Abstract

The photochemical system upon which the excitation of rod vision depends has been analyzed, and all its component processes brought into free solution. The only action of light in this system is to convert rhodopsin to the highly unstable lumi-rhodopsin. This bleaches in the dark via the intermediate meta-rhodopsin to a mixture of the carotenoid, retinene1, and the colorless protein, opsin. Retinene1 is reduced to vitamin A1 by the coenzyme, reduced cozymase, acting in concert with the enzyme retinene reductase or alcohol dehydrogenase. These are the degradative processes in the rhodopsin system.

The resynthesis of rhodopsin from these products is the basis of dark adaptation. A mixture of opsin and retinene1 forms rhodopsin spontaneously in the dark. The retinene reductase system, left to itself, reduces retinene1 to vitamin A1. In the presence of opsin, however, it oxidizes vitamin A1 to retinene1 as rapidly as retinene1, condenses with opsin to form rhodopsin.

A mixture of four substances in solution performs all the reactions of the rhodopsin system: vitamin A1, cozymase, alcohol dehydrogenase, and opsin. Opsin is the only one of these specific to the retina.

The synthesis of rhodopsin requires the presence of free sulfhydryl (−SH) groups in opsin. Conversely when rhodopsin bleaches, two such groups are liberated for each retinene1 formed. Based upon these changes, an artificial system has been devised in which the bleaching of rhodopsin results in an electrical variation. This may provide a model for the excitation process in rod vision.

© 1951 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. G. Wald, Documenta Ophth. 3, 94 (1949a).
    [Crossref]
  2. G. Wald, J. Gen. Physiol. 19, 351 (1935–36); J. Gen. Physiol. 21, 795 (1937–38).
    [Crossref]
  3. G. Wald and A.-B. Clark, J. Gen. Physiol. 21, 93 (1937–38).
    [Crossref]
  4. H. K. Hartline and P. R. McDonald, J. Cellular Comp. Physiol. 30, 225 (1947).
    [Crossref]
  5. R. C. C. St. George and G. Wald, Biol. Bull. 97, 248 (1949).
  6. G. Wald, Harvey Lectures 41, 117 (1945–46).
  7. Wald, Durell, and St. George, Science 111, 179 (1950).
    [Crossref] [PubMed]
  8. Ball, Goodwin, and Morton, Biochem. J. 42, 516 (1948).
  9. G. Wald and R. Hubbard, J. Gen. Physiol. 32, 367 (1948–1949).
    [Crossref]
  10. G. Wald, J. Gen. Physiol. 31, 489 (1947–1948).
    [Crossref]
  11. G. Wald, Science 109, 482 (1949); Biochim. et Biophys. Acta 4, 215 (1950a).
    [Crossref] [PubMed]
  12. A. F. Bliss, Biol. Bull. 97, 221 (1949).
  13. R. Hubbard and G. Wald, Proc. Nat. Acad. Sci. 37, 69 (1951).
    [Crossref]
  14. Hecht, Chase, Shlaer, and Haig, Science 84, 331 (1936).
    [Crossref] [PubMed]
  15. A. M. Chase and E. L. Smith, J. Gen. Physiol. 23, 21 (1939–1940).
    [Crossref]
  16. G. Wald and P. K. Brown, Proc. Nat. Acad. Sci. 36, 84 (1950).
    [Crossref]
  17. G. Wald and R. Hubbard, Proc. Nat. Acad. Sci. 36, 92 (1950).
    [Crossref]
  18. This is not its only function. The pigment epithelium contains also water-soluble, heat-labile factors, apparently protein, which aid the synthesis of rhodopsin (A. F. Bliss, Fed. Proc. 9, 12 (1950); reference 14). These have not yet been identified.
  19. G. Wald and P. K. Brown, Fed. Proc. 10, 266 (1951a). Also J. Gen. Physiol. to be published (1951–52). G. Wald and P. K. Brown, Science 113, 474 (1951b).
    [Crossref]
  20. I. M. Kolthoff and W. E. Harris, Ind. Eng. Chem., Anal. Ed.  18, 161 (1946).

