ITC is a sensitive and convenient method for detecting biomolecular interactions by measuring the heat that is released or absorbed upon binding . Measurements are conducted directly in solution, without modification of immobilization of the interacting species. We used ITC to study potential interactions between human plasma fibrinogen and LPS from E. coli O111:B4. Titration of fibrinogen into LPS resulted in strong endothermic injection heats with a clear sigmoidal saturation curve indicating a direct binding interaction (figure 6a). Assuming molecular weights of 340 kDa for fibrinogen and 20 kDa for monomeric LPS, we determined a binding stoichiometry (n) of approximately 0.135. This is consistent with each fibrinogen monomer binding to a micelle formed from approximately 75 LPS monomers. Reverse titrations were conducted, injecting LPS into plasma concentrations of fibrinogen (3 g l−1 = 8.8 µM). Titration of 2.5 µM LPS into fibrinogen yielded endothermic injection heats greater than those observed for titration of 2.5 µM LPS into buffer alone (figure 6b), again clearly indicating a direct binding of LPS to fibrinogen. Each injection added 125 ng of LPS into the instrument cell, increasing the LPS concentration by approximately 30 nM per injection. Although we expect that LPS binds fibrinogen at sub-nanomolar concentrations, interactions at these concentrations are below the detection limits of the ITC instrument. Figure 6.ITC analysis of the LPS–fibrinogen interaction. (a) Titration of 8.8 μM human plasma fibrinogen (black circles) or buffer (green open circles) into 100 µM of E. coli O111:B4 LPS. (b) Titration of 50 ng µl−1 LPS (2.5 µM) or buffer into 3 µg µl−1 fibrinogen (8.8 µM) or buffer as indicated. Experiments were conducted at 37°C in phosphate buffered saline. (Online version in colour.)