THE NEO-b ISOMER OF VITAMIN A AND RETINENE

BY PAUL K. BROWN AND GEORGE WALD*

(From the Biological Laboratories, Harvard University, Cambridge, Massachusetts)

(Received for publication, October 19, 1955)

Among the geometrical isomers of vitamin A and retinene, the neo-b isomer occupies a special position. As the precursor of rhodopsin and iodopsin, it plays a central r81e in rod and cone vision (1, 2). It is stored almost exclusively in the eyes of certain Crustacea (3) and is equally dis- tinctive chemically. The four unhindered geometrical isomers of vitamin A have been identified (4, 5); neo-b proved to be a fifth form (Fig. 1). Necessarily, therefore, it possesses a hindered cis linkage, the first such con- figuration to be found in nature. Apparently, also, it is monocis; hence, ei- ther 7- or 11-cis. The synthesis of an 11-cis isomer, which was not neo-b, led us to conclude that the latter must be 7-cis (6). In inferring this, how- ever, we were led astray by a prevalent misconception regarding the con- figuration of one of the components used: the presumed 11-cis isomer synthesized earlier was in fact 11,13-dicis (neo-c). What is apparently the 11-cis isomer has since been synthesized and is identical with neo-b (6).

The present paper describes the preparation and properties of neo-b vitamin A and retinene.

Preparation of Neo-b Retinenc

The first preparation of neo-b retinene yielded a small amount of crys- talline material, which, according to later work, contained about 70 per cent of this isomer (1). Later Dieterle and Robeson (7) prepared a crys- talline mixture of neo-a (13-cis) and neo-b retinene, from which the neo-b crystals were separated by hand and recrystallized.

Two new procedures are described below, a small scale chromatographic purification and a large scale fractional crystallization beginning with the oxidation of vitamin A.

Chromatographic PuriJication-Dry powdered alumina (Merck reagent, “suitable for chromatographic adsorption”) is weighed in a wide dish, 5 per cent of its weight of water is added, and the whole mixture is covered with a layer of petroleum ether. This mixture is stirred until smooth and poured

* This investigation was supported in part by the Rockefeller Foundation and the Office of Naval Research. We are greatly indebted to the Organic Research Labora- tory of Distillation Products Industries, Rochester, New York, for gifts of crystalline all-tram and neo-b retinene, and to Dr. Max Tishler of the Research Laboratories of Merck and Company, Rahway, New Jersey, for a gift of synthetic vitamin A.

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86G NEO-b ISOMER OF VITAMIN A AND RETINENE

into an upright glass tube to form a column. Onto this is poured a con- centrated solution of retinene in petroleum ether; a yellow zone forms high on the column. This is developed with 10 per cent benzene in petroleum ether. The lowest orange-yellow band is cut away and eluted with 1 per cent ethanol in petroleum ether.

Throughout this procedure the retinene is protected from light to pre- vent its isomerization. Mostly, it is kept in the dark or handled in red or orange light, which, since not absorbed by retinene, does not isomerize it. The column is wrapped in black cloth and viewed in a dim white light only when necessary.

After the first chromatography, the pigment consists of a mixture of neo-a and neo-b retinene. After being transferred to fresh petroleum ether, it is chromatographed a second time as before. A diffuse yellow zone forms on the column, of which the first fraction to run through is the pure neo-b

FIG. 1. Structure of all-tram retinene. The double bonds which are in cis con- figuration in the other geometrical isomers are indicated with arrows, the unhindered cis isomers below, and the hindered cis isomers above the diagram.

isomer. This is drawn off in successive small portions until the first con- tamination with neo-a retinene appears, which is marked by a fall in the ratio of extinctions at 251 and 362.5 rnp and a decrease in the changes caused by isomerization (see below). Neo-b retinene prepared in this way has nearly the same properties as the crystalline substance.

Oxidation of Vitamin A to Retinene-Vitamin A is oxidized to retinene by adsorption on manganese dioxide (8, 9), prepared by the procedure of Attenburrow et al. (10). The dry powder, weighing 6 to 7 times as much as the vitamin A to be oxidized, is packed in a glass tube to form a short column. Vitamin A alcohol, the synthetic oil or commercial crystals, both primarily all-trans, dissolved in hexane, is drawn through this column under light suction. A solution of retinene runs off as the filtrate. This is then taken up in petroleum ether and cooled to - 15”. It crystallizes on seeding with a few small crystals of all-trans retinene.