1951 (2)

R. Hubbard and G. Wald, Proc. Nat. Acad. Sci. 37, 69 (1951).
[Crossref]

G. Wald and P. K. Brown, Fed. Proc. 10, 266 (1951a). Also J. Gen. Physiol. to be published (1951–52). G. Wald and P. K. Brown, Science 113, 474 (1951b).
[Crossref]

1950 (4)

G. Wald and P. K. Brown, Proc. Nat. Acad. Sci. 36, 84 (1950).
[Crossref]

G. Wald and R. Hubbard, Proc. Nat. Acad. Sci. 36, 92 (1950).
[Crossref]

This is not its only function. The pigment epithelium contains also water-soluble, heat-labile factors, apparently protein, which aid the synthesis of rhodopsin (A. F. Bliss, Fed. Proc. 9, 12 (1950); reference 14). These have not yet been identified.

Wald, Durell, and St. George, Science 111, 179 (1950).
[Crossref] [PubMed]

1949 (4)

G. Wald, Documenta Ophth. 3, 94 (1949a).
[Crossref]

G. Wald, Science 109, 482 (1949); Biochim. et Biophys. Acta 4, 215 (1950a).
[Crossref] [PubMed]

A. F. Bliss, Biol. Bull. 97, 221 (1949).

R. C. C. St. George and G. Wald, Biol. Bull. 97, 248 (1949).

1948 (1)

Ball, Goodwin, and Morton, Biochem. J. 42, 516 (1948).

1947 (1)

H. K. Hartline and P. R. McDonald, J. Cellular Comp. Physiol. 30, 225 (1947).
[Crossref]

1946 (1)

I. M. Kolthoff and W. E. Harris, Ind. Eng. Chem., Anal. Ed.  18, 161 (1946).

1936 (1)

Hecht, Chase, Shlaer, and Haig, Science 84, 331 (1936).
[Crossref] [PubMed]

Ball,

Ball, Goodwin, and Morton, Biochem. J. 42, 516 (1948).

Bliss, A. F.

This is not its only function. The pigment epithelium contains also water-soluble, heat-labile factors, apparently protein, which aid the synthesis of rhodopsin (A. F. Bliss, Fed. Proc. 9, 12 (1950); reference 14). These have not yet been identified.

A. F. Bliss, Biol. Bull. 97, 221 (1949).

Brown, P. K.

G. Wald and P. K. Brown, Fed. Proc. 10, 266 (1951a). Also J. Gen. Physiol. to be published (1951–52). G. Wald and P. K. Brown, Science 113, 474 (1951b).
[Crossref]

G. Wald and P. K. Brown, Proc. Nat. Acad. Sci. 36, 84 (1950).
[Crossref]

Chase,

Hecht, Chase, Shlaer, and Haig, Science 84, 331 (1936).
[Crossref] [PubMed]

Chase, A. M.

A. M. Chase and E. L. Smith, J. Gen. Physiol. 23, 21 (1939–1940).
[Crossref]

Clark, A.-B.

G. Wald and A.-B. Clark, J. Gen. Physiol. 21, 93 (1937–38).
[Crossref]

Durell,

Wald, Durell, and St. George, Science 111, 179 (1950).
[Crossref] [PubMed]

Goodwin,

Ball, Goodwin, and Morton, Biochem. J. 42, 516 (1948).

Haig,

Hecht, Chase, Shlaer, and Haig, Science 84, 331 (1936).
[Crossref] [PubMed]

Harris, W. E.

I. M. Kolthoff and W. E. Harris, Ind. Eng. Chem., Anal. Ed.  18, 161 (1946).

Hartline, H. K.

H. K. Hartline and P. R. McDonald, J. Cellular Comp. Physiol. 30, 225 (1947).
[Crossref]

Hecht,

Hecht, Chase, Shlaer, and Haig, Science 84, 331 (1936).
[Crossref] [PubMed]

Hubbard, R.

