title: Dye-labeled benzodiazepines: development of small ligands for receptor binding studies using fluorescence correlation spectroscopy.
abstract: To investigate benzodiazepine receptor binding studies by fluorescence correlation spectroscopy (FCS), the four fluorophores fluorescein, tetramethylrhodamine, Oregon Green 488, and Alexa 532 were coupled to the benzodiazepine Ro 07-1986/602 (Ro). Binding assays to polyclonal antibodies to benzodiazepines and at the native benzodiazepine receptor on the membrane of rat hippocampal neurons were established to examine the dye-labeled ligands for their benzodiazepine character and their binding behavior. Both the fluorescein and the Oregon Green488 moiety led to a loss of the benzodiazepine receptor binding of the corresponding Ro derivatives. Antibody recognition and interactions to the receptor were observed for the tetramethylrhodamine derivative (K(D) = 96.0 +/- 9.5 nM) but with a high amount of nonspecific binding at the cell membrane of about 50%. In saturation experiments a K(D) value of 97.2 +/- 8.5 nM was found for the Alexa Fluor 532 derivative-antibody interaction. Investigation of the binding of this ligand to the benzodiazepine receptor in FCS cell measurements led to confirmation of high specific binding behavior with a K(D) value of 9.9 +/- 1.9 nM. A nonspecific binding of <10% was observed after coincubation with 1 microM of midazolam. The different properties of the labeled benzodiazepine derivatives and the requirements of the fluorophore in small dye-labeled ligands in FCS binding studies, at the membrane of living cells, are discussed.
authors.affiliation: Department of Pharmaceutical Biology, Philipps-University of Marburg, Deutschhausstrasse 17A, D-35032 Marburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Hegener
authors.forename: Randolf
authors.initials: R
authors.lastname: Jordan
authors.forename: Hanns
authors.initials: H
authors.lastname: Häberlein
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001569:Benzodiazepines / Q000138:chemical synthesis* / Q000276:immunology / Q000378:metabolism; D005452:Fluoresceins / Q000138:chemical synthesis* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006624:Hippocampus / Q000378:metabolism; D066298:In Vitro Techniques; D008024:Ligands; D011485:Protein Binding; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship
chemical_list: D000906:Antibodies; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008024:Ligands; D011963:Receptors, GABA-A; D001569:Benzodiazepines