Isomerization-10 gm. of crystalline all-trans retinene are dissolved in 1.5 liters of absolute ethyl alcohol and isomerized by standing in bright

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P. K. BROWN AND 0. WALD 867

sunlight for several hours with constant stirring. Thereafter the retinene is protected from isomerizing light. The alcohol is evaporated under suction at room temperature.

Fruc2ionaZ Crystallization-The residual oil is dissolved in 30 ml. of petroleum ether and passed through a sintered glass Corning F filter. After cooling to -X0, it is seeded with a few small crystals of all-trans retinene. Within 2 to 7 days a copious yield of crystals has formed. The mother liquor is poured off, and the crystals are washed rapidly with cold petroleum ether (- 15”).

The mother liquor and washings are mixed, concentrated to 10 to 15 ml., chilled to - 15”, and seeded with crystals of neo-a retinene. Within about 2 days this isomer has crystallized. The mother liquor is poured off, and the crystals are washed as before.

The mother liquor and washings are again mixed, concentrated to about 10 ml. of petroleum ether, and seeded with crystals of neo-b retinene.’ After 3 to 4 days at - 15’, this isomer has crystallized.

In this procedure the order of crystallization of neo-a and neo-b retinene can be reversed without difficulty. Also we have on occasion left both neo-a and neo-b retinene to complete their crystalliaations for periods approaching a month.

Recrystallization-The first crops of crystals were recrystallized two to four times. For this they were dissolved at room temperature in a small volume of petroleum ether, about 5 ml. per gm. of crystals. The solution was brought to 3-4” and seeded. If it did not crystallize soon, it was cooled to -15”. After crystallization, the mother liquor was poured off, and the crystals were washed with cold petroleum ether. When particularly well formed crystals were wanted, the seeding was performed at room temperature, and the solution was cooled gradually in a water bath.

Yields-By this procedure we have prepared almost 2 gm. of crystalline neo-b retinene. The yields from two runs are shown in Table I. With regard to the neo-b isomer, they are far from maximal. On isomerizing all-trans retinene with light in very dilute solution, about 5 y per ml., we find that about 25 per cent of the final product is the neo-b isomer. The more concentrated the solution, the less cis isomers we have obtained (1). In the experiments cited in Table I, in which concentrations range from 7 to 40 mg. per ml., our final yields of neo-b retinene represented only 1 to 3 per cent of the starting material. This may be owing to incomplete ex- posure of the retinene to light. In such highly concentrated solutions, in which retinene has extinctions of 10,500 to 60,000 in a layer 1 cm. deep, light of isomerizing wave lengths can penetrate only very slightly. Indeed,

1 We are grateful to Dr. C. D. Robeson for the crystalline neo-b retinene with which we performed our initial seeding.

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868 NE&b ISOMER OF VITAMIN A AND RETINENE

at the absorption maximum, the most efficient wave length for isomerization, the light falls to 10 per cent of its incident intensity after penetrating a layer only 0.16 to 1 cc deep. We tried to compensate for this difficulty by stirring, yet probably did not irradiate long enough even under these con- ditions to achieve maximal isomerization.

Physical Properties of Neo-b Retinene

Melting Point-The melting points of two preparations of neo-b retinene, three and four times recrystallized, were 60.3-61.9” and 61.3-62.7” (cor- rected). Dieterle and Robeson (7) have reported slightly higher values (63.5-64.4”)

Absorption Spectrum-The absorption of neo-b retinene in ethyl alcohol is shown in Fig. 2 and in hexane in Fig. 3.2 The positions of the band

TABLE I

Fractional Crystallization of Retinene Isomerate

From 10.8 gm. retinene From 60 gm. retinene

ISJ3llEI No. of Weight No. of

crystalliaations crystallizations Weight

All-trans. . . . . . . . . . . . . . . . . . 1 Neo-a........................... 2 Neo-b........................... 4

cm. m.

7.0 3 52.25 0.23 3 0.80 0.32 3 0.635

maxima in these solvents, and their specific extinctions, are presented in Table II.

The spectrum of neo-b retinene consists of three absorption bands: a main band at A,,, 376.5 rnH (ethyl alcohol), a subsidiary band at 254 rnlr, abnormally large as in other hindered cis compounds, and a small inflection at 290 rnp, which we think represents the “cis-peak” (6).