R. Hubbard and G. Wald, Proc. Nat. Acad. Sci. 37, 69 (1951).
[Crossref]

G. Wald and R. Hubbard, Proc. Nat. Acad. Sci. 36, 92 (1950).
[Crossref]

G. Wald and R. Hubbard, J. Gen. Physiol. 32, 367 (1948–1949).
[Crossref]

Kolthoff, I. M.

I. M. Kolthoff and W. E. Harris, Ind. Eng. Chem., Anal. Ed.  18, 161 (1946).

McDonald, P. R.

H. K. Hartline and P. R. McDonald, J. Cellular Comp. Physiol. 30, 225 (1947).
[Crossref]

Morton,

Ball, Goodwin, and Morton, Biochem. J. 42, 516 (1948).

Shlaer,

Hecht, Chase, Shlaer, and Haig, Science 84, 331 (1936).
[Crossref] [PubMed]

Smith, E. L.

A. M. Chase and E. L. Smith, J. Gen. Physiol. 23, 21 (1939–1940).
[Crossref]

St. George,

Wald, Durell, and St. George, Science 111, 179 (1950).
[Crossref] [PubMed]

St. George, R. C. C.

R. C. C. St. George and G. Wald, Biol. Bull. 97, 248 (1949).

Wald,

Wald, Durell, and St. George, Science 111, 179 (1950).
[Crossref] [PubMed]

Wald, G.

R. Hubbard and G. Wald, Proc. Nat. Acad. Sci. 37, 69 (1951).
[Crossref]

G. Wald and P. K. Brown, Fed. Proc. 10, 266 (1951a). Also J. Gen. Physiol. to be published (1951–52). G. Wald and P. K. Brown, Science 113, 474 (1951b).
[Crossref]

G. Wald and R. Hubbard, Proc. Nat. Acad. Sci. 36, 92 (1950).
[Crossref]

G. Wald and P. K. Brown, Proc. Nat. Acad. Sci. 36, 84 (1950).
[Crossref]

G. Wald, Science 109, 482 (1949); Biochim. et Biophys. Acta 4, 215 (1950a).
[Crossref] [PubMed]

G. Wald, Documenta Ophth. 3, 94 (1949a).
[Crossref]

R. C. C. St. George and G. Wald, Biol. Bull. 97, 248 (1949).

G. Wald and R. Hubbard, J. Gen. Physiol. 32, 367 (1948–1949).
[Crossref]

G. Wald, J. Gen. Physiol. 31, 489 (1947–1948).
[Crossref]

G. Wald, Harvey Lectures 41, 117 (1945–46).

G. Wald and A.-B. Clark, J. Gen. Physiol. 21, 93 (1937–38).
[Crossref]

G. Wald, J. Gen. Physiol. 19, 351 (1935–36); J. Gen. Physiol. 21, 795 (1937–38).
[Crossref]

Biochem. J. (1)

Ball, Goodwin, and Morton, Biochem. J. 42, 516 (1948).

Biol. Bull. (2)

R. C. C. St. George and G. Wald, Biol. Bull. 97, 248 (1949).

A. F. Bliss, Biol. Bull. 97, 221 (1949).

Documenta Ophth. (1)

G. Wald, Documenta Ophth. 3, 94 (1949a).
[Crossref]

Fed. Proc. (2)

This is not its only function. The pigment epithelium contains also water-soluble, heat-labile factors, apparently protein, which aid the synthesis of rhodopsin (A. F. Bliss, Fed. Proc. 9, 12 (1950); reference 14). These have not yet been identified.

