Purification of IRBP from the rainbow trout provides native two-repeat IRBP for binding studies

IRBP is a large glycolipoprotein found in the subretinal space of vertebrates. IRBP from mammals contains four self-homologous repeats with multiple retinoid-binding sites. Cloning and sequence analysis of IRBPs from zebrafish and goldfish showed that they are half the size of mammalian IRBP and contain two repeats. Purification of two-repeat IRBP would provide a simple system for the study of ligand binding to native IRBP. Purpose: To purify sufficient quantities of native two-repeat IRBP for ligand binding studies. Methods: Rainbow trout (Oncorhynchus mykiss) is a readily available teleost with large eyes. Soluble matrix was obtained from trout eyes by saline wash of eyecups and detached retinas. IRBP was identified by western blot. Although IRBP is routinely purified from mammalian eyes by affinity chromatography with con A, we found that trout IRBP did not bind con A therefore, we developed an alternative purification strategy. Matrix proteins were separated by ion-exchange chromatography. IRBP was further purified from these fractions by isoelectric focusing. Binding studies were performed by fluorometric titration with retinol. Results: The M_r of trout IRBP is 75kD, similar to that of goldfish. This suggests that trout IRBP contains two repeals. The failure of trout IRBP to bind con A indicates that glycosylation of teleost IRBP may be different from mammalian IRBP. Preliminary binding studies demonstrated saturable binding of retinol to our purified trout IRBP and suggest that trout IRBP contains a single retinoid-binding site. Conclusion: Two-repeat IRBP is a simple system that will allow us to characterize ligand binding to native IRBP. We have developed a protocol to purify sufficient quantities of teleost IRBP for binding studies.