Specific binding of low molecular weight ligands with direct optical detection.
The characterization of low molecular weight ligand interaction with receptor molecules is of importance for the investigation of biological processes and for drug research. We report on the investigation of the binding of low molecular weight ligands to immobilized receptors by label-free detection. Reflectometric interference spectroscopy, an optical transducer which allows the monitoring of a few picograms per square millimetre changes in surface coverage, was used to study two model systems. In both cases detection of the binding event was successful. High affinity binding of biotin to immobilized streptavidin was clearly detectable at receptor surface concentrations as low as 1-2 x 10(10) binding sites/mm2. Linear correlation between the receptor surface concentration and the response to biotin binding was observed. Using immobilized DNA, we investigated the binding of common intercalators with respect to kinetics and thermodynamics by evaluation of the association and the dissociation part of the binding curve. Bi-exponential increase and decrease of intercalator loading was observed, indicating complex interaction kinetics. The four structurally different intercalators showed significant distinction in binding kinetics and equilibrium signals. Improvement of experimental parameters is required to obtain more reliable kinetic data.
Piehler
J
J
Institute für Physikalische Chemie, Tübingen, Germany. jacob.piehler@uni-tuebingen.de
Brecht
A
A
Gauglitz
G
G
Maul
C
C
Grabley
S
S
Zerlin
M
M
D000327:Adsorption; D000470:Alkaloids / Q000032:analysis; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D053119:Benzophenanthridines; D015374:Biosensing Techniques; D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D003609:Dactinomycin / Q000032:analysis; D004317:Doxorubicin / Q000032:analysis; D007364:Intercalating Agents / Q000032:analysis*; D007546:Isoquinolines; D007700:Kinetics; D008024:Ligands; D008297:Male; D008970:Molecular Weight; D009621:Nogalamycin / Q000032:analysis; D010777:Photochemistry; D013057:Spectrum Analysis; D013094:Spermatozoa; D019809:Streptavidin; D014337:Trout
D000470:Alkaloids; D001426:Bacterial Proteins; D053119:Benzophenanthridines; D007364:Intercalating Agents; D007546:Isoquinolines; D008024:Ligands; D003609:Dactinomycin; D001710:Biotin; D004317:Doxorubicin; D004247:DNA; D019809:Streptavidin; C005705:sanguinarine; D009621:Nogalamycin