Antimony Chloride Test-We perform this test by adding 2.2 ml. of 20 per cent antimony chloride in chloroform to 1 ml. of the retinene solution in chloroform, in an absorption cell in a recording spectrophotometer. The spectrum is recorded at once, the pen drawing the absorption maximum about 15 seconds after the reagents are mixed.

All the geometrical isomers of retinene apparently yield the same blue product with antimony chloride, with A,, 666 rnp and E (1 per cent, 1 cm.) 3780 f 140 (11). Four tests with crystalline neo-b retinene yielded an average E (1 per cent, 1 cm.) of 3740.

z All the absorption spectra shown in this paper were measured with the Cary re- cording spectrophotometer. The figures show the records drawn by this instrument, mounted for publication.

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P. K. BROWN AND G. WALD 869

Reduction of Neo-b Retinene to Neo-b Vitamin A

The geometrical isomers of retinene are reduced quantitatively to the corresponding isomers of vitamin A by potassium borohydride (KBH4). A careful study of this process with all the available retinene isomers shows that it involves no appreciable isomerization; so, for example, the prod- ucts obtained have the same molar extinctions as given by Robeson et al.

TABLE II

Properties of Neo-b Vitamin A and Retinene The numbers in parentheses are values of E (1 per cent, 1 cm.). The molar ex-

tinctions are obtained by multiplying E (1 per cent, 1 cm.) by 28.6 in the case of vita- min A and by 28.4 in the case of retinene.

Amax bw) in Ethyl alcohol

Hexane

Antimony chloride product Rise in E,., of main band on

Reduction in ethyl alcohol Isomerization in hexane

I‘ “ ethyl alcohol Rhodopsin formation (calculated)

I‘ “ (observed) Fall in E,., of subsidiary band on

Isomerization in hexane “ “ ethyl alcohol

Vitamin A Retinene

319 (1220) 233 (370)

318 (1200) 233 (370)

618 (44OO)t

1.45 f 0.05

0.55 (at 233 rnp)

376.5 (878)* 290 (412) 254 (614) 362.5 (928) 282 (400) 251 (600) 666 (3740)

1.39 1.70 1.3 1.87 1.7

0.40 (at 251 rnp) 0.64 (“ 254 “ )

* Dieterle and Robeson (7) give the following values for the E (1 per cent, 1 cm.) of crystalline neo-b retinene in ethyl alcohol: at 376 nq.~, 857; at 255 n+, 595.

t Cawley et al. (15).

(5) for crystalline geometrical isomers of vitamin A. For these reasons we find that the reduction with borohydride can be used not only to pre- pare the single geometrical isomers of vitamin A, but also to relate their properties quantitatively to those of the corresponding retinenes and to identify the latter.

Procedure-Fig. 2 shows the reaction in its simplest form. To a solution of neo-b retinene in ethyl alcohol, in an absorption cell in the spectropho- tometer, a few grains (about 1 mg.) of potassium borohydride are added, and the solution is stirred, In this way the concentration remains un- changed and the reaction is completed within a few seconds. The re-

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870 NE&b ISOMER OF VITAMIN A AND RETINENE

duction can be performed equally well with the borohydride dissolved in ethyl alcohol, or with the borohydride in water, added to a solution of retinene in 2 per cent aqueous digitonin.

Properties of Neo-b Vitamin A

Absorption Spectrum-The absorption spectrum of neo-b vitamin A in ethyl alcohol is shown in Fig. 2 and in hexane in Fig. 4. The positions of the absorption bands and estimates of their specific extinctions are shown

FIG. 2. Absorption spectra of neo-b vitamin A and retinene in ethanol (6.8 y per ml.).

in Table II. Neo-b vitamin A possesses a main absorption band at X,,, about 319 rnp (ethyl alcohol) and a cis-peak at about 233 rnp. The differ- ence in position of these absorption bands, 86 rnp, is the same as in neo-b retinene.

Since neo-b vitamin A has not yet been crystallized, its absolute extinc- tion must be inferred from transformations which establish its quantitative relationships to substances of known absolute extinction. We make use of three such transformations: the reduction of neo-b retinene, already de- scribed, the antimony chloride reaction, and isomerization.

