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δ-Carotene
b-Carotene 1 J. W. Porter 2 and M. M. Murphey From the Department of Agricultural
Chemistry, Purdue University, Received November 27, 1950
3 Lafayette,
Indiana
b-Carotene was first reported by Winterstein (1) who found this pigment to be a constituent of Gonocaryum pyriforme fruit. Later Strain (2) reported d-carotene as a constituent of carrots. This pigment has also been reported to be present in twelve strains of Micrococcus pyogenes var. aureus (3) and in Neurospora crassa (4). In 1946 Porter and Zscheile (5) noted the presence of a-carotene in certain experimental selections of tomatoes. More recently Porter and Lincoln (6) have developed, by breeding and selecting for this pigment, tomato selections containing as much as 40 pg. of this compound/g. fresh fruit. This content of a-carotene, in contrast to traces in other sources, has made possible the analytical work reported in this paper. This work leads to a suggestion of a possible formula for this pigment. EXPERIMENTAL Extraction &Carotene was obtained from experimental tomato selections 4 that. contained between 20 and 40 fig. of this compound/g. fruit. The fruit, was ground in an electric meat grinder, and the resultant “soup” was dehydrated with an approximately equal quantity of 95% ethyl alcohol. The pulp was then separated from the alcohol solution by filtration of the liquid through several thicknesses of cheesecloth. The semidry 1Journal paper No. 423, Purdue University Agricultural Experiment Station. This work WBBsupported in part by a grant from the Nutrition Foundation, Inc. 2 Present address: Biology Division, General Electric Co., Richland, Washington. * Formerly Technical Assistant. 4 (a) Pritchard X Pritchard X I?, (Pritchard X Lycopersicwn hirsutum 127827). (b) Selection 4079-6003 from [Indiana Baltimore X F1 (Rutgers X L. hirsutum 126445)] was crossed with the hybrid (Marglobe Supreme X L. hirsutum 127827). The hybrid wan then selfed and selections were made in succeeding generations. 21
22
J.
W.
PORTER
AND
M.
M.
MURPHEY
pulp was extracted with acetone and hexane (approximately 3:2 proportions) in 5-gal. carboys for approximately 1 hr. The pulp and solvents were stirred with an electric stirrer. The hexane and acetone extract was then filtered through a Btichner funnel with suction. The tomato residue was again extracted once or twice more. This removed practically all of the color from the pulp. The combined hexane extracts were then washed several times with water to remove most of the acetone. The hexane extract was concentrated to a smaller volume, washed again several times with water, and stored at -20°C. until chromatographed.
Chromatographic Separations The hexane extract of tomato carotenoids was chromatographed on a column of MgO-Super Gel 6 (50: 50) and the column was developed with 10% acetone in hexane. &Carotene formed a broad band which clearly separated from other pigments. This compound occupies a position below r-carotene and above prolycopene, but it has never been observed to occur with the latter compound in tomatoes. The &carotene band was scraped from the column into a beaker containing 10% ethanol in hexane. The suspension was then poured into a second column into which about 1 in. of adsorbent had been packed as a filter. The &carotene was quickly eluted with suction and the addition of fresh solvent. The a-carotene solution was washed several times with distilled water, concentrated to a smaller volume, and then saponified with an equal volume of approximately 3 N sodium ethylate for 1 hr. The hexane solution was washed thoroughly with water and then saponified with 20% aqueous sodium hydroxide. The hexane solution was washed several times with water and then chromatographed on a column of MgOSuper Cel as before. Crystallization The washed hexane solution of a-carotene was concentrated to a small volume and an equal volume of absolute ethyl alcohol was added. The solution was then placed at -20°C. overnight. The crystals of &carotene were filtered, and redissolved in a minimum amount of carbon disulfide. The solution was then filtered and an equal volume of filtered hexane was added. Two volumes of filtered ethanol was added and the entire solution (25-50 ml.) was concentrated under a stream of nitrogen until crystals of &carotene appeared. The solution was then warmed until perfectly clear, and crystallization of Z-carotene, in very long red needles, was effected by slowly lowering the temperature of the solution to -20°C. Recrystallization of &carotene was effected at least once more for each%ample.
