I am also grateful to Dr. Alan F. Hofmann for guidance in the uses of detergents, and to Mr. It. Millecchia and Mr. P. Rosen for assistance in measuring the light ...
REVERSIBLE AND IRREVERSIBLE BLEACHING OF RHODOPSIN IN DETERGENT SOLUTIONS* By D. I\JAX SNODDERLY, .J}. THE ROCKEFELLER UNIVERSITY
Communicated by Floyd Ratliff, March 28, 1967
Most investigators studying rhodopsin in solution have used digitonin as the solubilizing agent. In solutions of this detergent, one can observe the bleaching of rhodopsin, as well as the reversal of bleaching by resynthesis from 11 cis retinal and opsin (e.g. see ref. 1) or by photoreversal from the intermediates of bleaching (e.g., see ref. 2). Other detergents can also solubilize rhodopsin (for review see ref. 3), but little information is available about bleaching and reversal of bleaching in solutions of these detergents. In an early study, Chase and Smith4 reported that rhodopsin did not regenerate in solutions of sodium deoxycholate, as it did in solutions of mixed bile salts (of unstated composition) or digitonin. The present paper describes the bleaching of rhodopsin in another detergent that prevents regeneration, cetyltrimethyl ammonium bromide (CH3-(CH2)15-N+-(CH3)3Br-, CTAB), and contrasts the behavior of rhodopsin in CTAB with that in digitonin. CTAB was chosen because of Bridges' reports that the corresponding chloride is an efficient solubilizing agent in which rhodopsin can be stored without appreciable loss of the pigment. The presence of 1 M sodium chloride in digitonin solutions retarded the bleaching of rhodopsin in the experiments to be described. Under these conditions, the photoreversal of bleaching could be observed during steady illumination at room temperature. In comparable CTAB solutions, however, rhodopsin bleaching was much more rapid, and no reversal of bleaching was observed either during or after illumination. Materials and Methods.-Preparation of solutions: A special effort was made to avoid treatment of the retina with protein denaturants such as alum or acids in the course of the purification procedure. Thus any difference of rhodopsin bleaching or reversal of bleaching in digitonin and in CTAB would have to be caused by the detergent alone, and not by action of the detergent on an already modified molecule. The retinas of large, dark-adapted bullfrogs (Rana catesbiana, about 500 gm) were removed by the method of Lythgoe5 and shaken in a cold, 0.9% solution of sodium chloride. The resulting slurry was filtered through 70-mesh wire gauze. Centrifugation of the filtrate for 10 min at 3200 X g sedimented the rods and retinal fragments, which were separated by Saito's method of flotation in 40% (w/v) sucrose in distilled water.6 The suspended rods were sedimented, after dilution with 2 vol of distilled water, by 30- to 45-min centrifugation at 12,800 X g. The pellet was then washed once with 0.9% sodium chloride and suspended in the appropriate detergent solution. Digitonin was dissolved by boiling the detergent in distilled water (2% w/v) and cooling the solution to 0C. The rods were dispersed in the solution at 0C for 2 hr, at which time the suspension was diluted with an equal volume of 0.2 M sodium phosphate buffer (pH 7.1 at 0.1 M) and centrifuged for 20 min at 12,800 X g. Even after 2 hr, though, the remaining pellet of material was still intensely colored with rhodopsin that had not been solubilized (cf. ref. 7, p. 820). CTAB solutions were 3% (w/v) in 0.1 M sodium phosphate buffer, pH 7.1, 220C. When the rods were first dispersed in the solution, the suspension appeared slightly cloudy under red light, but cleared almost immediately; at this point the rods apparently had been dissolved. Centrifugation of the solution yielded only a trace of sediment with no rhodopsin color. In several experiments a preparation of rods was first treated with digitonin as described above, and then suspended in CTAB; as much as four tenths of the total rhodopsin (measured in units of optical 1356
