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Some chemical and physical properties of papiliochrome II in the wings of Papilio xuthus
3. Insect Physiol.,1970, Vol. 16, pp. 1203 to 1228. PergamonPress. Printed in Great Britaitl
SOME CHEMICAL AND PHYSICAL PROPERTIES PAPILIOCHROME II IN THE WINGS OF
PAPILIO Y. UMEBACHI
OF
XUTHUS and K. YOSHIDA
Department of Biology, Faculty of Science, Kanazawa University, Kanazawa, Japan (Received 8 September 1969)
Abstract-The yellow pigments in the wings of Papilio xuthus were extracted and two-dimensionally chromatographed. Two major and two minor yellow pigments were found and named Papiliochrome IIa, IIb, IIIa, and IIIb respectively. Some chemical properties of these pigments were examined by colour tests. After being purified using DEAE cellulose, Dowex SO-W, and cellulose columns, IIa and IIb were examined for u.v., i.r., optical rotatory dispersion, and circular dichroism spectra. The decomposition products of these pigments were also examined for their chemical and physical properties. Moreover, ‘%Xabelled tryptophan, DOPA, and DOPAmine were injected into the prepupa and examined for their incorporation into the yellow pigments. It was inferred from the results of these experiments that Papiliochrome IIa and IIb are formed by a combination of DOPAmine derivative and kynurenine and are optically isomeric to each other. INTRODUCTION
AMONG the wing pigments
of butterflies, the pterins of the Pieridae (SCHBPF, 1964) of the Nymphalidae (BUTENANDT and SCHAFER, 1962) have been investigated in detail from the chemical and biochemical points of view. However, little work has been done on the pigments of other families. It was discovered by UMEBACHIand NAKAMUEW (1954) that the hot-water extract of the yellow scales of the papilionid butterflies contained a large quantity of kynurenine, an intermediate of tryptophan metabolism. Since then we have investigated the chemical properties and formation of the yellow pigments of this family (UMEBACHIand TAKAHASHI, 1956; UMEBACHI,1958,1959a, b, 1960,1961,1962; UMEBACHIand YAMADA,1964; UMEBACHI and KATAYAMA,1966). The yellow pigments of Pupilio mthus in particular have been used as the material for our chemical studies. In the previous paper (UMEBACHI,1961), two groups of yellow pigments were found in the yellow scales of this species and were named Papiliochrome II and III respectively. Both groups of pigments were readily decomposed by heating and yielded kynurenine and a phenolic compound. The present paper deals mainly with the more detailed chemical and physical properties of Papiliochrome II. It was proved that Papiliochrome II and III respectively contain two kinds of yellow pigments. They were named Papiliochrome IIa and IIb and IIIa and IIIb. and the ommatins
1203
1204
Y. UMEBACHI AND K. YOSHIDA
Papiliochrome IIa and IIb were separated, purified, and examined for their chemical and physical properties. From these experiments and the incorporation tests of 14C-labelled compounds, it was inferred that Papiliochrome IIa and IIb are formed by a combination of DOPAmine derivative and kynurenine. Interestingly enough, Papiliochrome IIa and IIb gave the same results regarding colour tests, decomposition products, U.V. and i.r. spectra, and the incorporation of i4C-labelled compounds. But, as to optical rotatory dispersion and circular dichroism, both pigments showed opposite curves, and thus it was suggested that Papiliochrome IIa and IIb are optically isomeric. MATERIALS
AND METHODS