Quantitative Light Absorption Curve Q-Carotene crystals were dried overnight under a high vacuum. A 2-3 mg. sample was weighed into a small platinum boat, dissolved in isoijctane, and then lightabsorption readings were taken with a Beckman spectrophotometer. Figure 1 shows the quantititative absorption curves obtained for the two best preparations of Gcarotene. Table I gives the specific extinction coefficients at the maxima and minima. 5 Adsorptive Magnesia No. 2641. Westvaco Chlorine Products Co., Newark, California. Johns-Manville Hyflo Super Cel.
&-CAROTENE
WAVE LENGTH,
FIG. 1. Quantitative light-absorption values given were obtained
ANALYTICAL
23
6
curve of S-carotene dissolved in isoiictane. on two separately prepared samples.
The
DETERMINATIONS
Melting Point The melting point of the orange- to red-colored needle-shaped &carotene as determined by the microscope hot-stage method of Zscheile and White 140.4-140.5°c.
crystals, (7), was
Carbon and Hydrogen Calculated for G0HS8: C, 89.15; H, 10.85. Found: C, 89.32, 90.02, 89.90, 89.60, 88.74, 88.85. H, 11.09, 10.52, 10.42, 10.28, 11.22, 11.02. Av. = C, 89.40; H, 10.76. TABLE Specific Extinction
Coeficienfs
Wavelength ii
= Slit width * Slit width
I
of &Carotene at the Maxim
In imiiatane
-&3@
$84
4720 s&560* 4400 JPm 3040 2800
182 315 178 mo 5.5 80
= 0.045 mm. = 0.054 mm.
and Minima
24
J.
W.
PORTER
AND
Molecular
M.
M.
MURPHEY
Weight
Molecular weights were determined in camphor by the Rast micro 0.260 mg. s-carotene in 3.480 mg. camphor (K = 38.4); A = 0.518 mg. &carotene in 3.344 mg. camphor (K = 38.1); A = 0.280 mg. s-carotene in 3.871 mg. camphor (K = 38.1); A = Calculated for CLoHss: 538; Found: 522, 537, 483.
method: 5.5’. 11.0”. 5.7”.
Hydrogen Absorption To 7.397 mg. a-carotene was added 3.26 ml. hydrogen (O”, 760 mm.). To 4.988 mg. &carotene was added 1.95 ml. hydrogen (O”, 760 mm.). Found: 10.6 and 9.6 double bonds.
C-Methyl Groups This determination was made by the method of Kuhn and Roth (8), and the larger number of distillations recomemnded by Ginger were made (9). &Carotene, 4.262 mg., required 1.35 ml. of 0.05 N Ba(OH)z for neutralization of the CH&OOH formed. 8Carotene, 5.200 mg., required 1.55 ml. of base, &Carotene, 4.663 mg., required 1.11 ml. of base. Blanks were each 0.48 ml. Found: 5.43, 5.54, and 5.70 C-methyl groups.
Isopropylidene
Groups
The method of Kuhn and Roth (10) was used. A 9.951-mg. sample of &carotene in 3 ml. acetic acid was ozonized for 2 hr. This sample yielded acetone equivalent to 1.80 ml. of 0.05 N iodine solution. b-Carotene, 9.969 mg., yielded acetone equivalent to 2.20 ml. of 0.05 N If. &Carotene, 10.034 mg., yielded acetone equivalent to 1.89 ml. of 0.05 N IZ. Found: 0.80, 0.97, and 0.82 moles of acetone.
Identification
of Acetone
Dibenzylidene acetone was prepared from 65 mg. &carotene by the procedure of Nash et al. (11). The product, recrystallized from boiling ethanol, melted at 112.1” and did not depress the melting point of an authentic sample of dibenzylidene acetone.
