TECHNICAL SODIUM BOROHYDRIDE
NOTE
REDUCTION
OF IODOPSIN
ROGER S. FAGER
Department of Physiology, University of Virginia Medical School. Charlottesville. Virginia 22901. U.S.A. MICHAEL KANDEL, THOMAS HEPPNER and EDWIN W. ABRAHAMSON
Department of Chemistry, Case Western Reserve University, Cleveland. Ohio 44106, U.S.A. (Receiced 6 Xovember 1972; in revised form 26 July 1974)
The chicken retina possesses two visual pigments (Wald, Brown and Smith, 1955). rhodopsin in the rods for low light intensity scotopic vision and iodopsin in the cones for higher intensity photopic vision. Both pigments have the same chromophore 11-cis, retinal, which isomerizes to the bans form upon light exposure, but since, at high light intensities, the cone pigment must be rapidly regenerated the chromophore binding site should be more readily accessible to the external solution than is that of rod pigments. This is demonstrated 11-cis retinal
by the fact that with externally added the chicken cone pigment regenerates
about two orders of magnitude faster than the chicken rod pigment. This accessibility is also illustrated by the fact that iodopsin is destroyed by several conditions which do not affect rhodopsin. Thus, whereas rhodopsin is stable over a broad pH range, the spectral integrity of iodopsin is destroyed by relatively small deviations from neutrality. Iodopsin is bleached by pCMB and by hydroxylamine which have no effect on the rhodopsin spectrum. We have found further evidence to the instability of iodopsin relative to rhodopsin and the easier access of the iodopsin chromophore to aqueous reagents. Chicken photoreceptors were prepared by the method of Wald, Brown and Smith (1955). and extracted with 2% digitonin from Nutritional Biochemicals buffered with C-1M pH 7.0 sodium phosphate. The iodopsin was measured by the difference spectrum before and after exhaustive bleaching with white light through a Kodak Wratten No. 1 red filter. The rhodopsin was measured in the same manner after a subsequent bleaching with white light. Typical yields were O-025 O.D.-ml,,, per retina for rhodopsin and 0009 O.D.-n&,, per retina for iodopsin. The bovine rhodopsin and squid rhodopsin chromophores solubilized in digitonin are both inaccessible to sodium borohydride reduction before light exposure. Therefore, we tried to see whether the more accessible chromophore of iodopsin could be reduced by borohydride. A 6-ml extract containing I.30 O.D.-ml ofrhodopsin and O-43O.D.-ml of iodopsin was brought ta 1 m&ml sodium borohydride at pH 7. The reduction was carried out at 3’C for 1,‘2hr. stirring in the dark. The sodium borohydride reducing reagent was flushed out using fresh additions of O-05 M phosphate buffer/ 1% digitonin, and concentrated on an Amicon XM1OOAfilter, which retains the micelles of both the rod and cone pigments.
After reduction and removal of the reducing agent the iodopsin and rhodopsin were measured by the same method as before reduction (see Figs. la and b). The iodopsin was completely reduced while there was no change in the rhodopsin. A great variety of detergents have been used to solubilize vertebrate rhodopsin without destroying its spectral integrity. However, only digitonin seems to be mild enough to maintain its full biochemical properties, e.g. regenerability and circular dichroism spectrum similar to the visual pigment in its membranebound form (Zorn and Futterman, 1971; Shichi. 197a. b). Similarly, only digitonin extracts squid rhodopsin (Hubbard and St. George, 1958) without disrupting its native spectrum.
Fig. 1. (a) Difference spectrum before and after red light exposure *C-O before borohydride reduction; +w after reduction. (b) Difference spectrum of red light exposed and white light exposed samples before (0) and after (0) borohydride reduction.
