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The isolation and identification of progoitrin from Brassica seed
ARCHIVES
OF
BIOCHEMISTRY
The Isolation
AND
and
BIOPHYSICS
99,
369-371
Identification
of Progoitrin
MONTE From the Division
of Endocrinology,
(1962)
Brassica
Seed’
A. GREER
Department of Medicine, School, Portland, Oregon Received
from
August
University
of Oregon Medical
20, 1962
The isolation and characterization of progoitrin obtained from Brassica seed are described. Progoitrin is converted to goitrin during enzymic, but not acid, hydrolysis. INTRODUCTION
Goitrin (L( -) - 5-vinyl-2-oxazolidinethione) is a potent antithyroid compound found in the aqueous extracts of the seeds of many species of crucifers and of the edible parts of some widely used by man (1). It is contained in clinically significant amounts in the fleshy root of rutabaga and turnip. Goitrin is not present as such in the plants or seeds, but as a thioglycoside precursor, progoitrin. Goitrin is formed by ring closure in the aglycone after enzymic hydrolysis of progoitrin. A preliminary report of the isolation and identification of progoitrin has been published previously (2). The isolation of the acetyl derivative of progoitrin (glucorapiferin) has also been reported by Schultz and Wagner (3). The purpose of this article is to present the details of the analytical results mentioned in the previous preliminary communication and to describe the method of progoitrin preparation currently used in our laboratory, a much simpler method than that originally employed. EXPERIMENTAL
ISOLATION Preliminary assay of Brassica seeds was useful in order to determine those lots with the highest content of progoitrin. This was accomplished by ’ I am indebted to Drs. M. G. Ettlinger and T. A. Geissman for valuable advice during the course of this investigation, which was supported by grants from the U. S. Public Health Serivce. 369
grinding a small quantity of seed in water in a Waring blendor and allowing the mixture to stand at room temperature for 1 hr. to permit transformation of progoitrin to goitrin, through the action of the thioglycosidase (myrosinase) present in the seeds. The mixture was then filtered, and the filtrate was extracted with an equal volume of peroxide-free ether to obtain the goitrin, which is equally soluble in ether and water. An aliquot of the ether extract was evaporated to dryness, and the residue was dissolved in water and read in appropriate dilution in a spectrophotometer at 240 rnF, the wavelength at which goitrin has maximum absorption. Progoitrin content can be calculated by dividing the goitrin content by 0.32, assuming stoichiometric transformation. In our experience, rutabaga and kale seed consistently have had the highest content of progoitrin from among various crucifer seeds tested over several years by this method, and of these Laurentian rutabaga was usually highest. When the appropriate seed lot had been selected, 1 kg. was ground dry in the Waring blendor to a fine consistency. The ground seed was slowly added to 6 1. of 75y0 acetone, previously heated to 5655”. The acetone was then further heated to a low-rolling boil, and the suspension was refluxed for 45 min., allowed to cool, and filtered. The filtrate was evaporated under reduced pressure at 39” in a continuous-flow flash evaporator to a volume of 306500 ml. The concentrate was extracted three times wit’h 56loo-ml. portions of peroxidefree ether. The ether extract was discarded, and the remaining material was concentrated to a volume of approximately 100 ml. in a rotary evaporator at 39” to insure complete removal of the ether. This concentrate was then filtered and passed through a 2.5 X 30 cm. anion-exchange column of purified Amberlite IR-4B in the chloride form and
370
GREER
O4000
, 2500 FREOUENCY
3000
3500
FIG. 1. Infrared
spectrum
PROPERTIES AND CHARACTERIZATION
Calculated Found
C
H
N
S
Na
32.11 32.44
4.38 4.59
3.41 3.39
15.56 15.27
5.59 5.52
Progoitrin has a maximum specific absorption at 227 rnp (c = 7700). Following enzymic cleavage, there is a spectral shift to a maximum at 240 rnp, corresponding to that of goitrin. An infrared spectrum was run in a KI pellet and is illustrated in Fig. 1. There is a broad stretching absorption at 3500 indicative of hydroxyl groups. C-H groups are indicated by absorption at 2925 and 2870. Ab-
I l500
of progoitrin
washed with distilled -rater until the eluate was completely colorless. This usually required approximately 3-4 1. water. The eluting agent was then changed to 0.1 X NaCl. Progoitrin has an absorption maximum at 227 mp so that its elution can be followed by reading aliquots in the spectrophotometer. When elution of the progoitrin was complete, or nearly complete, the total 0.1 N NaCl eluate was pooled and evaporated to dryness in a rotary evaporator at 39”. The dried residue, which consisted primarily of sodium chloride and progoitrin, was thrice extracted with 30 ml. of 95% ethanol. The combined alcoholic extracts were filtered, cooled to 5”, and seeded. Crystalline progoitrin began to form immediately. The progoitrin was recrystallized from 957, ethanol three times, thoroughly dried over Drierite, and kept in a vacuum desiccator. If kept under these conditions, it will remain stable at room temperature for many months, but it will decompose within a few weeks if exposed to air.