Optical Rotation The specific rotation of b-carotene determined in carbon disulfide using a photoflood lamp and Corning filters Nos. 242 and 244 was 317”.
Biological Activity A sample of 8.672 mg. (87% pure) was dissolved in 201.36 g. of Wesson oil to which 0.84 g: or-tocopherol had been added. One gram of this solution equaled 39.5 drops from a calibrated dropping pipet, and when the weight of b-carotene was calculated (7.545 me.), 1 drop was found to contain 0.949 pg. of this compound. The biological activity was tested by the usual vitamin A bioassay procedure. After a preliminary depletion period four lots of rats were given graded doses of U.S.P.
B-CAROTENE
25
vitamin A Reference Standard and two lots were fed 2.9 and 9.6 pg. &arotene/day for a period of 6 weeks. Whereas all animals receiving 1.0 pg. or more of the Reference Standard/day gained weight and survived the B-week period, none of those receiving b-carotene either gained or survived. Evidently, z-carotene is devoid of vitamin A activity. DISCUSSION
Little work has been done on the chemical nature of &carotene. The empirical formula C40H6ehas been quoted by Strain (12), who also states this compound contains 12 double bonds, 11 of which are in conjugation. In the present work the results of C-methyl determinations suggest an isoprenoid structure for this compound, and molecular-weight determinations are consistent with the assumption of 40 carbon atoms per molecule. The presence of one isopropylidene group per molecule is demonstrated by the results of analytical determinations by the method of Kuhn and Roth and the identification of acetone as the product formed on ozonolysis. The presence of a ring structure at the opposite end of the molecule is suggested by the results of measurements of the optical activity of b-carotene. The presence of 10 conjugated double bonds in the molecule is ascertained from the wavelength of the first light -absorption maximum (11). An examination of the light-absorption curves of the carotenoid hydrocarbons shows that they fall into three groups: b-carotene has broad maxima and shallow minima; a- and y-carotene have somewhat sharper maxima and deeper minima; lycopene, {-carotene (ll), tetrahydrolycopene (13), and phytofluene (14) have still sharper maxima and minima. The light-absorption curve of d-carotene compares very closely to lycopene in this respect. This fact is interpreted to mean that the conjugation in d-carotene does not extend into the ring structure at one end of the molecule. Certainly it does not extend into a ,&ionone ring for the compound has no vitamin A activity. As additional evidence of the restriction of the conjugated double bonds to an open chain, it has been found that the specific extinction coefficient of the longest wavelength maximum falls on a line drawn through the extinction coefficients of lycopene, tetrahydrolycopene, [-carotene, phytofluene, and phytoene (15). The number of isolated double bonds in the molecule is in question. Hydrogenation and C and H values, while erratic, do not indicate more than one such bond per molecule.
26
J. W. PORTER AND M. M. MURPHEY
The fact that S-carotene adsorbs only slightly below y-carotene on a chromatographic column suggests a close relationship between these compounds. d-Carotene also adsorbs below tetrahydrolycopene and above {-carotene. b-Carotene has been found to occur only in tomatoes with lycopene, y- and P-carotene (6). Since it has not been possible to develop a tomato selection homozygous for &carotene, the genes involved in lycopene, y- and p-carotene formation must also be acting in the formation of a-carotene. Therefore, it seems quite certain the structure of this compound must be closely related to those of these three pigments. On the basis of the evidence available on a-carotene it is suggested this compound may be dihydro-y-carotene. The exact location of the conjugated double bonds in b-carotene is not known with certainty. They are placed in agreement with those in lycopene and p-carotene largely on the basis of the adsorptive properties of this compound and its inheritance and occurrence in tomatoes with p- and y-carotenes and lycopene. The absence of an additional isolated double bond is not proven but hydrogenation values argue against this possibility. The position of d-carotene in a scheme postulated for the biosynthesis of carotenes has been set forth in another paper 0%. ACKNOWLEDGMENTS Hydrogen absorption, molecular weight, and carbon and hydrogen analyses were performed by Dr. A. J. Haagen-Smit of the California Institute of Technology, Pasadena, California. The biological assays were performed by Prof. S. M. Hauge. SUMMARY
The isolation and purification of d-carotene are reported and a quantitative light-absorption curve is given. Data on the physical, chemical, and biological properties of I-carotene and the occurrence and inheritance of this compound in tomatoes are utilized to suggest a tentative formula. The exact position of the conjugated double bonds and the number of isolated double bonds is not known with certainty. REFERENCES 1. WINTERSTEIN, A. H., 2. physiol. Chem. 219, 249 (1933). 2. STRAIN, H. H., J. Biol. Chem. 127, 191 (1939). 3. SOBIN, B., AND STAHLY, G. L., J. Bact. 44, 265 (1942).
