Exchange of retinol between IRBP and CRBP

During a bleach, all-trans-retinol passes from the photoreceptor outer segments to the retinal pigment epithelium (RPE), where retinol is found associated with cellular retinol-binding protein (CRBP). Interphotoreceptor retinoid-binding protein (IRBP) is thought to facilitate this exchange, but the transfer of retinol between IRBP and CRBP has not been explored. In this study we used a mixture of purified IRBP and CRBP as a model system to measure the amount and rate of retinol transfer between the two proteins. When retinol is transferred from IRBP to CRBP, its absorbance maximum shifts from 330 to 350 nm. By monitoring the increase in absorbance at 350 nm after mixing CRBP with IRBP-bound retinol, we measured the amount and time course of retinol transfer from IRBP to CRBP. To complement the absorbance measurements, the IRBP and CRBP in these mixtures were subsequently separated by size-exclusion HPLC and individually analysed for retinol content by scanning the effluent with a multiple-diode-array detector. As determined by measuring the change in absorbance at 350 nm, the mean percentage of IRBP-bound retinol transferred to CRBP was 103 +/- 11% (n = 9). The mean half-time of the transfer was 4.2 +/- 1.3 sec; time to reach equilibrium was 30-60 sec. IRBP that was separated from the mixture by HPLC contained little or no retinol, while the isolated CRBP was nearly saturated with retinol. No detectable transfer from CRBP to IRBP was observed. The distribution of retinol between these two proteins was consistent with the nearly 100-fold higher affinity of CRBP for retinol compared with IRBP. Both the degree and time-course of transfer support the idea that this difference in affinity contributes to the flow of retinol to the RPE during a bleach in vivo and, therefore, may play a role in the physiological regeneration of rhodopsin.