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density) remained in the rods after treatment with digitoniin and was solubilized by CTAB within 15 min. Initially the digitonin preparations of rhodopsin were optically unstable, and consistent Ineasurements could not be made. The stability was improved by the addition of sodium chloride to a concentration of 1 Al after the dilution with buffer (cf. ref. 8). This shifted the pH of the solultions to about 6.6. Subsequent storage of the preparations for 4 to 7 days at 40C produced a loss of optical density throughout the spectrum similar to that described by Crescitelli and Dartnall.9 A final centrifugation of the slightly aged preparations gave stable solutions with values of PXmin (ratio of extinction at maximum near 500 mu to extinction at minimum near 400 mU) of 0.48-0.54. Except for the greater stability and the slightly lower values of Pxmi., the aged solutions did not differ in any obvious way from fresh solutions. Although CTAB solutions of rhodopsin were stable without added salt, they too were made 1 M in sodium chloride in order that conditions for the two detergents should be identical. When the rods were isolated by sucrose flotation, the CTAB preparations of rhodopsin had P.min values of 0.35-0.44. The preparation of one CTAB solution of rhodopsin was expedited by omission of the sucrose flotations. This solution had a slightly higher Pxmin value (0.48) but was not detectably contaminated with hemoglobin as are similar digitonin preparations. At temperatures below 200C, CTAB solutions form gels, but rhodopsin can nevertheless be stored at 4°C in CTAB, the gel being warmed to room temperature for optical measurements. Another cationic detergent, Hyamine 1622 (diisobuitylphenoxyethoxyethyl dimethyl benzyl ammonium chloride, Rohm and Haas, Philadelphia, Pa.) was used for two preparations. It solubilized rhodopsin effectively, yielding solutions with PXmin 0.29 from rods isolated by sucrose flotations and PXmin 0.38 when the sucrose flotations were omitted. Hyamine forms clear, stable solutions in water from room temperature to 00C or below, and it would be worth further investigation. Irradiation of samples: Samples were irradiated by means of a microscope lamp with a tungsten filament, the light being passed through an interference filter. The intensity of the irradiating beam was measured with an Eppley thermopile and microvoltmeter with an accuracy of 45%. For the filter with X max 426 mjA, the intensity was 0.09 Mw/cm2; for that at 456 my, 0.05 Mw/cm2; and at 570 my, 0.20 or 0.25,uw/cm2 as noted in the figure legends. The half widths of these filters were, respectively, 15, 11, and 12 m~u. Since the distance from the source to the sample was not identical for all samples, the absolute intensity of irradiation varied as much as ±30% from these values. But the distance for any given sample was the same during irradiation with light at all wavelengths, so that the ratios of the intensities of illumination at the different wavelengths are accurate to i 5%. Spectrophotometry: Spectra were measured with a Bausch and Lomb 505 recording spectrophotometer over a 1-cm path, against distilled water as reference. At fast scanning speeds this machine shifts wavelength peaks 3 m~i toward the red; numbers quoted in the text are corrected for this error and for the solvent contribution due to use of a water reference cell, but the figures are unretouched tracings from the machine records.
Results.-Reversible bleaching of rhodopsin in digitonin: The spectra of Figure 1 show the photoreversal of bleaching of rhodopsin in digitonin. The presence of 1 111 sodium chloride stabilizes one or more of the intermediates and retards its decay so that after 3 '/2 hours of irradiation at 220C (curves 2 and A), the optical density is changing at a rate of less than 0.01 units per hour. With the intermediate(s) thus stabilized, steady illumination with light of 426 mA (curve 3) can be seen to photoregenerate a fraction of the rhodopsin.'0 Light at 456 mu will also photoregenerate a small amount of pigment, about one third as much as the 426-m/t light. One sample was still exhibiting photoregeneration after six hours of irradiation at 220C, which indicated that hydrolysis of the intermediates to retinal and opsin was not yet complete."I For comparison, see Matthews et al.'I who quote one hour as an adequate time for hydrolysis to retinal and opsin in digitonin solutions without the sodium chloride.
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FIG. 1.-(a) Photoreversal of rhodopsin bleaching in digitonin solution, 1 M in sodium chloride. (1) Bullfrog visual pigments in digitonin, 220C, pH 6.6. (2) Immediately after exposure to 570, m~ light, 0.25 Muw/cm2 for 3'/2 hr. (3) Immediately after subsequent exposure to 426-mMu light0.09Mw/cm2 for 1 hr. (b) Difference spectra of Fig. 1(a). (A) o o ai, Curve 1 -curve 2, peaks at 508 i 2 mMu and 387 ± 2 mg. (B) * * , Curve 2 -curve 3, rhodopsin (with probably a small amount of isorhodopsin) photoregenerated with peak at 511 ± 2 mM.