Progoitrin crystallizes in long white needles. The crystals begin to turn brown at 128-130” and to melt at 135-140”; specific’ optical rotation (01) = -22.3” (water). It is very soluble in water but sparingly soluble or insoluble in organic solvents such as ether. Elemental analysis (CllH18010NS2Na):
I 2000 (CM-'I
1 500
1000
run in KI pellet.2 TABLE
I
EFFECT OF VARIOUS CONCENTRATIONS OF HCl ON DISINTEGRATION OF PROGOITRIN IN AQUEOUS SOLUTION Concentration
HCI
AT 100°C. Per cent progoitrin remaining
30 min.
90 min.
N
2 1
0.5 0.1
0.05 0.01
0.005 0
1.1 7.9
0 0
22.8 75.0 77.5 88.5 91.0 87.4
0 21.8 39.8 65.5 77.8
sorption at 1620 indicates C=cI or C=X linkage, that at 1280 indicates C-O, that at 1060 indicates sulfate, and that at 700 is consistent with C-S-. The infrared spectrum is thus consistent with the structure advanced herein.* Optimal pH for enzymic hydrolysis was determined by dissolving 200 pg. progoitrin in 3 ml. of phosphate or acetate buffer to which 0.4 ml. of a purified myrosinase preparation from kale seed was added (4). Reaction rates at 23” were ascertained by following the decrease in absorption at 277 mp and the increase in absorption at 240 rnN by taking readings every 2 min. over a 20-min. period. Optimal pH was found to be 5.4, although later studies have shown 7.4 to be best. A 2-mg. sample of progoitrin was incubated with 2.5 ml. of purified myrosinase and 2.5 ml. of pH 5.4 phosphate buffer at 37” for 24 hr. The solution was extracted with an equal volume of ether, and an aliquot of the ether extract was evaporated to dryness. The residue was dissolved in water and read in the ultraviolet spectrophotometer at ap2 I am indebted to Dr. J. H. Fellman frared spectrum and its interpretation.
for the in-
PROGOITRIN
FROM BRASSICA
propriate dilution. The quantity of goitrin formed was calculated from the optical density reading at 240 mp; this was found to be 94@hof the theoretical yield. To st,udy the effect of acid hydrolysis, 400 pg. progoitrin was dissolved in 5 ml. HCl ranging in concentration from 0.005 to 2.0 N. Complete spectra from 215 to 250 rnp were determined after 0, 30, and 90 min. of heating at 100” in a boiling water bath, In no instance was there any evidence for 6he formation of goitrin. With 2 S HCI all spectrophotometric characteristics of progoitrin had disappeared in 30 min. There was progressively less destruction of progoitrin with decreasing concent-ration of HCI, until by the time 0.005 i%’ had been reached, there was little difference between boiling in acid or water (Table I). A 30.mg. sample of progoitrin was hydrolyzed by heating at 100” in 5 ml. of 1 A: HCI for 2 hr. Sulfate was det,ermined as the barium salt precipit,ated, and glucose by the method of Somogyi (5). In addition, glucose was qualit,atively identified by paper chromatography in two solvent systems (1.propanol-et,hyl acetate-water = 7:1:2 and Ibutanol-acetic acid-water = 4: 1:5).
