Application of the continuous variation method to cooperative interactions: mechanism of Fe(II)-ferrozine chelation and conditions leading to anomalous binding ratios.

The method of continuous variation, often known as the Job plot, has long been used for determining the stoichiometry of two interacting components. The correct binding ratio, n, is generally obtained when the total concentration of the reactants, C(o), is much greater than the dissociation constants involved. For non-cooperative binding systems, the stoichiometry varies between one and n as C(o) increases; whereas for positive cooperative systems, values larger than n may be observed at low C(o). In this report, we present examples to illustrate how the changing apparent stoichiometries as a function of C(o) can provide clues for differentiating various binding mechanisms. To test these concepts, we examined the chelation of Fe(II) with ferrozine in the range of C(o)=7 to 210 microM with Fe(II) expressed in molar concentration or in terms of its binding equivalents (three in this case). The results were analyzed according to several models and found to be most consistent with the mechanism of one-step complex formation or infinite cooperativity with a K(d) of 8 microM.