Materials The male adults of Pap&o xuthus were used in the experiments other than the incorporation tests of 14C-labelled compounds. Most of them were obtained through the Okura Biological Institute. Some of them were collected during their larval stage in Kanazawa and raised to the adult stage in the laboratory. For the incorporation tests of i*C-labelled compounds, they were injected during their pharate pupal stage with the compounds which will be mentioned later and then raised to the adult stage. Extraction of yellow pigments The yellow pigments of the wings are soluble in water and 80% ethanol and are insoluble in ethyl ether and chloroform (UMEBACHI, 1961). Therefore, the yellow pigments were extracted from the wings or yellow scales with water or 80% ethanol. After centrifugation, the supernatant fluid, which is referred to below as a crude extract of the yellow pigments, was used as the material for the chromatographic separation and purification. Paper chromatography Toyo No. 51A filter paper was used. The chromatogram was run by the ascending method with 80% methanol or the upper layer of n-butanol-glacial acetic acid-water (4 : 1 : 5) mixture (BAW). For two-dimensional separation, the former solvent was used for the first direction, and the latter for the second direction. After development, the filter paper was inspected under U.V. rays and then subjected to one of the following colour tests: (1) Ninhydrin test (NIN). (2) Ehrhch’s test for indole structure and aromatic ammo compounds usingp-dimethylaminobenzaldehyde (DAB) (DALCLIESH, 1952). After the reagent was sprayed, the filter paper was warmed. (3) The test for aromatic amines using Tsuda’s reagent (fi-diethylaminoethyl-anaphthylamine oxalate) (TSU) (UMEBACHIand TSUCHITANI,1955). (4) Aniline hydrogen phthalate test for reducing sugars (AHP) (CRAMER,1954). (5) Be&dine test for reducing sugars (BEZ) (HORROCKS,1949). (6) Resorcinol test for sugars (RES) (LBDERERand LEDERBR,1957).
YELLOW
PIGMBN’ISIN
WINGS OF PAPILIO
XUTHUS
1205
(7) 2,3,5-Triphenyltetrszoliumchloride test for reducing sugars, o-diphenols, and other reducing substancea (TTC) (TREVELYAN et OZ., 1950: PITTARD et uZ., 1961). (8) Silver nitrate test for reducing sugars, o-diphenols, and other reducing substances (SIN) (SWAIN, 1953). (9) Millon’s test for phenolic compounds (MIL) (CRAMJXR,1954). (10) Ferric chloride test for phenolic compounds (FEC) (CRAMER, 1954). After the reagent was sprayed, the filter paper was warmed. (11) Phosphomolybdic acid-ammonia test for phenolic compounds (PMA) (RILEY, 1950). (12) Diazotized p-nitroaniline test for phenolic compounds (DNT) (SWAIN, 1953; COULSONand EVANS,1958). (13) or-Nitroso-fi-naphthol test for p-substituted phenolic compounds (NNP) (A&RR and CROCKER,1952). (14) Evans’s test for o-diphenols (EVA) (COULSONand EVANS,1958). (15) Sodium molybdate test for o-diphenols (SMO) (PRIDHAM,1959). (16) Potassium ferricyanide test for adrenaline and related compounds (PFC) (SENOH and WITKOP, 1959; SENOHet al., 1959). (17) Ethylenediamine-ammonia test for o-diphenols (EDA) (Soet al., 1963; FURNEAUXand MCFARLANE, 1965). DOPA and DOPAmine give a whitish yellow and yellow fluorescence respectively. Noradrenaline and adrenaline show an orsngeish yellow fluorescence. (18) Elson-Morgan’s test for glucosamine (ELM). Of the reagents a, b, and c used for detecting glucosamine, only the last two reagents were used. These two reagents were reported to be useful as a test for the presence of iV-acetylated derivative (PARTRIDGE,1948). Although this test is not suitable for o-diphenols, the following results were obtained for DOPA derivatives by us. DOPA and DOPAmine did not give a red but rather, an orangeish brown colour, while N-acetyl-DOPAmine gave a red colour. (19) 2,6-Dichloroquinone chlorimide test for phenols (DCQ). This is generally said to be a test for phenols in which the position puru to the hydroxyl group is unsubstituted (GIBBS, 1927). But, according to SENOH et al. (1959), 6-hydroxy-DOPAmine is also positive. (20) The test for peptides, amines, amides, and other N-H-containing compounds using tert-butylhypochlorite and potassium iodide (BHC) (MAZUR et al., 1962). A 1 : 1 mixture of saturated o-tolidine in 2% acetic acid and 0.05 N KI was used instead of the 1 y0 KI and 1 y0 starch solution. Tyrosine and DOPA are negative, while DOPAmine and kynurenine give a dark blue colour.