On reduction in ethanol, the maximal extinction (E,,,) of neo-b retinene rises on the average 1.39 times (Fig. 2). Since neo-b retinene possesses E

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P. K. BROWN AND G. WALD 871

(1 per cent, 1 cm.) 878, neo-b vitamin A appears to have E = 1.39 X 878 = 1220.

Antimony Chloride Test-On mixing with antimony chloride, all the geometrical isomers of vitamin A apparently yield the same blue product, with X,,, about 618 rnp and E (1 per cent, 1 cm.) about 4400. When we add 2.2 ml. of 20 per cent antimony chloride in chloroform to 1 ml. of a solution of neo-b vitamin A in chloroform, having E,,, 0.425, we record, within 15 seconds, a maximal extinction at 618 rnp of 0.524. Since this procedure dilutes the original solution 3.2 times, the vitamin A in the same concentration would have had the extinction 0.42513.2 = 0.133. The E (1 per cent, 1 cm.) of neo-b vitamin A in chloroform therefore appears to be about (0.133/0.524) X 4400 = 1120.

Isomerization of Neo-b Retinene and Vitamin A

One of the most characteristic properties of neo-b vitamin A or retinene is its behavior on isomerization. Retinene is isomerized by simple expo- sure to light, but isomerizes much more rapidly in the presence of traces of iodine. Vitamin A requires the presence of iodine to isomerize in vis- ible light. When carefully performed, both reactions yield quantitatively accurate results. Fig. 3 shows the isomerization of neo-b retinene and Fig. 4 that of neo-b vitamin A.

Procedure-To a solution containing, retinene or vitamin A in hexane (2 to 5 y per ml.), 1 drop of a stock solution of iodine in hexane is added, bringing the final iodine concentration to about 0.05 y per ml. in the case of retinene, or 0.4 y per ml. in the case of vitamin A. This mixture is irradiated for 1 to 2 minutes with white light of intensity of about 30 ft. candles.

It is difficult to specify these conditions accurately, and in each new instance we check for completion of the reaction. The end point in the case of the neo-b isomer is marked by failure of the extinction to rise on further irradiation. The iodine concentration and irradiation needed to reach this point vary with the hexane employed, presumably because traces of aromatic hydrocarbons absorb the iodine. In tissue extracts the presence of contaminants which take up iodine may make it necessary to use more iodine or light to complete the isomerization.

In such a homopolar solvent as hexane, all the geometrical isomers of retinene and vitamin A isomerize almost entirely to the all-trans condition. With neo-b retinene, the extinction of the main band rises sharply, X,,, shifting from 363 to 367 rnp (Fig. 3). Simultaneously the absorption bands at lower wave lengths fall in extinction until obliterated. The ex- tinction of the main band rises on the average by a factor of 1.70, while that in the region of the subsidiary band at 250 rnp falls to about 0.40 of its original value.

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872 Sno-b ISOMER OF VITAMIX A .4ND RETINENE

The B (1 per cent, 1 cm.) of all-trans retinene in hexane is 1680. Isom- erization with iodine and light lowers this value by a factor of 0.93. The E (1 per cent, 1 cm.) of isomerized retinene is therefore 1560; it appears to be the same regardless of the retinene isomer used as starting material.

0.8

Wavelength-mp

Flo. 3. Absorption spectra of neo-b retinene in hexane and of the product of its isomerization by light in the presence of iodine.

Hence the observation that the E,,, of neo-b retinene rises 1.70 times on isomerization implies that its E (1 per cent, 1 cm.) in hexane is 1560/1.70 = 920. This is almost exactly equal to the value determined directly (928).

The corresponding experiment with neo-b vitamin A yields a similar result (Fig. 4). E,,, of the main band rises 1.45 f 0.05 times, X,,, shift- ing meanwhile from 318 to 325 rnp. Simultaneously the extinction in the

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P. K. BROWN AND G. WALD 873

region of the cis-peak at 322 rnk falls to 0.55 times its original value. The absorption spectrum also changes characteristically in shape: the main band of neo-b vitamin A is broad and relatively symmetrical, whereas that

0.8

250 300 350 400 Wavelength-n-p

FIG. 4. Absorption spectra of neo-b vitamin A in hexane and of the product of its isomerization by light in the presence of iodine.

of the isomerate forms a sharper peak and bears a distinct inflection on its short wave length shoulder.

