Photoaffinity labeling of human IRBP with all-trans-retinoic acid

Interphotoreceptor retinoid-binding protein (IRBP), found only in photosens itive tissues, is a large similar to 135-kDa glycoprotein that contains a f ourfold repeat structure. IRBP may function as a buffer and prevent retinoi d toxicity and retinoid degeneration. Here we asked (i) whether each repeat of IRBP possesses the capability of photo-crosslinking all-trans-retinoic acid (RA), (ii) within Repeat 1 whether a single retinoic acid-binding doma in exists, and (iii) whether protease and CNBr digestion of Repeat 1 bound RA indicate the exact location of the binding site. H-3-RA cross-linked to all four repeats, consistent with the current model of multiple binding sit es in IRBP. Acetone precipitation was effective in removing unbound H-3-RA LysC and tryptic digestion of the RA-Repeat 1 detected 18- and 5-kDa bands, respectively. CNBr digestion showed two bands about 9 and 11 kDa in size. Our data suggests a single binding site near positions 151-160 in the cente r of Repeat 1. (C) 2001 Academic Press.