Mj. Janssen et al., SOLUBILIZED BENZODIAZEPINE RECEPTORS FOR USE IN RECEPTOR ASSAYS, Journal of pharmaceutical and biomedical analysis, 14(8-10), 1996, pp. 989-996
In the development of non-radioactive receptor assays for benzodiazepi
nes, employing fluorescent ligands, it was observed that the fluoresce
nce measurements were hampered by the background fluorescence of the r
eceptor preparation. This receptor preparation is a brain tissue homog
enate in which the benzodiazepine receptors are membrane-bound. To min
imize the influence of the receptor material on the fluorescence detec
tion, the benzodiazepine receptors were solubilized with 0.5% sodium d
eoxycholate. The binding characteristics of the receptors were examine
d after solubilization and compared with membrane-bound receptors. The
K-d and B-max values for membrane-bound receptors were 1.20 nM and 1.
01 pM mg(-1) protein and for solubilized receptors they were 4.1 nM an
d 0.54 pM mg(-1) protein respectively. Inhibition curves with the benz
odiazepine antagonist flumazenil and the agonist lorazepam revealed th
at their affinities for the solubilized receptor as compared to the me
mbrane-bound receptor were also reduced from 0.67 nM to 3.2 nM and fro
m 1.49 nM to 8.4 nM respectively. The detection limits for the two ben
zodiazepines, however, were not affected by the solubilization. Furthe
rmore, three different methods to separate the fraction of free labell
ed ligand and the fraction bound to the solubilized receptor were comp
ared, namely polyethylene glycol precipitation/filtration, ion exchang
e filtration and charcoal adsorption. Polyethylene glycol precipitatio
n/filtration gave the highest yield for the bound fraction and the bes
t reproducibility.