Physicochemical parameters affecting the charcoal adsorption assay for quantitative retinoid-binding measurement.
The different parameters affecting the accuracy and reliability of the dextran-coated charcoal adsorption assay for characterization of retinoic acid receptors ligand binding activity were investigated. Using dextran-coated charcoal (DCC) at a final 10 mg/ml concentration, an efficient adsorption of free [3H]retinoic acid was observed with a yield in the range 99.2 to 99.8% for ligand concentrations varying from 10(-9) to 10(-4) M. Nonspecific adsorption of retinoic acid reached 50% to polystyrene and silanized glass and 70% to uncoated glass. Results obtained by the DCC method and by gel-filtration assay were correlated; however, the DCC assay appeared easier to perform and gave more reproducible results. When a careful measurement of free retinoid concentration was performed, the apparent equilibrium dissociation constant (KD) of retinoic acid was 3.1 +/- 0.4 nM and the KD of CD367, a synthetic retinoid, was 1.8 +/- 0.3 nM. Optimal pH for the binding of [3H]retinoic acid or [3H]CD367 was in the range 7.5 to 8.5. Under the conditions described for the adsorption assay, bound retinoid measurement was linearly related to the protein concentration between 0.05 and 0.25 mg/ml. At a lower protein concentration, addition of bovine serum albumin exerted a stabilizing effect on retinoid binding, allowing an accurate measurement of the number of specific binding sites. Using retinoic acid as ligand, bacterial extracts often resulted in a level of nonspecific binding in the range 10-25%. It could be lowered (4-10%) when resorting to [3H]CD367.(ABSTRACT TRUNCATED AT 250 WORDS)
Sablonnière
B
B
Laboratoire de Biochimie Structurale, CJF INSERM 92-03, Faculté de Médecine, Lille, France.
Dallery
N
N
Grillier
I
I
Formstecher
P
P
Dautrevaux
M
M
D000327:Adsorption; D002021:Buffers; D002606:Charcoal / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003911:Dextrans / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D012176:Retinoids / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013535:Suspensions; D014212:Tretinoin / Q000737:chemistry / Q000378:metabolism; D014316:Tritium
D002021:Buffers; D003911:Dextrans; D018168:Receptors, Retinoic Acid; D012176:Retinoids; D013535:Suspensions; D014316:Tritium; C064545:4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid; D002606:Charcoal; D014212:Tretinoin