Abstract: A simple and rapid method is presented for determination of the association constants and stoichiometries describing ligand macromolecule interactions. Based on flow injection analysis and electrochemical detection by amperometry, the only requirements for direct measurements are that the ligand have redox properties and that these properties change upon binding to the macromolecule. Bound ligand may then be measured in the presence of free ligand. Detection limits are of the order of 2 pmol of ligand or less, a level that should provide access to previously unmeasurable systems. For the exemplary system, chlorpromazine and human orosomucoid, K0ass was determined as 0.39 x 10^6 M-1 with 0.76 chlorpromazine binding sites of this affinity per orosomucoid molecule.

Abstract: Transferrin, haptoglobin, IgG, IgA, IgM and orosomucoid in plasma, and albumin in urine were determined by flow injection analysis on a FIA Star (Tecator) system. Samples were diluted up to 200-fold with carrier solution and a 40 µL portion was injected into carrier solution of 0.05 M phosphate - 0.1 M NaCl buffer (pH 7.4) containing 4 or 7% of polyethylene glycol and mixed with antibody solution; detection was at 405 nm. The results are presented for each protein; the method is not suitable for IgG, IgA and IgM and can be used for orosomucoid if a kinetic setup is applied.