Monitoring of unbound protein in vesicle suspensions with off-null ellipsometry

Peter L.A. Giesen*, George M. Willems, H. Coenraad Hemker, Marc C.A. Stuart, Wim Th. Hermens

*Corresponding author for this work

    Research output: Contribution to journalArticleAcademicpeer-review

    68 Downloads (Pure)

    Abstract

    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 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 obtained. For factor Xa, these values were K(d) = 49.6 +/- 6.3 nM and GAMMA(max) = (23.0 +/- 1.4) . 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.
    Original languageEnglish
    Pages (from-to)125-131
    Number of pages7
    JournalBiochimica et Biophysica Acta-biomembranes
    Volume1147
    Issue number1
    DOIs
    Publication statusPublished - 8 Apr 1993

    Cite this