It is possible to demonstrate the reversibility of rhodopsin bleaching in digitonin 0.0 through several cycles. In Figure byo03^s driving the regenerated fraction of rhodopsin 2 are shown the spectra when the solution is irradiated alternately with 57O-m~s changes in optical density at long wavelengths are and 01 426-mM light at 30 C. The N N~~~~~~~~~~~0 essentially reversible, while the changes at shorter wavelengths are more complex. The first exposure to 426 my causes a greater density loss, with a peak at longer wavelengths (curve B), than the second 426-mu. irradiation (curve D). However,
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FIG. 2. (a) Photic cycling of rhodopsin. (1) Bullfrog visual pigments in digitonin, 30c, pH 0.202mw/cm2 for 9 hr. (3) After 6.6, 1 M in sodium chloride. (2) After 570-my irradiation, subsequent exposure to 426-mM light, 0.09mw/cm2 for 2 hr. (4) After additional exposure to 570 mMu, 0.25 Muw/cm2 for 1 hr. (5) After final 426-me irradiation, 0.09 Muw/cm2 for 2 hr. (b) Difference spectra for Fig. 2(a). (B)(a), Curve 2-curve 3. 42-mM, photoregenerates rhodopsin. (B') 000,O Corrected for bleaching of blue-sensitive pigment. (C) A A A\, Curve 3 -curve 4. Second 570-nd/8 exposure. (D) X X X, Curve 4 - curve 5. Final 426-m(i exposure photore-
generates rhodopsin again, completing two cycles.
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if one assumes that the first exposure to 426 m~i bleached the blue-sensitive pigment of the frog retina6' 12, 13 and converted it to a product absorbing in the ultraviolet, curve B can be corrected for this extra density loss. Curve B' is constructed by assuming that the sample contains 6 per cent as much blue-sensitive pigment as rhodopsin (as found by Dartnall6 under comparable conditions) and subtracting this contribution from curve B. After this correction, the difference spectra B' and D are more nearly alike. Irreversible bleaching of rhodopsin in CTAB: The bleaching of rhodopsin is more rapid in CTAB at 220C than in digitonin. Figure 3a shows that bleaching is almost complete within 30 minutes; within one hour the peak at 387 i 2 my corresponding to the product is completely stable. Furthermore the gain in optical density after bleaching matches well the spectrum of retinal. Subsequent irradiation of the solution (see Fig. 4a) with 426-mL light produced only a loss of optical density, nearly identical to that caused by irradiation of crystalline all-trans retinal in CTAB under the same conditions (Fig. 4b). Thus the hydrolysis of the intermediates to retinal and opsin must be complete. The CTAB preparation of visual pigments nevertheless exhibits a slightly greater loss than pure retinal in the region around 440 m~i after the irradiation with blue light. This additional density loss may be due to the breakdown of an extraneous photosensitive pigment formed artifactually from retinal that has combined with proteins in the solution (see ref. 1), or it may be due to a small amount of the bluesensitive pigment normally found in digitonin preparations of pigments from frog retinas. The amount of blue-sensitive pigment that would account for the difference between the two curves of Figure 4b is about half that found in digitonin preparations, which suggests that CTAB may have destroyed part of the bluesensitive pigment, and perhaps all of it.'4 During a period of darkness following the irradiation with blue light, there is no regeneration of rhodopsin in CTAB solutions. Although irradiation of rhodopsin with blue light stimulates subsequent regeneration in digitonin solutions,4 this does not seem to be true for CTAB. 10-
1,0-
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FIG. 3.-(a) Rapid bleaching of rhodopsin to retinal and opsin in CTAB. (1) Bullfrog visual pigments in CTAB, 22 C, pH 6.6, 1 M in sodium chloride. (2) After exposure to 570-xnji light, 0.25 MAw/cm2 for 30 min (3) After additional 30 min of 570-mg light. (b) Difference spectrum of Fig. 3 (a). cn oc, Curve 1 - curve 3, peaks at 506 ±t 2 mju, 387 4d 2 mjs. X X X, Spectrum of crystalline all trans retinal in CTAB, appropriately scaled.
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PROC. N. A. S.
1.0
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FIG. 4.-(a) Loss of rhodopsin regenerability in CTAB. (1) Bullfrog rhodopsin in CTAB
220C, pH 6.6, 1 M in sodium chloride. (2) After 570-mg irradiation, 0.25 ,uw/cm'. (3) After
426-nmi irradiation, 0.09 ,gw/cm2, for 2 additional hr. (b) Difference spectra for 426 mju irradiation. A A A, Curve 2 - curve 3. Optical density loss of product of rhodopsin bleaching. 0 0 0, Optical
density loss of crystalline all-trans retinal in CTAB when irradiated with 426-m/ light.
