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 retinoic acid binding protein II (CRABP-II) are small, cytoplasmic proteins which bind all-trans-retinoic acid with high affinity. Both of these proteins belong to a family of intracellular proteins which bind amphiphilic lipids, including fatty acids, bile salts, and retinoids. Because CRABP-I and -II exhibit different tissue distributions and differential transcriptional regulation, they are proposed to serve different functions. The binding properties of mouse CRABP-I and -II purified from Escherichia coli were examined to further understand their role in intracellular retinoic acid processing. Fluorescence titrations were performed using nanomolar protein concentrations, near the obtained dissociation constants, and analyzed by direct mathematical fitting to raw data, in order to extend the range and accuracy of binding constant determination. The apparent dissociation constants, K'd, of mouse CRABP-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 previously 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 +/- 1 nM and 15 +/- 11 nM. Both CRABPs bound 9-cis-retinoic acid with a K'd of roughly 200 nM, and neither exhibited significant binding of 13-cis-retinoic acid.
Norris
A W
AW
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.
Cheng
L
L
Giguère
V
V
Rosenberger
M
M
Li
E
E
D017255:Acitretin / Q000378:metabolism; D000818:Animals; D004926:Escherichia coli; D051379:Mice; D008968:Molecular Conformation; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014212:Tretinoin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; C082927:retinoic acid binding protein I, cellular; C082928:retinoic acid binding protein II, cellular; D014212:Tretinoin; D017255:Acitretin