&CAROTENE
27
4. HAXO, F., Arch. Biochem. 20, 400 (1949). 5. PORTER, J. W., AND ZSCHEILE, F. P., Arch. Biochem. 10, 537 (1946). 6. PORTER, J. W., AND LINCOLN, R. E., Arch. Biochem. 27, 390 (1950). 7. ZSCHEILE, F. P., AND WHITE, J. W., JR., Ind. Eng. Chem., Anal. Ed. 12, 436
(1940). 8. KUHN, R., AND ROTH, H., Ber. 66, 1274 (1933). 9. GINGER, L. J., J. Biol. Chem. 156, 453 (1944). 10. KUHN, R., AND ROTH, H., Ber. 65, 1285 (1932). 11. NASH, H. A., QUACKENBUSH, F. W., AND PORTER, J. W., J. Am. Chem. Sot. 70,
3613 (1948). 12. STRAIN, H. H., Chromatographic Adsorption Analysis. Interscience Publishers, Inc., New York, 1942. 13. TROMBLY, H. H., AND PORTER, J. W., unpublished. 14. WALLACE, V., AND PORTER, J. W., unpublished. 15. PORTER, J. W., unpublished.
Chemistry, Purdue University, Received November 27, 1950
3 Lafayette,
Indiana
b-Carotene was first reported by Winterstein (1) who found this pigment to be a constituent of Gonocaryum pyriforme fruit. Later Strain (2) reported d-carotene as a constituent of carrots. This pigment has also been reported to be present in twelve strains of Micrococcus pyogenes var. aureus (3) and in Neurospora crassa (4). In 1946 Porter and Zscheile (5) noted the presence of a-carotene in certain experimental selections of tomatoes. More recently Porter and Lincoln (6) have developed, by breeding and selecting for this pigment, tomato selections containing as much as 40 pg. of this compound/g. fresh fruit. This content of a-carotene, in contrast to traces in other sources, has made possible the analytical work reported in this paper. This work leads to a suggestion of a possible formula for this pigment. EXPERIMENTAL Extraction &Carotene was obtained from experimental tomato selections 4 that. contained between 20 and 40 fig. of this compound/g. fruit. The fruit, was ground in an electric meat grinder, and the resultant “soup” was dehydrated with an approximately equal quantity of 95% ethyl alcohol. The pulp was then separated from the alcohol solution by filtration of the liquid through several thicknesses of cheesecloth. The semidry 1Journal paper No. 423, Purdue University Agricultural Experiment Station. This work WBBsupported in part by a grant from the Nutrition Foundation, Inc. 2 Present address: Biology Division, General Electric Co., Richland, Washington. * Formerly Technical Assistant. 4 (a) Pritchard X Pritchard X I?, (Pritchard X Lycopersicwn hirsutum 127827). (b) Selection 4079-6003 from [Indiana Baltimore X F1 (Rutgers X L. hirsutum 126445)] was crossed with the hybrid (Marglobe Supreme X L. hirsutum 127827). The hybrid wan then selfed and selections were made in succeeding generations. 21
22
J.
W.
PORTER
AND
M.
M.