Discussion.-Stabilization of rhodopoin and its intermediates of bleaching by inorganic salts: Inorganic salts have been previously observed to stabilize rhodopsin and its intermediates of bleaching in digitonin solutions. Lythgoe and Quilliam'5 reported that 2 Il sodium chloride substantially retarded the thermal decomposition of rhodopsin at 360C; Bridges"6 found that 3.3 M ammonium sulfate stabilized an orange intermediate that decayed only when the solution was diluted to half strength. Since there is no spectroscopic evidence in my experiments for the stabilization of an orange intermediate, it seems logical to assume that metarhodopsin II (X max 380 mA,"1 called M\IRH380 by Ostroy, Erhardt, and Abrahamson)"7 is the principal intermediate that is stabilized by the 1 M sodium chloride. If metarhodopsin II is the intermediate that is stabilized and is responsible for the photoregeneration of rhodopsin in the experiments of Figures 1 and 2, we should expect less regeneration with light of longer wavelengths, which is only weakly absorbed by metarhodopsin II (cf. ref. 18). Since only one third as much rhodopsin is regenerated with light of 456 m/A as with light of 426 m/A, our expectation is fulfilled. Two additional observations by other authors are consistent with the suggestion that metarhodopsin II is stabilized by sodium chloride. According to Ostroy et al."7 the decay of metarhodopsin II (MRH380) is probably the rate-limiting step in the thermal stages of bleaching at physiological temperatures. Thus the stabilization of metarhodopsin II would be expected to prolong the bleaching of rhodopsin, as was observed. In addition, Matthews et al." reported that neutral salts shift the equilibrium between metarhodopsin I and metarhodopsin II toward the latter. This shift might be another manifestation of the stabilization of metarhodopsin II. Significance of photoregeneration with steady light: The photoreversal of rhodopsin bleaching in digitonin solution has been demonstrated in the past by the use of intense flashes of light (e.g. ref. 2) or by steady illumination at low temperatures (e.g., refs. 18 and 19). The present studies merely emphasize that, under conditions where the intermediates are relatively stable, steady illumination at room
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temperature can also produce measurable photoregeneration. Furthermore, Reuterl4, 20 has presented evidence that steady illumination can cause photoregeneration in the frog retina. Thus this phenomenon may have physiological significance. One must realize, though, that photoregeneration in the retina (unlike that in most preparations of rhodopsin) may be limited by the vitamin A cycle. Dartnall21, 22 has reported that rhodopsin in aged frog rods bleaches to a stable intermediate with an estimated X max of 460-470 m1A (cf. MRH465 of Ostroy et al.;'7 465-mA compound of Matthews et al."). In fresh preparations of rods that still have active enzymes, however, the intermediate disappears and a vitamin A peak appears. This implies that the lifetime of the intermediate in vivo, and hence its participation in the photoregeneration of rhodopsin, may be reduced by the vitamin A cycle. CTAB as a solubilizing agent for visual pigments: The data already presented show that CTAB accelerates the decomposition to retinal and opsin of the intermediates of rhodopsin bleaching. This accounts for the absence of photoregeneration during illumination with blue light, but it does not prove that photoreversal of bleaching is impossible in CTAB. It remains to be seen whether intense flashes of light, for example, could cause some reversal of bleaching by forcing the shortlived intermediates to absorb light. Nevertheless it seems clear that CTAB blocks the reversal of bleaching (i.e., resynthesis from retinal and opsin) once hydrolysis has taken place, and this together with the other properties of the detergent suggests at least two possible applications: (1) If one wished to study regeneration of rhodopsin in the retina (in the manner of Reuter140' 23) without possible complications arising from regeneration in the solutions used for measurement, OTAB might be a valuable tool.24 Its fast and quantitative solubilizing action should also be helpful in accurate determinations of the rhodopsin content of the retina. (2) The rapidity of bleaching in CTAB could facilitate studies of reactions that are slow in digitonin-for example, the decay of MRH38o06 (metarhodopsin II). Summary.-The reversibility of rhodopsin bleaching in solution depends on the detergent used to solubilize the pigment. In digitonin solution the presence of 1 M sodium chloride retarded the bleaching of rhodopsin (probably through the stabilization of metarhodopsin II), which made it possible to observe photoreversal of bleaching during steady illumination at room temperature. However, in cetyltrimethylammonium bromide (CTAB) (also 1 M in sodium chloride) the intermediates of bleaching decayed much more rapidly, and no regeneration was observed either during or after illumination. Since CTAB quantitatively solubilizes rhodopsin and prevents regeneration in solution, it may be useful as a solubilizing agent in studies of pigment content and regeneration in the retina. I would like to thank Dr. D. D. Dziewiatkowski for providing laboratory facilities and encouragment during these experiments, and Dr. D. C. Mauzerall and Dr. F. P. Woodford for their helpful advice and criticism during preparation of this report. I am also grateful to Dr. Alan F. Hofmann for guidance in the uses of detergents, and to Mr. It. Millecchia and Mr. P. Rosen for assistance in measuring the light intensities quoted.