The E (1 per cent, 1 cm.) of all-trans vitamin A in hexane is 1820 (12). On isomerization with iodine and light, Em,, falls by a factor of 0.94. The E (1 per cent, 1 cm.) of vitamin A isomerate is therefore 1710. Since the Em,,, of neo-b vitamin A rises 1.45 times on isomerization, its E (1 per cent, 1 cm.) in hexane is about 171011.45 = 1180. This is close to the value determined by the reduction of neo-b retinene in alcohol (1220).

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874 NEO-b ISOMER OF VITAMIN A AND RETINENE

In such a polar solvent as ethyl alcohol, retinene isomerizes in the light to yield a much larger proportion of cis isomers than in hexane. An alco- hol solution of neo-b retinene, exposed to white light (no iodine), exhibits the following changes: the extinction of the main band (376.5 mp) rises about 1.3 times, with little change in wave length, while the extinction of the subsidiary band (254 mp) falls to about 0.64 of its former height, a considerable subsidiary band remaining in the final product.

Synthesis of Rhodopsin

Another characteristic property of neo-b retinene is the synthesis of rhodopsin on incubation in the dark with the protein of the retinal rods, opsin (1, 11). The preparation of cattle opsin has been described (13). With this and neo-b retinene, the synthesis of rhodopsin presents little more complication than any other calorimetric reaction. When neo-b retinene is added in excess, the reaction measures opsin; when opsin is in excess, it measures neo-b retinene.

An example of such an experiment is shown in Fig. 5. A solution of cattle opsin in 2 per cent aqueous digitonin was used as the control; the spectrum of opsin or of the opsin moiety of rhodopsin therefore does not appear in the record. The same concentration of opsin was mixed with neo-b retinene in 2 per cent digitonin, and the spectrum was recorded at once (Curve 1). Although completed within 20 seconds after mixing, this spectrum already displayed an inflection at about 500 rnp, owing to the formation of a trace of rhodopsin. The spectra were recorded periodically thereafter in the dark, at the times shown in the legend of Fig. 5. The concentration of rhodopsin (X,,, 498 mp) rose regularly, while that of neo-b retinene (X,,, 380 rnp) fell. The reaction was complete in about 2 hours (22.5”). The small absorption band at about 350 rnp remaining in the final product (Curves 8 and 9) is primarily the @band of rhodopsin. When opsin is in excess, as here, the synthesis of rhodopsin almost entirely re- moves neo-b retinene from solution.

The molar extinction of cattle rhodopsin in aqueous digitonin at 498 rnp is 40,600 (14). Retinene has about 0.87 times the extinction in aqueous digitonin that it displays in alcohol. In digitonin solution, therefore, neo-b retinene has E (1 per cent, 1 cm.) about 0.87 X 878 = 764 and a molar extinction of 21,700. We therefore expect the ratio of the extinction of rhodopsin formed to that of neo-b retinene employed to be 40,600/21,700 = 1.87.

The highest value of this ratio that we have observed experimentally is 1.7. This is achieved, for example, in Fig. 5. We find this same discrep- ancy with our preparations and with crystals obtained from Dieterle and Robeson. It implies at best an “activity” of about 90 per cent. At the

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P. K. BROWN AND G. WALD 875

end of such syntheses of rhodopsin in solution, we find a small residue of retinene, inactive in forming further rhodopsin, the presence of which is

T

0 400 sbo 600 Wavelength-mp

FIQ. 5. Synthesis of rhodopsin. Cattle opsin is mixed with neo-b retinene, in 2 per cent aqueous digitonin solution, and the mixture is incubated in the dark (pH 7.0, 22.5’). The absorption spectrum was recorded periodically, Curve 1 at 0.3, Curve 2 at 2.5, Curve 3 at 5, Curve 4 at 10, Curve 5 at 18, Curve 6 at 30, Curve 7 at 60, Curve 8 at 120, and Curve 9 at 180 minutes. Initial concentrations, opsin, 29 pmoles per liter; retinene, 21 rmoles per liter.

exposed by adding hydroxylamine (0.17 M). This has no effect upon a natural preparation of rhodopsin, but in such synthetic preparations it causes a small lowering of absorption in the region of 415 rnp, and a rise of extinction at about 365 rnp, associated with the formation of retinene oxime (cf. (14)). It is not yet known whether this residue represents a

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876 NEo-b ISOMER OF VITAMIN A AND RETINENE

contamination of neo-b retinene with perhaps a trace of neo-a or is caused by some isomerization of the neo-b isomer in the 2 to 3 hours of incuba- tion with opsin.