Calculated Found
Sulfate
Glucose
24.00 22.19
45.00 41.36
that
progoitrin has closely approxibut with an addi-
3 I am indebted to Dr. Martin G. Ettlinger determination of the hydroxylamine content this sample of progoitrin.
oso,-
N-OSO,-
A-S-C,H,,O,
tl -Q-C6H1,05
!!CH,CII=CH,
hH?--CH=CH,
I
II
tl -S-C!sH,,Os
DISCUSSION
When eneymic
drolysis of progoitrin occurs, the aglycone is released. Since it possesses a reactive secondary alcohol group, it immediately cyclimes to form goitrin. The formula originally proposed by Gadamer (7) for the structure of sinigrin is (I). St,udies by Ettlinger and Lundeen (8) have shown, however, that Gadamer’s structure is incorrect and that sinigrin actually undergoes a Lossen rearrangement on enzymic hydrolysis. The correct formula for sinigrin would thus be (II). One would expect the structural configuration of progoitrin to be similar to that of sinigrin. The demonstration of hydroxylamine in progoitrin provides further evidence of the similarity of structure. The correct formula for progoitrin thus can be written as (III).
N-OSO,-
Determination of hydroxylamine in progoitrins indicated a content about 30-40% of bheoretical ba.sed on the Ettlinger-Lundeen st,ructure for mustard oil glycosides, but it is likely t,hat the samples on which this determination was run had undergone some deterioration. No C-methyl groups were found in progoitrin as det,ermined by t
These results indicate a molecular configuration mating that of sinigrin, tional CHOH group.
371
SEED
L~-cH--CH=CH~ I OH III REFERENCES 1. ASTWO~D, E. B., GREER, M. A., AND ETTLINGER, M. G., J. Biol. Chenl. 181, 121 (1949). 2. GREER, M. A., J. .iZm. C’hern. Sot. 78, 1260 (1956). 3. SCHULTZ, O.-E., .~ND WAGXER, W., .4rch.
Pham. 289, 597 (195G). 4. WREDE, F. in “Die Methoden
hy-
5. 6.
for of
7. 8.
der Fermentforschung” (E. Bamann and K. MyrbLck, eds.), Vol. 5, p. 1834. Thieme, Leipzig, 1940. SOMOGYI, M., J. Lab. Clin. Xed. 26, 1220 (1941). KUHN, It., .~XD ROTH, H., Ber. 66B, 1274 (1933). GADI\MER, J., Arch. Pharm. 235, 44 (1897). ETTLINGER, M. G., AXD I,(-XDEEX, A. J., J. .4nl. Chem. Sot. 78, 4172 (1956).
OF
BIOCHEMISTRY
The Isolation
AND
and
BIOPHYSICS
99,
369-371
Identification
of Progoitrin
MONTE From the Division
of Endocrinology,
(1962)
Brassica
Seed’
A. GREER
Department of Medicine, School, Portland, Oregon Received
from
August
University
of Oregon Medical
20, 1962
The isolation and characterization of progoitrin obtained from Brassica seed are described. Progoitrin is converted to goitrin during enzymic, but not acid, hydrolysis. INTRODUCTION
Goitrin (L( -) - 5-vinyl-2-oxazolidinethione) is a potent antithyroid compound found in the aqueous extracts of the seeds of many species of crucifers and of the edible parts of some widely used by man (1). It is contained in clinically significant amounts in the fleshy root of rutabaga and turnip. Goitrin is not present as such in the plants or seeds, but as a thioglycoside precursor, progoitrin. Goitrin is formed by ring closure in the aglycone after enzymic hydrolysis of progoitrin. A preliminary report of the isolation and identification of progoitrin has been published previously (2). The isolation of the acetyl derivative of progoitrin (glucorapiferin) has also been reported by Schultz and Wagner (3). The purpose of this article is to present the details of the analytical results mentioned in the previous preliminary communication and to describe the method of progoitrin preparation currently used in our laboratory, a much simpler method than that originally employed. EXPERIMENTAL
ISOLATION Preliminary assay of Brassica seeds was useful in order to determine those lots with the highest content of progoitrin. This was accomplished by ’ I am indebted to Drs. M. G. Ettlinger and T. A. Geissman for valuable advice during the course of this investigation, which was supported by grants from the U. S. Public Health Serivce. 369
