True affinity labeling

True Affinity Labeling. Effect of Reagent Concentration. The reactions to be considered are given by Equation 1. 

Inspection of Equation 8 reveals that the kinetics of true affinity labeling are first order as long as [R] does not change significantly during the reaction. Furthermore, the observed first-order rate constant, k0bs, is a hyperbolic function of [R], i.e. 

As in the previous section on true affinity labeling we have the material balance equation... 

Comparing Equation 29 with Equation 9 shows that the two expressions for kobe differ only in the constants in the numerators of the right-hand sides. Both mechanisms predict first-order kinetics for the loss of site activity and identical dependence of the observed first-order rate constant, kobBy on the [R]. The similarity of Equations 9 and 29 demonstrates that the documentation of saturation kinetics as evidenced by linear Kitz-Wilson or Eadie-Hofstee plots or by the critria of the direct linear plot does not prove that true affinity labeling is involved necessarily in a site-inactivating reaction. 

This expression for kohB is of the same mathematical form as the analogous expression for the effects of [R] and [L] in true affinity labeling (see Equation 19). In other words, protection by [L] is quantitatively the same for both mechanisms and thus cannot differentiate between them. 

Criteria for Distinguishing Between True Affinity Labeling and Bimolecular Self-Protection Mechanisms. In the case of a labeling reaction that exhibits saturation kinetics and the appropriate protection effects... 

Meloche has presented a concise analysis of this two-step process. The treatment emphasizes the main experimental criteria for true affinity labeling rate saturation effect on the rate of inactivation of the enzyme by the affinity labeling reagent, protection against activation by substrate or competitive inhibitors, and the stoichiometric incorporation of one reagent molecule per binding site. 

This type of kinetic analysis, together with careful analyses of reagent incorporation, represents the most common way of assessing tiie basic criteria for true affinity labeling. It will become evident in subsequent chapters in this volume that there are variations, exceptions, and... 

Many of the criteria for ascertaining whether a reagent is acting as a true affinity label are universally applicable and are described in a previous chapter of this volume. ... 

The two examples discussed above show that, at least in certain cases, direct or photosensitized irradiation of solutions containing a receptor (in this case, E. coli ribosomes) and a radioactive but not especially photolabile native ligand can yield true affinity labeling results. Several of the problems that arise in the course of utilizing this approach are discussed below. 

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