Retinoids absorption spectra

Saari has made the bold statement that Three derivatives of 11-cis-retinaldehyde serve as the chromophores of all known visual pigments. They are complexed with a protein component (an opsin), and the resulting protein-retinoid interactions determine the spectral sensitivity of the visual pigment. 85 No reference is given for this statement nor is any explanation of how these complexes exhibit an absorption spectrum in the visual region. However, except for the substitution of all-trans for 11 -cis in the above quotation and a slight modification to the retinoid involved, this work agrees completely with the statement and provides an explanation for how it is applied. 

Wald, et. al97,98. performed a set of experiments during the 1940 s that purported to demonstrate the formation of rhodopsin from either retinene, (now known as retinal) or Vitamin A, and a native protein. While their work involved materials showing a peak absorption at 500 nm, this is the wavelength of peak isotropic absorption of a large number of dipolar retinoids. Such a peak is not exclusive to the chromophoies of vision. Neither is it relevant to the anisotropic absorption spectrum of the chromophores of vision. 

As in the case of the P/D Equation, the absorption spectrum of any retinoid can be described in terms of the absorption cross section of the material. 

As discussed above, there are three pertinent situations with respect to the absorption spectrum of the retinoids in the visual range. For most of the retinoids, there is no significant visible spectrum absorption. The dominant mode of molecular absorption is due to bulk excitation and this absorption is most prominent in the ultraviolet. The relevant visual band absorption cross section is zero. 

For those retinoids rigidized by conjugation and containing two heavy atoms, the molecules may exhibit a significant absorption spectrum in the visible due to dipole-molecular excitation. This absorption spectrum, peaking at 502 nm, is shown by the solid black fine. It can be described in terms of a finite isotropic absorption cross section. 

Figure 5.5.10-5 CR Absorption spectrum of putative frog rhodopsin as a function of bleaching level. Curve 1 is unbleached. The material was prepared as a red powder and then placed in dilute solution. From Wolken, 1966. (A) Intrinsic ultraviolet peak of a retinoid. (B) intrinsic isotropic absorption peak of a retinol (al) complexed with opsin. From wolken, 1966.

Ma, Znoiko, et. al. also provide data on the effect of adding hydroxylamine to one of their samples at 4° C. The change of the absorption spectrum with time is reminiscent of other tests involving the change in pH of a chromophore. It once again demonstrates that the chromophores of vision involve a retinoid with the complexity and form of an indicator. Within the context of this work, the chromophore, rhodonine(9) shifted its spectral peak from its functional (anisotropic) peak at 432 nm to its intrinsic (isotropic) peak at 350-360 nm. 

Details of HPLC of retinoids (4-6) can be found in Chapter 2 of this book Here we give a descnption of purification of a retinoid to be used as a standard. For HPLC purification, a concentrated solution of the retinoid is injected. How much of the solution is to be injected and appropriate concentration of the solution will depend on the impurities present in the sample, their resolution during HPLC, the column size, the solvent used, and other conditions. These can be determined by trials A reasonably volatile solvent or solvent mixture that can be removed easily under argon or nitrogen or in a rotary evaporator should be selected. Depending on the amount of the retinoid required, several injections may be performed, and the appropriate peak collected each time. Solvent is evaporated from the pooled fractions, the sample reconstituted in an appropnate solvent, and the concentration determined by recording the absorption spectrum. Small quantities of HPLC standards or retinoids for tissue culture studies may be readily purified by this procedure, using standard analytical-scale columns. 

Dissolve the appropriate methyl ester in methanol, and saponify as previously described for preparation of retinol from retinyl acetate After saponification, add water, and then acidify with dilute glacial-acetic acid make sure the solution is acidic to litmus paper. (In aqueous-alkaline solution, retinoid carboxylic acids remain as sodium salts, and are not extracted by organic solvents.) Extract the retinoid-carboxylic acid with diethyl ether two or three times (Note that hexane is not a good solvent for these polar retinoids.) Then wash the ether extract with water, and dry it over anhydrous sodium sulfate. Alternatively, if the volume is small, vortex and centrifuge the sample, remove any water, and evaporate the solvent The retinoid-carboxylic acids usually are obtained as yellow solids. Do not add any (not even a trace) HCl to 5,6-epoxy retinoids, because they instantaneously undergo isomerization to 5,8-epoxy retinoids, this change in structure is readily confirmed by the change m absorption spectrum (Table 1). 

The peculiar spectral properties of CRBP-bound retinol and RME are indicative of specific ligand-protein interactions (see Fig. 2) Instead, other holo-retinoid-binding proteins exhibit less characteristic spectra. For example the absorption spectrum of the complex of retinol with plasma retinol-binding protein (RBP) is characterized by a single, well-shaped peak centered at approx 328 nm On the other hand, the absorption spectra of some CRBP-bound retinoids, like CRBP-bound a l-trans retinal (12,13), do not resemble those of CRBP-bound retinol and RME. 

Some retinoids may not cause changes in protein fluorescence when protein bound. This is even possible, but unlikely, for retinoids with an absorption spectrum that overlaps with the protein-emission spectrum... 

In rigorously identifying a retinoid or carotenoid species, however, its chromatographic behavior and its absorption spectrum are not sufficient (340). Additional physiochemical data, e.g., mass spectra, NMR spectra, and information on chemical derivatives, are needed (340). 

Second, the absorption characteristic of each Rhodonine chromophore is highly directional (15R). This anisotropic absorption is only observed for radiation applied perpendicular to the surface of the film, i.e., parallel to the axis of the Outer Segment. The peak absorption wavelength for resonant absorption by these chromophores is nominally either 342,437, 532 or 625 nm. The chromophore is not polarization sensitive for excitation along this axis. For radiation applied along other axes, such as transverse to the axis of the OS, only the intrinsic absorption characteristic due to conjugate absorption and shared by all retinoids of the Vitamin A Group will be observed. This intrinsic spectrum has a nominal spectral peak at 502 nm at 37C. 

It is significant to note the shape of the anisotropic spectrum of the ultraviolet absorber, Rhodonine (11) measured by these workers, does not correspond to the isotropic absorption characteristic of the dilute retinoids. The anisotropic spectrum reflects resonant conjugate absorption in the Rhodonines. Note also the lack of any absorption characteristic with a peak near 502 nm. 

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