Technical Note
741
On the other hand. the tendency of dipitonin to flocculate makes it unacceptable for column chromat-
ography for purification of the visual pigments. Therefore. we have attempted to solubilize iodopsin in a variety of detergents. When chicken photorcceptorr are extracted with l”, CTAB. I”,, Emulophogene BC 720, 1”” Triton X-100. or 1”: sodium cholate. no iodopsin appears in the extract; the iodopsin is clearly destroyed. This is confirmed b? the fact that if the same detergents in the same concentration are added to a digitonin extract containing both pigments the iodopsin is rapidly destroyed while the rhodopsin is unaffected. This further underscores the instability of cone pigments relative to rhodopsin. We tried another approach to chromatographing the chicken visual pigments. namely, rolubilizing digitonin by addition of a smaller amount of a second detergent. Emulphogene, Triton. cholate and CTAB all effect such a solubilization. However, even at the minimum amount of the second deter_qent (lii+l::2 per cent) to stabilize a 1:; digitonin solution. iodopsin was denatured.
Similar
experiments
with
invertebrate
~squid] had shown that detergent mixtures with Triton. Emulphogene and CTAB denatured it. while digitonin-cholate (I”,, digitonin: I ?‘,J cholate: 0.01 \I sodium phosphate. pH 7.0. 3’0 enabled chromatographic purification on Sephadex. Therefore iodopsin is even less stable than this invertebrate visual pigment.
REFEREXCES
Hubbard R. and St. George C. C. (19581The rhodopoin system of the squid. J. grn. Ph!kL 44. 502-528. Shichi H. (1971a) Biochemistry of visual pigments--II: Phospholipid requirement for regenerarion of bovine rhodopsin. J. hiol. Chrm. 246,671-681. Shichi H. (197 I bl Circular dichroism of bovine rhodopsin. Phorochrm
Phorohiol.
13, 499-502.
Wald G., Brown P. K. and Smith P. S. ( 1955) Iodopsin. J. yen. Physiol. 38,623-681. Zorn &I. and Futterman S. (1971) Properties of rhodopsin dependent on associated phospholipid. J. biol. Chum 246, 881-886.
NOTE
REDUCTION
OF IODOPSIN
ROGER S. FAGER
Department of Physiology, University of Virginia Medical School. Charlottesville. Virginia 22901. U.S.A. MICHAEL KANDEL, THOMAS HEPPNER and EDWIN W. ABRAHAMSON
Department of Chemistry, Case Western Reserve University, Cleveland. Ohio 44106, U.S.A. (Receiced 6 Xovember 1972; in revised form 26 July 1974)
The chicken retina possesses two visual pigments (Wald, Brown and Smith, 1955). rhodopsin in the rods for low light intensity scotopic vision and iodopsin in the cones for higher intensity photopic vision. Both pigments have the same chromophore 11-cis, retinal, which isomerizes to the bans form upon light exposure, but since, at high light intensities, the cone pigment must be rapidly regenerated the chromophore binding site should be more readily accessible to the external solution than is that of rod pigments. This is demonstrated 11-cis retinal
by the fact that with externally added the chicken cone pigment regenerates
about two orders of magnitude faster than the chicken rod pigment. This accessibility is also illustrated by the fact that iodopsin is destroyed by several conditions which do not affect rhodopsin. Thus, whereas rhodopsin is stable over a broad pH range, the spectral integrity of iodopsin is destroyed by relatively small deviations from neutrality. Iodopsin is bleached by pCMB and by hydroxylamine which have no effect on the rhodopsin spectrum. We have found further evidence to the instability of iodopsin relative to rhodopsin and the easier access of the iodopsin chromophore to aqueous reagents. Chicken photoreceptors were prepared by the method of Wald, Brown and Smith (1955). and extracted with 2% digitonin from Nutritional Biochemicals buffered with C-1M pH 7.0 sodium phosphate. The iodopsin was measured by the difference spectrum before and after exhaustive bleaching with white light through a Kodak Wratten No. 1 red filter. The rhodopsin was measured in the same manner after a subsequent bleaching with white light. Typical yields were O-025 O.D.-ml,,, per retina for rhodopsin and 0009 O.D.-n&,, per retina for iodopsin. The bovine rhodopsin and squid rhodopsin chromophores solubilized in digitonin are both inaccessible to sodium borohydride reduction before light exposure. Therefore, we tried to see whether the more accessible chromophore of iodopsin could be reduced by borohydride. A 6-ml extract containing I.30 O.D.-ml ofrhodopsin and O-43O.D.-ml of iodopsin was brought ta 1 m&ml sodium borohydride at pH 7. The reduction was carried out at 3’C for 1,‘2hr. stirring in the dark. The sodium borohydride reducing reagent was flushed out using fresh additions of O-05 M phosphate buffer/ 1% digitonin, and concentrated on an Amicon XM1OOAfilter, which retains the micelles of both the rod and cone pigments.
