Job plot

Abstract: Publisher Summary An important element in the study of protein-ligand or protein-protein interaction is the binding stoichiometry of the reactants. This chapter discusses the determination of binding stoichiometry by continuous variation method known as the job plot method. Determination of stoichiometry is based on the measurement of complex formation at various combinations of mole fractions of the reactants. The chapter primarily concerned with the use of enzymic activity as an indicator of complex formation. In this sense, changes in enzymic activity are the same as other measurable parameters such as fluorescence or absorbance changes. The advantage of measuring activity is the relative ease of carrying out the experiments. It is, however, applicable only to ligands (or regulatory proteins) that are not consumed during the enzymic reaction—namely, the effectors, cofactors, and nonconsumable substrates. For purposes of comparison, other approaches that can utilize enzymic activity as an indicator of enzyme-ligand complex formation, such as the use of modified Klotz equation, are also discussed.

Abstract: Continuous variation method, known as Job plot, is the most commonly applied method for the determination of stoichiometry of complex chemical entities for over 100 years. Although, the method was proven successful in the analysis of very stable metal–ligand complexes, we demonstrate that its use in supramolecular chemistry often provides false results. We support this statement with multiple simulations as well as cases studies of several real host–guest systems. We propose an alternative, general method relying on the analysis of residual distribution in titration data fitting. The latter method is more convenient compared to the Job plot and unlike it gives correct results in all real cases studied.

Abstract: In chloroform-d molecular tweezer 1 forms a 1:l complex (Job plot) with 9-propyladenine (4). Changes in the UV-visible absorption spectrum of 1 upon addition of 4 and the changes 1 and 4 induce in each other's IH NMR spectrum are consistent with those of a complex comprised of hydrogen bonds and a-stacking interactions. The microenvironment around the carboxylic acid group in 1 markedly alters its complexation behavior relative to a simple carboxylic acid such as butyric acid (Lancelot, G. J. Am. Chem. SOC. 1977, 99,7037-7042). The association constants for the 1-4 and butyric acid-5 complexes are 25000 M-' (298 K) and 160 M-' (303 K), respectively. Butyric acid prefers a type 1 hydrogen bonding pattern while 1 adopts a type 7 pattern. The nucleotide base selectivities follow the order G > C > A > U for butyric acid and A > G >> C > U for 1. The presence of protic solvents markedly decreases the strength of the complex between 1 and 4. Two analogues of 1 have also been studied, molecular tweezer 2 and 3. Both lack the dimethylamino substituent found in 1, while 3 has a spacer unit that is fully oxidized. The association constants for the 2-4 and 3-4 complexes are 14000 and 120000 M-I, respectively.

Abstract: The method of continuous variation (often referred to as Job’s method) is an easy and common method for the determination of the reactant stoichiometry of chemical equilibria. The traditional interpretation of Job plots has been limited to complex association equilibria of the type nA + mB ⇌ AnBm, while little focus has been placed upon displacement type reactions (e.g., A + B ⇌ C + D), which can give Job plots that look quite similar. We developed a novel method that allows the user to accurately distinguish between 1:1 complex association, 2:2 complex association, and displacement reactions using nothing more than a pocket calculator. This method involves preparing a Job plot of the system under investigation (using regularly spaced mole fractions), normalizing the measured quantities (such as the concentration of AnBm or C for the above reactions) to their maximum value (i.e., at mole fraction 0.5), and determining the sum of the normalized values. This sum is then compared with theoretically predicted...