Specific binding of peanut agglutinin to GM1-doped solid supported lipid bilayers investigated by shear wave resonator measurements |
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Authors: | Andreas Janshoff Claudia Steinem Manfred Sieber H-J Galla |
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Institution: | Institute of Biochemistry, Westf?lische Wilhelms-University, Wilhelm-Klemm-Strasse 2, D-48149 Münster, Germany, DE
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Abstract: | This study deals with the specific interaction between the lectin peanut agglutinin (PNA) from Arachis hypogaea and the ganglioside GM1 which was incorporated in a solid supported lipid bilayer immobilized on a gold electrode placed on top of an AT-cut quartz
crystal. Bilayer formation was reached by self-assembly processes. The first monolayer consists of octanethiol attached to
the gold surface via chemisorption and the second monolayer was immobilized by vesicle fusion on the preformed hydrophobic
surface. We managed to keep unspecific binding to a minimum by using a phospholipid matrix consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Lectin binding to ganglioside GM1 containing membranes was determined by a decrease of the resonant frequency of the quartz crystal. The minimum amount of
receptor within the membrane which is necessary to obtain a complete protein monolayer was found to be less than 2 mol%. The
adsorption isotherm of PNA to GM1 was recorded and analyzed to be of Langmuir type, exhibiting a binding constant of PNA to the ganglioside of 8.3 ⋅ 105 M–1. The good agreement of the calculated Langmuir adsorption isotherm with the obtained experimental data implies that protein
multilayers are not formed and that interactions between the adsorbents can be neglected. Furthermore, the association constants
of two different saccharides, β-Galp-(1 → 3)-GalNAc exhibiting a strong binding to PNA in solution, and β-D-galactose with a much lower affinity were estimated by determining the equilibrium concentration of PNA attached to the
surface. Moreover we were able to remove the attached lectin monolayer by digestion of the protein with pronase causing an
increase in the resonant frequency which almost reversed the frequency shift to lower frequencies during adsorption. An even
more complex system was built up by the use of digoxigenin-labeled PNA which also binds to the solid supported membrane containing
the receptor GM1. The immobilized lectin was recognized by anti-digoxigenin-Fab-fragments, which is measurable by a further decrease of the resonant frequency. For all binding processes we found larger
frequency shifts for a complete protein monolayer than predicted by Sauerbrey's equation, clearly showing that in addition
to mass loading viscoelastic changes occur at the lipid-protein interface.
Received: 22 July 1996 / Accepted: 12 September 1996 |
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Keywords: | Quartz crystal microbalance (QCM) Impedance spectroscopy Solid supported lipid bilayers Lectins Gangliosides |
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