MURPHEY
pulp was extracted with acetone and hexane (approximately 3:2 proportions) in 5-gal. carboys for approximately 1 hr. The pulp and solvents were stirred with an electric stirrer. The hexane and acetone extract was then filtered through a Btichner funnel with suction. The tomato residue was again extracted once or twice more. This removed practically all of the color from the pulp. The combined hexane extracts were then washed several times with water to remove most of the acetone. The hexane extract was concentrated to a smaller volume, washed again several times with water, and stored at -20°C. until chromatographed.
Chromatographic Separations The hexane extract of tomato carotenoids was chromatographed on a column of MgO-Super Gel 6 (50: 50) and the column was developed with 10% acetone in hexane. &Carotene formed a broad band which clearly separated from other pigments. This compound occupies a position below r-carotene and above prolycopene, but it has never been observed to occur with the latter compound in tomatoes. The &carotene band was scraped from the column into a beaker containing 10% ethanol in hexane. The suspension was then poured into a second column into which about 1 in. of adsorbent had been packed as a filter. The &carotene was quickly eluted with suction and the addition of fresh solvent. The a-carotene solution was washed several times with distilled water, concentrated to a smaller volume, and then saponified with an equal volume of approximately 3 N sodium ethylate for 1 hr. The hexane solution was washed thoroughly with water and then saponified with 20% aqueous sodium hydroxide. The hexane solution was washed several times with water and then chromatographed on a column of MgOSuper Cel as before. Crystallization The washed hexane solution of a-carotene was concentrated to a small volume and an equal volume of absolute ethyl alcohol was added. The solution was then placed at -20°C. overnight. The crystals of &carotene were filtered, and redissolved in a minimum amount of carbon disulfide. The solution was then filtered and an equal volume of filtered hexane was added. Two volumes of filtered ethanol was added and the entire solution (25-50 ml.) was concentrated under a stream of nitrogen until crystals of &carotene appeared. The solution was then warmed until perfectly clear, and crystallization of Z-carotene, in very long red needles, was effected by slowly lowering the temperature of the solution to -20°C. Recrystallization of &carotene was effected at least once more for each%ample.
Quantitative Light Absorption Curve Q-Carotene crystals were dried overnight under a high vacuum. A 2-3 mg. sample was weighed into a small platinum boat, dissolved in isoijctane, and then lightabsorption readings were taken with a Beckman spectrophotometer. Figure 1 shows the quantititative absorption curves obtained for the two best preparations of Gcarotene. Table I gives the specific extinction coefficients at the maxima and minima. 5 Adsorptive Magnesia No. 2641. Westvaco Chlorine Products Co., Newark, California. Johns-Manville Hyflo Super Cel.
&-CAROTENE
WAVE LENGTH,
FIG. 1. Quantitative light-absorption values given were obtained
ANALYTICAL
23
6
curve of S-carotene dissolved in isoiictane. on two separately prepared samples.
The
DETERMINATIONS
Melting Point The melting point of the orange- to red-colored needle-shaped &carotene as determined by the microscope hot-stage method of Zscheile and White 140.4-140.5°c.
crystals, (7), was
Carbon and Hydrogen Calculated for G0HS8: C, 89.15; H, 10.85. Found: C, 89.32, 90.02, 89.90, 89.60, 88.74, 88.85. H, 11.09, 10.52, 10.42, 10.28, 11.22, 11.02. Av. = C, 89.40; H, 10.76. TABLE Specific Extinction
Coeficienfs
Wavelength ii
= Slit width * Slit width
I
of &Carotene at the Maxim
In imiiatane
-&3@
$84
4720 s&560* 4400 JPm 3040 2800
182 315 178 mo 5.5 80
= 0.045 mm. = 0.054 mm.
and Minima
24
J.
W.
PORTER
AND
Molecular
M.
M.