grinding a small quantity of seed in water in a Waring blendor and allowing the mixture to stand at room temperature for 1 hr. to permit transformation of progoitrin to goitrin, through the action of the thioglycosidase (myrosinase) present in the seeds. The mixture was then filtered, and the filtrate was extracted with an equal volume of peroxide-free ether to obtain the goitrin, which is equally soluble in ether and water. An aliquot of the ether extract was evaporated to dryness, and the residue was dissolved in water and read in appropriate dilution in a spectrophotometer at 240 rnF, the wavelength at which goitrin has maximum absorption. Progoitrin content can be calculated by dividing the goitrin content by 0.32, assuming stoichiometric transformation. In our experience, rutabaga and kale seed consistently have had the highest content of progoitrin from among various crucifer seeds tested over several years by this method, and of these Laurentian rutabaga was usually highest. When the appropriate seed lot had been selected, 1 kg. was ground dry in the Waring blendor to a fine consistency. The ground seed was slowly added to 6 1. of 75y0 acetone, previously heated to 5655”. The acetone was then further heated to a low-rolling boil, and the suspension was refluxed for 45 min., allowed to cool, and filtered. The filtrate was evaporated under reduced pressure at 39” in a continuous-flow flash evaporator to a volume of 306500 ml. The concentrate was extracted three times wit’h 56loo-ml. portions of peroxidefree ether. The ether extract was discarded, and the remaining material was concentrated to a volume of approximately 100 ml. in a rotary evaporator at 39” to insure complete removal of the ether. This concentrate was then filtered and passed through a 2.5 X 30 cm. anion-exchange column of purified Amberlite IR-4B in the chloride form and
370
GREER
O4000
, 2500 FREOUENCY
3000
3500
FIG. 1. Infrared
spectrum
PROPERTIES AND CHARACTERIZATION
Calculated Found
C
H
N
S
Na
32.11 32.44
4.38 4.59
3.41 3.39
15.56 15.27
5.59 5.52
Progoitrin has a maximum specific absorption at 227 rnp (c = 7700). Following enzymic cleavage, there is a spectral shift to a maximum at 240 rnp, corresponding to that of goitrin. An infrared spectrum was run in a KI pellet and is illustrated in Fig. 1. There is a broad stretching absorption at 3500 indicative of hydroxyl groups. C-H groups are indicated by absorption at 2925 and 2870. Ab-
I l500
of progoitrin
washed with distilled -rater until the eluate was completely colorless. This usually required approximately 3-4 1. water. The eluting agent was then changed to 0.1 X NaCl. Progoitrin has an absorption maximum at 227 mp so that its elution can be followed by reading aliquots in the spectrophotometer. When elution of the progoitrin was complete, or nearly complete, the total 0.1 N NaCl eluate was pooled and evaporated to dryness in a rotary evaporator at 39”. The dried residue, which consisted primarily of sodium chloride and progoitrin, was thrice extracted with 30 ml. of 95% ethanol. The combined alcoholic extracts were filtered, cooled to 5”, and seeded. Crystalline progoitrin began to form immediately. The progoitrin was recrystallized from 957, ethanol three times, thoroughly dried over Drierite, and kept in a vacuum desiccator. If kept under these conditions, it will remain stable at room temperature for many months, but it will decompose within a few weeks if exposed to air.
Progoitrin crystallizes in long white needles. The crystals begin to turn brown at 128-130” and to melt at 135-140”; specific’ optical rotation (01) = -22.3” (water). It is very soluble in water but sparingly soluble or insoluble in organic solvents such as ether. Elemental analysis (CllH18010NS2Na):
I 2000 (CM-'I
1 500
1000
run in KI pellet.2 TABLE
I
EFFECT OF VARIOUS CONCENTRATIONS OF HCl ON DISINTEGRATION OF PROGOITRIN IN AQUEOUS SOLUTION Concentration
HCI
AT 100°C. Per cent progoitrin remaining
30 min.
90 min.
N
2 1
0.5 0.1
0.05 0.01
0.005 0
1.1 7.9
0 0
22.8 75.0 77.5 88.5 91.0 87.4
0 21.8 39.8 65.5 77.8
sorption at 1620 indicates C=cI or C=X linkage, that at 1280 indicates C-O, that at 1060 indicates sulfate, and that at 700 is consistent with C-S-. The infrared spectrum is thus consistent with the structure advanced herein.* Optimal pH for enzymic hydrolysis was determined by dissolving 200 pg. progoitrin in 3 ml. of phosphate or acetate buffer to which 0.4 ml. of a purified myrosinase preparation from kale seed was added (4). Reaction rates at 23” were ascertained by following the decrease in absorption at 277 mp and the increase in absorption at 240 rnN by taking readings every 2 min. over a 20-min. period. Optimal pH was found to be 5.4, although later studies have shown 7.4 to be best. A 2-mg. sample of progoitrin was incubated with 2.5 ml. of purified myrosinase and 2.5 ml. of pH 5.4 phosphate buffer at 37” for 24 hr. The solution was extracted with an equal volume of ether, and an aliquot of the ether extract was evaporated to dryness. The residue was dissolved in water and read in the ultraviolet spectrophotometer at ap2 I am indebted to Dr. J. H. Fellman frared spectrum and its interpretation.