G. Wald and P. K. Brown, Fed. Proc. 10, 266 (1951a). Also J. Gen. Physiol. to be published (1951–52). G. Wald and P. K. Brown, Science 113, 474 (1951b).
[Crossref]

Harvey Lectures (1)

G. Wald, Harvey Lectures 41, 117 (1945–46).

Ind. Eng. Chem. (1)

I. M. Kolthoff and W. E. Harris, Ind. Eng. Chem., Anal. Ed.  18, 161 (1946).

J. Cellular Comp. Physiol. (1)

H. K. Hartline and P. R. McDonald, J. Cellular Comp. Physiol. 30, 225 (1947).
[Crossref]

J. Gen. Physiol. (5)

A. M. Chase and E. L. Smith, J. Gen. Physiol. 23, 21 (1939–1940).
[Crossref]

G. Wald, J. Gen. Physiol. 19, 351 (1935–36); J. Gen. Physiol. 21, 795 (1937–38).
[Crossref]

G. Wald and A.-B. Clark, J. Gen. Physiol. 21, 93 (1937–38).
[Crossref]

G. Wald and R. Hubbard, J. Gen. Physiol. 32, 367 (1948–1949).
[Crossref]

G. Wald, J. Gen. Physiol. 31, 489 (1947–1948).
[Crossref]

Proc. Nat. Acad. Sci. (3)

G. Wald and P. K. Brown, Proc. Nat. Acad. Sci. 36, 84 (1950).
[Crossref]

G. Wald and R. Hubbard, Proc. Nat. Acad. Sci. 36, 92 (1950).
[Crossref]

R. Hubbard and G. Wald, Proc. Nat. Acad. Sci. 37, 69 (1951).
[Crossref]

Science (3)

Hecht, Chase, Shlaer, and Haig, Science 84, 331 (1936).
[Crossref] [PubMed]

G. Wald, Science 109, 482 (1949); Biochim. et Biophys. Acta 4, 215 (1950a).
[Crossref] [PubMed]

Wald, Durell, and St. George, Science 111, 179 (1950).
[Crossref] [PubMed]

Cited By

OSA participates in Crossref's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (11)

Fig. 1
Fig. 1

Spectra of rhodopsin and of the product of its bleaching in aqueous solution. Bullfrog rhodopsin in solution in 2 percent aqueous digitonin at pH 5.55. The spectrum of rhodopsin consists of three absorption bands. On bleaching, the α- and β-bands of the carotenoid prosthetic group are replaced by the spectrum of retinene1, with an absorption maximum at about 385 mμ; the protein γ-band remains unchanged (from reference 1).

Fig. 2
Fig. 2

The absorption spectrum of frog rhodopsin in aqueous solution, compared with the action spectrum for the bleaching of rhodopsin in solution, and with the spectral sensitivity of human rod vision in the aphakic (lensless) eye. The bleaching spectrum is from Schneider, Goodeve, and Lythgoe, Proc. Roy. Soc. (London), Ser. A 170, 102 (1939) and Goodeve, Lythgoe, and Schneider, Proc. Roy. Soc. (London), Ser. B 130, 380 (1941–42); at low wavelengths it descends toward a point at 254 mμ, not shown in this figure. The human spectral sensitivity data are from Wald (Science 101, 653 (1945)), since revised in the ultraviolet. Both the bleaching spectrum and the visual sensitivity data have been quantized; in both cases “sensitivity” represents the reciprocal of the numbers of quanta at various wavelengths needed to produce a constant effect. (Modified from Wald, reference 1.)

Fig. 3
Fig. 3

Absorption spectra of rhodopsins from various animals, and of porphyropsin from the yellow perch. The bullfrog preparations (average of 3) were made by R. Hubbard and P. K. Brown; those of cattle and squid by R. C. C. St. George; and the yellow perch preparation by P. K. Brown. (From Wald, science 113, 287 (1951).)

Fig. 4
Fig. 4

Bleaching of rhodopsin at low temperatures in a glycerol-water mixture (2:1). The absorption spectrum was measured at 23°C, and again at −45°C. The solution was then exposed to bright white light at the low temperature until all changes were complete (lumi-rhodopsin). It was then warmed to −15° in darkness, left at this temperature until all changes were completed, and was re-cooled to −55° prior to re-measurement (meta-rhodopsin). Finally the solution was warmed to room temperature in the dark, and the spectrum of the final product was measured. This was a mixture of regenerated rhodopsin and retinene1+opsin in roughly equal amounts. All these spectra have been corrected for changes in volume of the solvent with the changing temperatures (from reference 8).

