MEASUREMENT OF SUBNANOMOLAR RETINOIC ACID-BINDING AFFINITIES FOR CELLULAR RETINOIC ACID-BINDING PROTEINS BY FLUOROMETRIC TITRATION

Cellular retinoic acid binding protein I(CRABP-I) and cellular retinoi c acid binding protein II (CRABP-II) are small, cytoplasmic proteins w hich bind all-trans-retinoic acid with high affinity. Both of these pr oteins belong to a family of intracellular proteins which bind amphiph ilic lipids, including fatty acids, bile salts, and retinoids. Because CRABP-I and -II exhibit different tissue distributions and differenti al transcriptional regulation, they are proposed to serve different fu nctions. The binding properties of mouse CRABP-I and -II purified from Escherichia coli were examined to further understand their role in in tracellular retinoic acid processing. Fluorescence titrations were per formed using nanomolar protein concentrations, near the obtained disso ciation constants, and analyzed by direct mathematical fitting to raw data, in order to extend the range and accuracy of binding constant de termination. The apparent dissociation constants, K'(d), of mouse CRAB P-I and CRABP-II binding all-trans-retinoic acid were determined to be 0.4 +/- 0.3 nM and 2 +/- 1 nM respectively, stronger binding than pre viously reported. The K'(d) of mCRABP-I and mCRABP-II. complexing with acitretin, a pharmacologically active synthetic retinoid used in the treatment of psoriasis, was 3 +/- nM and 15 +/- 11 nM. Both CRABPs bou nd 9-cis-retinoic acid with a K'(d) of roughly 200 nM, and neither exh ibited significant binding of 13-cis-retinoic acid