After reduction and removal of the reducing agent the iodopsin and rhodopsin were measured by the same method as before reduction (see Figs. la and b). The iodopsin was completely reduced while there was no change in the rhodopsin. A great variety of detergents have been used to solubilize vertebrate rhodopsin without destroying its spectral integrity. However, only digitonin seems to be mild enough to maintain its full biochemical properties, e.g. regenerability and circular dichroism spectrum similar to the visual pigment in its membranebound form (Zorn and Futterman, 1971; Shichi. 197a. b). Similarly, only digitonin extracts squid rhodopsin (Hubbard and St. George, 1958) without disrupting its native spectrum.
Fig. 1. (a) Difference spectrum before and after red light exposure *C-O before borohydride reduction; +w after reduction. (b) Difference spectrum of red light exposed and white light exposed samples before (0) and after (0) borohydride reduction.
Technical Note
741
On the other hand. the tendency of dipitonin to flocculate makes it unacceptable for column chromat-
ography for purification of the visual pigments. Therefore. we have attempted to solubilize iodopsin in a variety of detergents. When chicken photorcceptorr are extracted with l”, CTAB. I”,, Emulophogene BC 720, 1”” Triton X-100. or 1”: sodium cholate. no iodopsin appears in the extract; the iodopsin is clearly destroyed. This is confirmed b? the fact that if the same detergents in the same concentration are added to a digitonin extract containing both pigments the iodopsin is rapidly destroyed while the rhodopsin is unaffected. This further underscores the instability of cone pigments relative to rhodopsin. We tried another approach to chromatographing the chicken visual pigments. namely, rolubilizing digitonin by addition of a smaller amount of a second detergent. Emulphogene, Triton. cholate and CTAB all effect such a solubilization. However, even at the minimum amount of the second deter_qent (lii+l::2 per cent) to stabilize a 1:; digitonin solution. iodopsin was denatured.
Similar
experiments
with
invertebrate
~squid] had shown that detergent mixtures with Triton. Emulphogene and CTAB denatured it. while digitonin-cholate (I”,, digitonin: I ?‘,J cholate: 0.01 \I sodium phosphate. pH 7.0. 3’0 enabled chromatographic purification on Sephadex. Therefore iodopsin is even less stable than this invertebrate visual pigment.
REFEREXCES
Hubbard R. and St. George C. C. (19581The rhodopoin system of the squid. J. grn. Ph!kL 44. 502-528. Shichi H. (1971a) Biochemistry of visual pigments--II: Phospholipid requirement for regenerarion of bovine rhodopsin. J. hiol. Chrm. 246,671-681. Shichi H. (197 I bl Circular dichroism of bovine rhodopsin. Phorochrm
Phorohiol.
13, 499-502.
Wald G., Brown P. K. and Smith P. S. ( 1955) Iodopsin. J. yen. Physiol. 38,623-681. Zorn &I. and Futterman S. (1971) Properties of rhodopsin dependent on associated phospholipid. J. biol. Chum 246, 881-886.
