as many curves as feasible for each 

 type of graph. For type A plotting, several curves for different inhibitor 

 concentrations, and for type E plotting several curves at different sub- 

 strate concentrations, will increase the accuracy and reliability of the 

 methods. 



(4) Do not use logarithmic coordinates in the methods of plotting 

 described above; this seems obvious but examples from the literature can 

 be cited. Although the intercepts may be easily interpretable, the slopes 

 are not; in fact, linearity would be only fortuitous, for when a plot of A 

 against B is linear, plots of A against log B or of log A against log B are not. 



(C) Interpretation 



(1) Note deviations from linearity carefully; a straight line drawn through 

 points that are obviously not linear may simplify the problem a good deal 

 but provide meaningless results. Any deviation can be indicative of a dif- 

 ferent mechanism from the one assumed on the basis of a straight line. The 

 deviations may not relate directly to the inhibition but be due to extra- 

 neous factors, but this must be proved. 



