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.