Fig. 5
Fig. 5

Bleaching of rhodopsin in a gelatine film. The spectrum was first measured in a film dried over calcium sulfate. The film was then exposed to the instantaneous illumination of a photoflash lamp, and the spectrum recorded within the first minute thereafter (lumi-rhodopsin). After about 1 hour in the dark at room temperature, the spectrum was re-measured (meta-rhodopsin). These changes were complete; a second exposure to a photoflash lamp produced no further effect. The film was then soaked in M/15 neutral phosphate buffer for 10 minutes and re-dried, all in darkness. The spectrum of the final product shows a mixture of regenerated rhodopsin and retinene1+opsin in roughly equal amounts (from reference 8).

Fig. 6
Fig. 6

A photograph of black and white stripes, made with a gelatine film of rhodopsin. The dry film was first exposed to the pattern, forming a “latent image” composed of rhodopsin and meta-rhodopsin. It was then “developed” by wetting in the dark with an aqueous solution of hydroxylamine; this permitted the meta-rhodopsin to bleach to a mixture of retinene1 and opsin. The hydroxylamine prevented any regeneration of rhodopsin during the latter process, by itself condensing with retinene1 to form the colorless retinene1 oxime. This picture was made by P. K. Brown and O. Starobin. (From Wald, Sci. Amer. 183, 32 (1950b).)

Fig. 7
Fig. 7

The action of the retinene reductase system on retinene1 and retinene2. Each preparation contained the retinene reductase protein or apoenzyme, extracted from frog retinas; reduced cozymase (DPN-H2); and retinene1 or retinene2. The control mixtures yielded on extraction the spectra of the retinenes; the experimental mixtures, having been incubated for 2 hours at 23°C, yielded the spectra of the vitamins A (from Wald, reference 12).

Fig. 8
Fig. 8

The synthesis of rhodopsin from opsin and retinene1. A solution of frog opsin was mixed with retinene1 and placed in the dark. The extinction at 500 mμ, shown at the left, rose rapidly as rhodopsin was synthesized (25°C, pH 6.3). At A the product was exposed to daylight for 20 minutes; it bleached to B. The difference in absorption spectrum before and after bleaching (AB) is shown at the right; it has the maximum at about 498 mμ characteristic of regenerated rhodopsin (from Wald and Brown, reference 17).

Fig. 9
Fig. 9

Known components of the rhodopsin system. The intermediate steps in the bleaching of rhodopsin may not all be retraced when retinene1 and opsin recombine to form rhodopsin. The bulk of the rhodopsin system lies within the outer segments of the rods, but it is supplemented with vitamin A1, respiratory factors and oxygen from the blood circulation and the pigment epithelium (from Hubbard and Wald, reference 14).

Fig. 10
Fig. 10

The synthesis of rhodopsin in a system of known components. The upper curve shows the difference spectrum of rhodopsin synthesized in solution by incubating together vitamin A1, cozymase, liver alcohol dehydrogenase, and frog opsin. The lower curve shows the rhodopsin formed in an identical mixture lacking only the alcohol dehydrogenase (from Hubbard and Wald, reference 14).

Fig. 11
Fig. 11

Amperometric silver titration of rhodopsin. On first adding silver nitrate to the rhodopsin preparation in the dark, no current was registered, the sulfhydryl groups present initially removing the silver ions. When all the initial sulfhydryl had been bound, further addition of silver nitrate caused the current to rise linearly. On bleaching the rhodopsin with light, the current fell again, due to the liberation of new sulfhydryl groups. Adding more silver ions restored the current to its former value and beyond. The horizontal distances (0.19, 0.20 ml) measure the silver ion equivalent of the −SH groups liberated on bleaching rhodopsin. Two such groups appear for each retinene1 formed (from Wald and Brown, reference 20).

Equations (1)

Equations on this page are rendered with MathJax. Learn more.

C 19 H 27 CHO retinene 1 + DPN - H 2 retinene reductase C 19 H 27 CH 2 OH + DPN vitamin A 1 .