[27] Determination of binding stoichiometry by the continuous variation method: The job plot

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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.