for the in-
PROGOITRIN
FROM BRASSICA
propriate dilution. The quantity of goitrin formed was calculated from the optical density reading at 240 mp; this was found to be 94@hof the theoretical yield. To st,udy the effect of acid hydrolysis, 400 pg. progoitrin was dissolved in 5 ml. HCl ranging in concentration from 0.005 to 2.0 N. Complete spectra from 215 to 250 rnp were determined after 0, 30, and 90 min. of heating at 100” in a boiling water bath, In no instance was there any evidence for 6he formation of goitrin. With 2 S HCI all spectrophotometric characteristics of progoitrin had disappeared in 30 min. There was progressively less destruction of progoitrin with decreasing concent-ration of HCI, until by the time 0.005 i%’ had been reached, there was little difference between boiling in acid or water (Table I). A 30.mg. sample of progoitrin was hydrolyzed by heating at 100” in 5 ml. of 1 A: HCI for 2 hr. Sulfate was det,ermined as the barium salt precipit,ated, and glucose by the method of Somogyi (5). In addition, glucose was qualit,atively identified by paper chromatography in two solvent systems (1.propanol-et,hyl acetate-water = 7:1:2 and Ibutanol-acetic acid-water = 4: 1:5).
Calculated Found
Sulfate
Glucose
24.00 22.19
45.00 41.36
that
progoitrin has closely approxibut with an addi-
3 I am indebted to Dr. Martin G. Ettlinger determination of the hydroxylamine content this sample of progoitrin.
oso,-
N-OSO,-
A-S-C,H,,O,
tl -Q-C6H1,05
!!CH,CII=CH,
hH?--CH=CH,
I
II
tl -S-C!sH,,Os
DISCUSSION
When eneymic
drolysis of progoitrin occurs, the aglycone is released. Since it possesses a reactive secondary alcohol group, it immediately cyclimes to form goitrin. The formula originally proposed by Gadamer (7) for the structure of sinigrin is (I). St,udies by Ettlinger and Lundeen (8) have shown, however, that Gadamer’s structure is incorrect and that sinigrin actually undergoes a Lossen rearrangement on enzymic hydrolysis. The correct formula for sinigrin would thus be (II). One would expect the structural configuration of progoitrin to be similar to that of sinigrin. The demonstration of hydroxylamine in progoitrin provides further evidence of the similarity of structure. The correct formula for progoitrin thus can be written as (III).
N-OSO,-
Determination of hydroxylamine in progoitrins indicated a content about 30-40% of bheoretical ba.sed on the Ettlinger-Lundeen st,ructure for mustard oil glycosides, but it is likely t,hat the samples on which this determination was run had undergone some deterioration. No C-methyl groups were found in progoitrin as det,ermined by t
These results indicate a molecular configuration mating that of sinigrin, tional CHOH group.
371
SEED
L~-cH--CH=CH~ I OH III REFERENCES 1. ASTWO~D, E. B., GREER, M. A., AND ETTLINGER, M. G., J. Biol. Chenl. 181, 121 (1949). 2. GREER, M. A., J. .iZm. C’hern. Sot. 78, 1260 (1956). 3. SCHULTZ, O.-E., .~ND WAGXER, W., .4rch.
Pham. 289, 597 (195G). 4. WREDE, F. in “Die Methoden
hy-
5. 6.
for of
7. 8.
der Fermentforschung” (E. Bamann and K. MyrbLck, eds.), Vol. 5, p. 1834. Thieme, Leipzig, 1940. SOMOGYI, M., J. Lab. Clin. Xed. 26, 1220 (1941). KUHN, It., .~XD ROTH, H., Ber. 66B, 1274 (1933). GADI\MER, J., Arch. Pharm. 235, 44 (1897). ETTLINGER, M. G., AXD I,(-XDEEX, A. J., J. .4nl. Chem. Sot. 78, 4172 (1956).