MURPHEY
Weight
Molecular weights were determined in camphor by the Rast micro 0.260 mg. s-carotene in 3.480 mg. camphor (K = 38.4); A = 0.518 mg. &carotene in 3.344 mg. camphor (K = 38.1); A = 0.280 mg. s-carotene in 3.871 mg. camphor (K = 38.1); A = Calculated for CLoHss: 538; Found: 522, 537, 483.
method: 5.5’. 11.0”. 5.7”.
Hydrogen Absorption To 7.397 mg. a-carotene was added 3.26 ml. hydrogen (O”, 760 mm.). To 4.988 mg. &carotene was added 1.95 ml. hydrogen (O”, 760 mm.). Found: 10.6 and 9.6 double bonds.
C-Methyl Groups This determination was made by the method of Kuhn and Roth (8), and the larger number of distillations recomemnded by Ginger were made (9). &Carotene, 4.262 mg., required 1.35 ml. of 0.05 N Ba(OH)z for neutralization of the CH&OOH formed. 8Carotene, 5.200 mg., required 1.55 ml. of base, &Carotene, 4.663 mg., required 1.11 ml. of base. Blanks were each 0.48 ml. Found: 5.43, 5.54, and 5.70 C-methyl groups.
Isopropylidene
Groups
The method of Kuhn and Roth (10) was used. A 9.951-mg. sample of &carotene in 3 ml. acetic acid was ozonized for 2 hr. This sample yielded acetone equivalent to 1.80 ml. of 0.05 N iodine solution. b-Carotene, 9.969 mg., yielded acetone equivalent to 2.20 ml. of 0.05 N If. &Carotene, 10.034 mg., yielded acetone equivalent to 1.89 ml. of 0.05 N IZ. Found: 0.80, 0.97, and 0.82 moles of acetone.
Identification
of Acetone
Dibenzylidene acetone was prepared from 65 mg. &carotene by the procedure of Nash et al. (11). The product, recrystallized from boiling ethanol, melted at 112.1” and did not depress the melting point of an authentic sample of dibenzylidene acetone.
Optical Rotation The specific rotation of b-carotene determined in carbon disulfide using a photoflood lamp and Corning filters Nos. 242 and 244 was 317”.
Biological Activity A sample of 8.672 mg. (87% pure) was dissolved in 201.36 g. of Wesson oil to which 0.84 g: or-tocopherol had been added. One gram of this solution equaled 39.5 drops from a calibrated dropping pipet, and when the weight of b-carotene was calculated (7.545 me.), 1 drop was found to contain 0.949 pg. of this compound. The biological activity was tested by the usual vitamin A bioassay procedure. After a preliminary depletion period four lots of rats were given graded doses of U.S.P.
B-CAROTENE
25
vitamin A Reference Standard and two lots were fed 2.9 and 9.6 pg. &arotene/day for a period of 6 weeks. Whereas all animals receiving 1.0 pg. or more of the Reference Standard/day gained weight and survived the B-week period, none of those receiving b-carotene either gained or survived. Evidently, z-carotene is devoid of vitamin A activity. DISCUSSION
Little work has been done on the chemical nature of &carotene. The empirical formula C40H6ehas been quoted by Strain (12), who also states this compound contains 12 double bonds, 11 of which are in conjugation. In the present work the results of C-methyl determinations suggest an isoprenoid structure for this compound, and molecular-weight determinations are consistent with the assumption of 40 carbon atoms per molecule. The presence of one isopropylidene group per molecule is demonstrated by the results of analytical determinations by the method of Kuhn and Roth and the identification of acetone as the product formed on ozonolysis. The presence of a ring structure at the opposite end of the molecule is suggested by the results of measurements of the optical activity of b-carotene. The presence of 10 conjugated double bonds in the molecule is ascertained from the wavelength of the first light -absorption maximum (11). An examination of the light-absorption curves of the carotenoid hydrocarbons shows that they fall into three groups: b-carotene has broad maxima and shallow minima; a- and y-carotene have somewhat sharper maxima and deeper minima; lycopene, {-carotene (ll), tetrahydrolycopene (13), and phytofluene (14) have still sharper maxima and minima. The light-absorption curve of d-carotene compares very closely to lycopene in this respect. This fact is interpreted to mean that the conjugation in d-carotene does not extend into the ring structure at one end of the molecule. Certainly it does not extend into a ,&ionone ring for the compound has no vitamin A activity. As additional evidence of the restriction of the conjugated double bonds to an open chain, it has been found that the specific extinction coefficient of the longest wavelength maximum falls on a line drawn through the extinction coefficients of lycopene, tetrahydrolycopene, [-carotene, phytofluene, and phytoene (15). The number of isolated double bonds in the molecule is in question. Hydrogenation and C and H values, while erratic, do not indicate more than one such bond per molecule.
