Molecular properties of bovine interphotoreceptor retinol-binding protein.
Interphotoreceptor retinol-binding protein (IRBP) is a large retinol-carrying glycoprotein, located only in the interphotoreceptor (or subretinal) space of vertebrate eyes. It has recently been purified to apparent homogeneity. The present report presents its sedimentation, spectroscopic, and binding properties. The molecular weight of bovine IRBP, determined by sedimentation equilibrium, is 133,000. The sedimentation coefficient is 5.8S. The Stokes radius, 56 A, obtained from gel-filtration chromatography, is much larger than that expected for a globular protein of the same molecular weight. These results indicate that IRBP is asymmetric (it can modeled as a prolate ellipsoid of revolution with axial ratio of about 8:1) and explain the overestimates of molecular weight obtained in previous studies based on size-exclusion methods. The molar absorption coefficients for IRBP (at 280 nm) and for bound retinol are both unaffected by ligand dissociation. Fluorescence of the holoprotein displays neither fine structure nor energy transfer from tryptophan to bound retinol. Circular dichroism suggests a secondary structure containing approximately 15% alpha-helix and approximately 20% beta-structure, unchanged by the presence of ligand. The binding of retinol creates a positive, extrinsic Cotton effect at 330 nm, proportional to the amount of retinol bound. The apparent dissociation constant for all-trans-retinol is 1.3 X 10(-6) M. This relatively loose binding implies that, if required during the visual cycle, IRBP should be able to transfer its ligand to other binding proteins in the neural retina and retinal pigment epithelium.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005136:Eye Proteins; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D012177:Retinol-Binding Proteins / Q000032:analysis*; D014801:Vitamin A / Q000378:metabolism; D000099214:Retinol-Binding Proteins, Interstitial
D005136:Eye Proteins; D012177:Retinol-Binding Proteins; D014801:Vitamin A; D000099214:Retinol-Binding Proteins, Interstitial