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* The preparation of this report was supported in part by a research grant (NB 864) from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. 1 Wald, G., and P. K. Brown, "Synthesis and bleaching of rhodopsin," Nature, 177, 174-176 (1956). 2 Williams, T. P., "Photoreversal of rhodopsin bleaching," J. Gen. Physiol., 47, 679-689 (1964). 3 Bridges, C. D. B., "Cationic extracting agents for rhodopsin and their mode of action," Biochem. J., 66, 375-383 (1957). 4Chase, A. M., and E. L. Smith, "Regeneration of visual purple in solution," J. Gen. Physiol., 23, 21-39 (1939). 5 Lythgoe, R. J., "The absorption spectrum of visual purple and of indicator yellow," J. Physiol., 89, 331-358 (1937). 6 Dartnall, H. J. A., The Visual Pigments (London: Methuen and Co., 1957). 7 Wald, G., "On rhodopsin in solution," J. Gen. Physiol., 21, 795-832 (1937-38). 8 Colowick, S. P. and N. 0. Kaplan, eds., Methods in Enzymology (New York: Academic Press, 1955), vol. 2, p. 686. 9 Crescitelli, F., and H. J. A. Dartnall, "Photosensitive pigment of the carp retina," J. Physiol., 125, 607-627 (1954). 10 There is probably a small amount of isorhodopsin generated as well,18 but I shall continue, for simplicity's sake, to refer to the regenerated fraction as rhodopsin. 11 Matthews, R. G., R. Hubbard, P. K. Brown, and G. Wald, "Tautomeric forms of metarhodopsin," J. Gen. Physiol., 47, 215-240 (1963). 12 Donner, K. O., and T. Reuter, "The spectral sensitivity and photopigment of the green rods in the frog's retina," Vision Res., 2, 357-372 (1962). 13 Chapman, R. M., "Spectral sensitivity of single neural units in the bullfrog retina," J. Opt. Soc. Am., 51, 1102-1112 (1961). 14 The blue-sensitive pigment is known to be preferentially destroyed by another agent, hydroxylamine, while rhodopsin remains intact. See (a) Dartnall, H. J. A., "The visual pigment of the green rods," Vision Res., 7, 1-16 (1967); and (b) Reuter, T., "The synthesis of photosensitive pigments in the rods of the frog's retina," Vision Res., 6, 15-28 (1966). 15 Lythgoe, R. J., and J. P. Quilliam, "The thermal decomposition of visual purple," J. Physiol., 93, 24-38 (1938). 16 Bridges, C. D. B., "Bleaching of visual purple in solutions of inorganic salts," Nature, 178, 860-861 (1956). 17 Ostroy, S. E., F. Erhardt, and E. W. Abrahamson, "Protein configuration changes in the photolysis of rhodopsin. II. The sequence of intermediates in thermal decay of cattle metarhodopsin in vitro," Biochim. Biophys. Acta, 112, 265-277 (1966). 18 Hubbard, R., and A. Kropf, (1958). "The action of light on rhodopsin," these PROCEEDINGS, 44, 130-139 (1958). 19 Yoshizawa, T., and G. Wald, "Pre-Lumirhodopsin and the bleaching of visual pigments," Nature, 197, 1279-1286 (1963). 20 Reuter, T., "Formation of isorhodopsin in the frog's eye during continuous illumination," Nature, 204, 784-785 (1964). 21 Dartnall, H. J. A., "Visual pigments before and after extraction from visual cells," Proc. Roy. Soc. (London), Ser. B, 154, 250-266 (1961). 22 Dartuall, H. J. A., "The photobiology of visual processes," in The Eye, ed. 11. Davson (New York: Academic Press, 1962), vol. 2. 23 Reuter, T., "Kinetics of rhodopsin regeneration in the eye of the frog," Nature, 202, 11191120 (1964). 24 An article by Bliss ("Properties of the pigment layer factor in the regeneration of rhodopsin," J. Biol. Chem., 193, 525-531 (1951)) anticipated part of this suggestion. Bliss used deoxycholate to block the synthesis of rhodopsin, but not to solubilize the pigment.