DISCUSSION

The properties of neo-b vitamin A and retinene are summarized in Table II. Some of them, particularly the synthesis of rhodopsin and the mag- nitude of the changes in extinction which accompany isomerization and the antimony chloride test, are unique among the geometrical isomers of vitamin A and retinene and readily identify and measure the neo-b isomer in mixtures and tissue extracts (cf. (3)).

Our measurements on crystalline neo-b retinene agree well with those of Dieterle and Robeson. These authors, however, on reducing neo-b retinene with lithium aluminum hydride and sodium borohydride, obtained a product whose properties depart appreciably from those we find in neo-b vitamin A: E (1 per cent, 1 cm.) 940 (322 mp) and 270 (233 mp) in eth- anol. Not knowing the details of their procedure, we cannot explain this discrepancy, other than to suggest that our potassium borohydride is a gentler reducing agent than those used by Dieterle and Robeson, less in- clined to produce side effects, and that such a procedure as illustrated in Fig. 2 avoids such losses as almost inevitably accompany the manipulation of vitamin A. In our experience with other geometrical isomers of retinene, this procedure yields a quantitatively accurate result.

SUMMARY

The neo-b (11-cis) isomer of vitamin A and retinene, the precursor of rhodopsin and iodopsin, is a hindered cis polyene, the first such structure to appear in nature. Its preparation and properties are described, partic- ularly properties useful in its identification and measurement in mixtures and tissue extracts: reduction of the retinene to vitamin A with potassium borohydride, isomerization with iodine and light, the antimony chloride reaction, and the synthesis of rhodopsin.

BIBLIOGRAPHY

1. Hubbard, R., and Wald, G., J. Gen. Physiol., 36,269 (1952-53). 2. Wald, G., Brown, P. K., and Smith, P. H., J. Gen. Physiol., 38,623 (1954-55). 3. Wald, G., and Burg, S. P., Federation PTOC., 14,366 (1955). 4. Robeson, C. D., Blum, W. P., Dieterle, J. M., Cawley, J. D., and Baxter, J. G.,

J. Am. Chem. Sot., 17.4120 (1955). 5. Robeson, C. D., Cawley, J. D., Weisler, L., Stern, M. H., Eddinger, C. C., and

Chechak, A. J., J. Am. Chem. Sot., 77, 4111 (1955). 6. Wald, G., Brown, P. K., Hubbard, R., and Oroshnik, W., Proc. Nat. Acad. SC.,

41, 438 (1955). Oroshnik, W., J. Am. Chem. Sot., 78, 2651 (1956). Oroshnik, W., Brown, P. K., Hubbard, R., and Wald, G., Proc. Nat. Acad. SC., in press.

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7. Dieterle, J. M., and Robeson, C. D., Science, 120,219 (1954). 8. Ball, S., Goodwin, T. W., and Morton, R. A., B&hem. J., 40.59 (1946). 9. Wald, G., J. Gen. Physiol., 31,489 (1947-48).

10. Attenburrow, J., Cameron, A. F. B., Chapman, J. H., Evans, R. M., Hems, B. A., Jansen, A. B. A., and Walker, T., J. Chem. Sot., 1094 (1952).

11. Hubbard, R., Gregerman, R. I., and Wald, G., J. Gen. Physiol., 36,415 (1952-53). 12. Boldingh, J., Cama, H. R., Collins, F. D., Morton, R. A., Gridgeman, N. T.,

Isler, O., Kofler, M., Taylor, R. J., Welland, A. S., and Bradbury, T., Nature, 168, 598 (1951).

13. Wald, G., and Brown, P. K., J. Gen. Physiol., 36,797 (195162). 14. Wald, G., and Brown, P. K., J. Gen. Physiol., 37, 189 (1953-54). 15. Cawley, J. D., Robeson, C. D., Weisler, L., Shantz, E. M., Embree, N. D., and

Baxter, J. G., Science, 107, 346 (1948).

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Paul K. Brown and George WaldAND RETINENE

THE NEO-b ISOMER OF VITAMIN A

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