Modification of Job’s method for determining the stoichiometry of protein–protein complexes

Abstract

These reactions can be easily monitored by spectroscopic techniques, which do not require isolating a stable compound, i.e., physically separating bound and free interacting species, particularly when the amount of complex formed can be selectively measured. A typical protein–ligand titration is usually carried out according to the limiting reagent method, using a concentrated ligand solution to avoid excessive dilution. Namely, several small volumes of ligand solution are added to a protein solution of fixed concentration to monitor signal modifications originated from complex formation [1]. However, this procedure is impracticable when both species are proteins, because a very high concentration of either protein to act as titrant can be difficult to obtain. In such cases, it may be useful to resort to Job s [2] method of continuous variation, originally devised to determine the stoichiometry of inorganic complexes. The purpose of this note is to point out that this method can be successfully employed under conditions more favorable than those traditionally adopted to monitor complex formation. In Job s method different amounts of stock solutions of A and B are mixed, varying the mole ratio of reactants in such a compensatory manner that their total molar concentration is kept constant. Under given circumstances, the maximum amount of complex will form in the solution in which the two species are present in the correct combination ratio, provided that the mixing ratio has been varied from 0 to some value certain to be larger than n=m [2–4]. Usually, this is achieved by preparing a series of mixture solutions such that the total moles of reactants and the total volume across the set of solutions are fixed. It is evident that depending on the number of different mixtures prepared this procedure may require considerable amounts of reagents. Considering, however, that the main requisite of Job s method is that the total molar concentration of interacting species be kept constant [2–4], it can be easily verified that it is sufficient to mix different volumes from two stock solutions of the two species, each with the same concentration C0, to make the total concentration equal C0 throughout the experiment. To satisfy this condition it is not necessary that even the volume of the solutions be unchanged while systematically varying the relative amounts of the two species. In other words, using two such solutions, a titration can be carried out in much the same manner as the limiting reagent method, adding increasing aliquots of one species to any volume of the other species, regardless of the total volume. Incidentally, such a procedure allows us to save some material. As an example, we report here experimental details on the binding between basic fibroblast growth factor (bFGF) and BB homodimer of platelet-derived growth factor (PDGF-BB), as monitored by fluorescence spectroscopy. Concerning this aspect, it is opportune to consider that the primary restraint in the application of Job s method is that the signal used to detect complex formation is suitable to measure the amount of product formed. If, for example, one or both reactants also make Analytical Biochemistry 313 (2003) 170–172