MONITORING OF UNBOUND PROTEIN IN VESICLE SUSPENSIONS WITH OFF-NULL ELLIPSOMETRY

In studies on the binding of proteins to small unilamellar phospholipi d vesicles (SUV), the concentration of unbound protein usually remains unknown, because the vesicles cannot be separated from the bulk solut ion. In the present study, this limitation was overcome by addition of a supported planar phospholipid bilayer to the cuvette containing a v esicle suspension. Ellipsometric measurement of the protein adsorption velocities on this bilayer allowed determination of the concentration s of unbound protein. At high protein concentrations the adsorption is rapidly completed and the usual null-ellipsometry is too slow to obta in well-defined initial adsorption rates. Therefore, an off-null techn ique was developed, allowing measurement of the adsorbed protein mass at time intervals of 20 ms. Binding of prothrombin and coagulation fac tor Xa was measured in SUV suspensions prepared from a 20% dioleoylpho sphatidylserine (DOPS) and 80% dioleoylphosphatidylcholine (DOPC) phos pholipid mixture. For prothrombin, a dissociation constant K(d) = 140 +/- 27 nM (mean +/- S.E.) and maximal surface concentration GAMMA(max) = (8.9 +/- 0.8) . 10(-3) Mole of protein per mole of lipid, were obta ined. For factor Xa, these values were K(d) = 49.6 +/- 6.3 nM and GAMM A(max) = (23.0 +/- 1.4) . 10(-3) mole of protein per mole of lipid. Th ese binding parameters are similar to those obtained earlier for plana r bilayers. Apparently, the binding of factor Xa and prothrombin is no t dependent on surface curvature.