26
J. W. PORTER AND M. M. MURPHEY
The fact that S-carotene adsorbs only slightly below y-carotene on a chromatographic column suggests a close relationship between these compounds. d-Carotene also adsorbs below tetrahydrolycopene and above {-carotene. b-Carotene has been found to occur only in tomatoes with lycopene, y- and P-carotene (6). Since it has not been possible to develop a tomato selection homozygous for &carotene, the genes involved in lycopene, y- and p-carotene formation must also be acting in the formation of a-carotene. Therefore, it seems quite certain the structure of this compound must be closely related to those of these three pigments. On the basis of the evidence available on a-carotene it is suggested this compound may be dihydro-y-carotene. The exact location of the conjugated double bonds in b-carotene is not known with certainty. They are placed in agreement with those in lycopene and p-carotene largely on the basis of the adsorptive properties of this compound and its inheritance and occurrence in tomatoes with p- and y-carotenes and lycopene. The absence of an additional isolated double bond is not proven but hydrogenation values argue against this possibility. The position of d-carotene in a scheme postulated for the biosynthesis of carotenes has been set forth in another paper 0%. ACKNOWLEDGMENTS Hydrogen absorption, molecular weight, and carbon and hydrogen analyses were performed by Dr. A. J. Haagen-Smit of the California Institute of Technology, Pasadena, California. The biological assays were performed by Prof. S. M. Hauge. SUMMARY
The isolation and purification of d-carotene are reported and a quantitative light-absorption curve is given. Data on the physical, chemical, and biological properties of I-carotene and the occurrence and inheritance of this compound in tomatoes are utilized to suggest a tentative formula. The exact position of the conjugated double bonds and the number of isolated double bonds is not known with certainty. REFERENCES 1. WINTERSTEIN, A. H., 2. physiol. Chem. 219, 249 (1933). 2. STRAIN, H. H., J. Biol. Chem. 127, 191 (1939). 3. SOBIN, B., AND STAHLY, G. L., J. Bact. 44, 265 (1942).
&CAROTENE
27
4. HAXO, F., Arch. Biochem. 20, 400 (1949). 5. PORTER, J. W., AND ZSCHEILE, F. P., Arch. Biochem. 10, 537 (1946). 6. PORTER, J. W., AND LINCOLN, R. E., Arch. Biochem. 27, 390 (1950). 7. ZSCHEILE, F. P., AND WHITE, J. W., JR., Ind. Eng. Chem., Anal. Ed. 12, 436
(1940). 8. KUHN, R., AND ROTH, H., Ber. 66, 1274 (1933). 9. GINGER, L. J., J. Biol. Chem. 156, 453 (1944). 10. KUHN, R., AND ROTH, H., Ber. 65, 1285 (1932). 11. NASH, H. A., QUACKENBUSH, F. W., AND PORTER, J. W., J. Am. Chem. Sot. 70,
3613 (1948). 12. STRAIN, H. H., Chromatographic Adsorption Analysis. Interscience Publishers, Inc., New York, 1942. 13. TROMBLY, H. H., AND PORTER, J. W., unpublished. 14. WALLACE, V., AND PORTER, J. W., unpublished. 15. PORTER, J. W., unpublished.