Figure 2.
Validation of assays for release of cis-retinal from CRALBP. Cellular retinaldehyde-binding protein (CRALBP) ·9-cis-retinal (12 μM) was incubated in the dark with 2 mM NH2OH in the presence or absence of 4 M urea. Incubation of the protein with 4 M urea resulted in a progressive loss of absorbance at 410 nm and an increase in absorbance at 355 nm (A). The retinoid was recovered by extraction and high performance liquid chromatography (HPLC) analysis as 9-cis-retinal oxime (B). During 60 min of incubation without urea, there was minimal shift of the ligand UV absorption maximum (C). The retinoid was recovered by extraction and HPLC analysis as 9-cis-retinal and an unidentified retinoid, possibly a di-cis-retinal (D). The results shown in the panels are representative of at least six independent repeats of the experiment.