Retinal extraction

Figure 1(13 Chromatograms of the reaction mixture of (A) crude brain extract under glutamic acid decarboxylase (GAD) assay conditions 0.17 mg of crude enzyme protein plus assay mixture was incubated for 1 hour (B) Crude retinal extract under GAD assay conditions 0.30 mg of crude enzyme protein plus assay mixture was incubated for 2 hours. (From Pahuja et al., 1981.)...

Nagao, A. et al., Stoichiometric conversion of all trans-P-carotene to retinal by pig intestinal extract, Arch. Biochem. Biophys., 328, 57, 1996. 

Fig. 2.162. Absorption spectra of Amphiopl expressed in HEK293s cells (a) and the HPLC patterns of retinal oximes (b). Absorption spectra and the HPLC patterns were measured before (a, curve 1, and b, top trace) and after irradiation at 520 nm for 2 min (a, curve 2, and b, middle trace). The HPLC pattern of retinal oximes extracted from a mixture of irradiated and non-irradiated bovine rhodopsin in equal amounts is indicated as a reference (b, bottom trace). The absorption maxima of the original pigment and its phoroproduct are shown in panel (a). Reprinted with permission from M. Koyanagi et al. [334].

In brief, the rats are anesthetized, followed by an injection of 0.2 mL of the test solution into the common carotid artery. The injection solution consists of a HEPES buffered Ringer s solution (containing 141 mM NaCl, 4 mM KC1, 2.8 mM CaCl2, and 10 mM HEPES, pH 7.4) which contains both the test substrate (e.g., a [3H]-labeled compound, about 10 /xCi) and a reference compound, which is highly extracted by the tissue (e.g., 0.1 /xCi [14C]n-butanol) in the presence or absence of transport inhibitors. If a [14C]-labeled compound is used as a test substrate, [3H]H20 can be selected as a reference compound. Rats are decapitated at 15 s after injection and the retina is removed. The retina is dissolved in 2 N NaOH and subsequently neutralized with 2 N HC1. The radioactivity is measured by liquid scintillation spectrometry. The RUI value, an index of the retinal distribution characteristics of the [3H] test substrate, is estimated using the following relationship ... 

In our series, full retinal reapplication was achieved in 16 of 17 patients (95%). There was only one case of major emulsification immediately after surgery. This was consistent with Wolfs results [47], who reported no case of emulsification with Oxane Hd . Only once did we note silicone passage into the anterior chamber. Wolf reported the post-operative passage of heavy silicone oil (1.03 g/cm ) into the anterior chamber in two aphatic patients despite superior peripheral iridectomy. The incidence of post-operative glaucoma was lower in our series than that reported by Wolf [47] and other publications on standard silicone oil complications [16,17]. Heavy oil removal was uneventful in all patients in Wolfs series, who used 1.03 g/cm density silicone oil. Likewise, in all our patients operated on with the same density (1.03 g/cm ) heavy silicone oil (17 eyes), active extraction with the extraction module of the vitrectomy apparatus posed no problem. 

Resonance Raman Spectroscopy. A review of the interpretation of resonance Raman spectra of biological molecules includes a consideration of carotenoids and retinal derivatives. Another review of resonance Raman studies of visual pigments deals extensively with retinals. Excitation profiles of the coherent anti-Stokes resonance Raman spectrum of j8-carotene have been presented. Resonance Raman spectroscopic methods have been used for the detection of very low concentrations of carotenoids in blood plasma and for the determination of carotenoid concentrations in marine phytoplankton, either in situ or in acetone extracts. ... 

The dynamics of proton binding to the extra cellular and the cytoplasmic surfaces of the purple membranes were measured by the pH jump methods [125], The purple membranes selectively labeled by fluorescein Lys-129 of bacteri-orhodopsin were pulsed by protons released in the aqueous bulk from excited pyranine and the reaction of the protons with the indicators was measured. Kinetic analysis of the data implied that the two faces of the membrane differ in then-buffer capacities and in their rates of interaction with bulk protons. The extracellular surfaces of the purple membrane contains one anionic proton binding site per protein molecule with pA" 5.1. This site is within a Coulomb cage radius from Lys-129. The cytoplasmic surface of the purple membrane bears four to five pro-tonable moieties that, due to close proximity, function as a common proton binding site. The reaction of the proton with this cluster is at a very fast rate (3 X 1010 M-1 sec ). The proximity between the elements is sufficiently high that even in 100 mM NaCl, they still function as a cluster. Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates of the cytoplasmic surface, and two to three of them assume positions that almost bar their reaction with bulk protons. Quantitative evaluation of the dynamics of proton transfer from photoactivated bacteriorhodopsin to the bulk has been done by using numerical... 

Protein-lipid interaction in retinal-rod outer disc membranes in sonicated vesicles is suggested from comparison of the T1 data of these vesicles with those of extracted liposome preparations from the same source (Brown et al., 1976). Chloroplast thylakoids form micellar structures in chloroform and bilayer structures in water. It was shown by 13C relaxation (Johns et al., 1977) that T1 data are sensitive to this change in secondary structure. As in the... 

Berman discusses the difficulty of extracting the proteins of vision from the retinal outer segments based on the assumption that the chromophores of vision are an integral part of the protein rhodopsin235. She discusses the various extraction techniques based on this assumption and the resulting percentage compositions reported in the literature. 

Fig. 8. A nonisomerizing procedure for the extraction of retinals from their binding sites in visual pigments [90],...

In order to ascertain the nature of the bound chromophore, the pigments can be hexane-washed at low temperature to eliminate excess unbound retinal, and the bound retinal can then be extracted by shaking with methylene chloride procedure [90] (Fig. 8), which denatures the protein but does not isomerize the chromophore. Another procedure in which isomerization of the chromophore is unlikely to occur used ethanol denaturation at 0°C to identify 1 l-cfs-retinal as the chromophore in rhodopsin [91]. 

Fig. 13. Absorption (a) and circular dichroism (b) spectra of 3-diazoacetoxy-9-alv-retinaI in hexane, at room temperature (R. Sen, unpublished results). From the incubation of an enantiomeric mixture of retinals, the opsin preferentially bound one of the enantiomers (at position 3) as shown by the circular dichroism spectrum (in hexane), (b), of the chromophore extracted by the methylene chloride procedure. In (b) and (c) are shown the UV spectrum and the HPLC trace of the extracted chromophore.

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