Monitoring of unbound protein in vesicle suspensions with off-null ellipsometry.
In studies on the binding of proteins to small unilamellar phospholipid vesicles (SUV), the concentration of unbound protein usually remains unknown, because the vesicles cannot be separated from the bulk solution. In the present study, this limitation was overcome by addition of a supported planar phospholipid bilayer to the cuvette containing a vesicle suspension. Ellipsometric measurement of the protein adsorption velocities on this bilayer allowed determination of the concentrations of unbound protein. At high protein concentrations the adsorption is rapidly completed and the usual null-ellipsometry is too slow to obtain well-defined initial adsorption rates. Therefore, an off-null technique was developed, allowing measurement of the adsorbed protein mass at time intervals of 20 ms. Binding of prothrombin and coagulation factor Xa was measured in SUV suspensions prepared from a 20% dioleoylphosphatidylserine (DOPS) and 80% dioleoylphosphatidylcholine (DOPC) phospholipid mixture. For prothrombin, a dissociation constant Kd = 140 +/- 27 nM (mean +/- S.E.) and maximal surface concentration gamma max = (8.9 +/- 0.8) x 10(-3) mole of protein per mole of lipid, were obtained. For factor Xa, these values were Kd = 49.6 +/- 6.3 nM and gamma max = (23.0 +/- 1.4) x 10(-3) mole of protein per mole of lipid. These binding parameters are similar to those obtained earlier for planar bilayers. Apparently, the binding of factor Xa and prothrombin is not dependent on surface curvature.
Giesen
P L
PL
Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands.
Willems
G M
GM
Hemker
H C
HC
Stuart
M C
MC
Hermens
W T
WT
D000327:Adsorption; D000818:Animals; D002417:Cattle; D015951:Factor Xa / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry; D008563:Membrane Lipids / Q000032:analysis / Q000378:metabolism*; D010743:Phospholipids / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding; D011516:Prothrombin / Q000378:metabolism*
D008051:Lipid Bilayers; D008563:Membrane Lipids; D010743:Phospholipids; D011516:Prothrombin; D015951:Factor Xa