An improved assay procedure and a new chemically stable ligand for cytosolic retinoic acid binding protein

A high through-put method to assay for binding of retinoids to their cytosolic binding protein is described. The protein-bound retinoid is quantified following its complete separation from the free ligand by a single gel filtration chromatography step on Sephadex G-25 columns. The method allows a single person to process up to 100 samples per day. A new, substituted benzo[b]thiophene carboxylic acid derivative (tritiated) is proposed as an alternative ligand for cytosolic retinoic acid binding protein. This new analog (CD270), which contains no olefinic double bonds, is characterized by its chemical stability to light and atmospheric oxidation. This molecule shows binding characteristics similar to those of retinoic acid, both in terms of binding affinity and binding specificity (Kd of about 2 nM), and offers the additional advantage that the unbound molecule is absorbed on Sephadex G-25, while the protein-bound ligand is unaffected by the carbohydrate gel matrix. When the affinities of retinoic acid and several analogs were determined by competition binding experiments, similar results were obtained when either tritiated retinoic acid or tritiated CD270 were used as the labeled ligand. The tritiated, heterocyclic retinoic acid analog, CD270, is thus proposed as an alternative ligand for cytosolic retinoic acid binding protein.