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A. L. Lyubarsky, E. N. Pugh, Jr.; Over 98% of 11-cis Retinal in the Dark-Adapted Mouse Eye Is Bound to Rod and Cone Opsins. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3246.
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(1) To determine the fraction of 11-cis retinal (11cRAL) that is not bound to rod and cone opsins (Non-Opsin-bound 11cRAL = NOB-11cRAL) in a dark-adapted mouse eye. (2) To determine whether the hypothetical NOB-11cRAL can recombine with free opsin.
(1) To exclude regeneration of 11cRAL experiments were carried out with euthanized Balb/c mice, or excised eyes or sonicated retina preparations. (2) Since the absorbance spectra for 11cRAL and rhodopsin differ by ~ 120 nm (max = 380 nm, 500 nm, respectively), we attempted to identify NOB-11cRAL by its immunity to isomerization by orange (>570 nm) light capable of isomerizing >99.9% of the rhodopsin chromophore ("full bleach"). (3) To test the ability of the hypothetical NOB-11cRAL to recombine in situ with free opsin, excised, dark-adapted albino mouse eyes were illuminated to "pre-bleach" ~8% of rhodopsin, incubated at 37 oC for 15 min and then deeply bleached with orange light. We anticipated that NOB-11cRAL would recombine with the free opsin formed during the pre-bleach and, thus, become isomerized on subsequent illumination. To exclude that at-RAL formed on pre-bleach stayed bound to opsin and impeded 11cRAL recombination, HPLC retinoid profiles of the eyes of live mice allowed to recover after a full bleach were measured: >60% of at-RAL was reduced in the initial 15 min.
(1) Under conditions preventing photoreversal to 11cRAL (dark-adapted sonicated retina preparation in 100 mM hydroxylamine), 500 s orange illumination bleaching rhodopsin at initial rate of ~ 1% s-1 yielded a non-isomerizable fraction of 11cRAL of ~ 2% of the total retinal (~ the amount of mouse cone UV-pigment); thus, NOB-11cRAL did not exceed 2% of total retinal. (2) Intense orange illumination for 5 - 10 s that bleached rhodopsin at a rate ~ 50% s-1 left similar amounts of non-isomerized 11cRAL in the retina and in the whole eye preps (6.7 ± 0.5 and 5.0 ± 1 percent of total retinoid), indicating no detectable amount of NOB-11cRAL in the RPE. (3) The pre-bleach level did not affect the amount of non-isomerized 11cRAL.
The fraction NOB-11cRAL in a dark-adapted mouse eye does not exceed our detection limit of ~ 2% of the total retinal, and so at least 98% of the total 11cRAL corresponds to the chromophore bound to rod and cone opsins.
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