Human plasma albumin, displacement

Even complicated, charged macromolecules like proteins can be succesfully displaced. As an example we give in Figure 6 the displacement isotherm for human plasma albumin from silica by morpholine (21). Of course, in this case where charge effects and a variety of segment/surface interactions play a role, our simple Equation 5 does not apply. Nevertheless, for practical work it is important to realize that most macromolecules, often thought to be irreversibly adsorbed, can be removed completely from the adsorbent surface by the concerted action of a large number of small molecules. 

Figure 6. Displacement isotherm of Human Plasma Albumin from silica in aqueous morpholine solutions at pH = 8.5.

Kraak et al. (38) reported the first ACE application to study drug binding to a plasma protein. They used the model system warfarin-human serum albumin (HSA) to compare the suitability of the Hummel-Dreyer, frontal analysis, and vacancy peak methods. A more methodologically intended paper from Erim and Kraak (39) used VACE to study the displacement of warfarin from bovine serum albumin (BSA) by furosemide and phenylbutazone. They concluded that VACE is especially suited to examining competitive properties of simultaneously administered compounds toward a given protein-drug system. 

A series of experiments were made with heparin-PVA bead columns. Thrombin (100 U crude bovine, II50 U purified human or 100 U purified bovine) was loaded onto the columns and the excess removed with PBS. Antithrombin III ( 2000-2500 U purified human) was then loaded, and the columns eluted with PBS, (w/v) bovine serum albumin, or plasma fractions. In some cases heat defibrinated or arvinized plasma was used instead of purified human antithrombin III as a crude source of antithrombin III, as well as a displacing agent. The inactivation of thrombin by anti thrombin III was shown in a separate experiment using the chromogenic substrate assay. 

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