Preparation
of DEAE
cellulose column
One hundred ml of DEAE cellulose was suspended in about 500 ml of distilled water, and the fine and coarse powders were repeatedly removed by decantation. For the purpose of the present experiment, other pretreatment was not necessary. A 2 x 15 cm column of the DEAE cellulose was prepared, washed with about 175 ml of 0.2 N HCl and finally washed with about 750 ml of distilled water. Preparation of Dowex-50 W column Dowex-50 W X4 (200-400 mesh) was used. One hundred ml of dry resin was suspended in distilled water, and the particle size was made uniform by repeating decantation. The resin suspension in water was filtered through a glass filter and washed with 2 N NaOH until the filtrate turned alkaline. After washing with distilled water until neutralization, the resin was washed with 4 N HCl until the
1206
Y. UMJIBACHI AND K. YOSHIDA
resin turned pale in colour. It was washed again with distilled water and then with 2 N NaOH until the filtrate became colourless. These washing procedures were carried out in the same glass filter. Next, the resin was suspended in 3 vol. of 1 N NaOH in a beaker and heated in a water-bath at 60 to 70°C for about 3 hr. After standing at room temperature for 1 hr, the supernatant fluid was removed by decantation and hot 1 N NaOH was again added. This procedure was repeated five times. Next the resin was washed with distilled water, about 100 ml of 4 N HCl, and about 100 ml of 2 N HCl successively. After washing with distilled water until neutralization, the resin was suspended in 2 vol. of 2 M pyridine and stirred at room temperature for 1 to 2 hr. After that, the resin was filtered through a glass filter and washed with distilled water until the filtrate became neutral. Finally, the resin was suspended in 2 vol. of O-1 M pyridine-formic acid buffer (pH 3.1) and equilibrated by storing in the cold for at least 2 weeks. A O-9 x 10 cm column of the resin was prepared and washed with 500 ml of the above-mentioned buffer. Preparation
of cellulose column
About 100 ml of cellulose powder (Toyo, 100-200 mesh) was suspended in about 1 1. of 0.1 N HCl in 99% ethanol and boiled in a water-bath under reflux for 20 hr. After that, the powder was filtered through a glass filter and dried. Then the powder was suspended in BAW solvent and a 0.9 x 80 cm column was prepared. Ultra-violet
absorption spectra
These were determined in the range from 240 to 400 nm with a Hitachi model The solvent was 0.067 or O-1 M Sorensen’s phosphate 139 spectrophotometer. buffer (pH 7.0) 99% methanol, or 1O-3 N HCl. Infra-red These Bunko DS was mixed a pressure
absorption spectra were determined in the range from 4000 to 650 cm-l with a Nihon 301 i.r. spectrophotometer. The powder of the purified yellow pigment with KBr powder, and a micro-pellet (diameter, 5 mm) was made under of 4 to 6 tons.
Optical rotatory
dispersion (ORD)
and circular dichroism (CD) spectra
These measurements were made in the range from 450 to 250 nm with a Nihon Bunko ORD/UV-5 spectropolarimeter equipped with a CD attachment. The solvent was 99% methanol. Injection experiments
of 14C-labelled compounds
The labelled compounds used were as follows: (1) nL-Tryptophan (alanine-2- 1%). Specific activity, 0.45 mc/mM. Commercial source, Tracerlab. (2) nL-Tryptophan (ring-Z i4C). Specific activity, 21 mc/mM. Commercial source, Commissariat a l’$nergie Atomique Livraison de Molecules Marquees.
YELLOW
PIGMENTS
IN WINGS
OF PAPILIO XVTHVS
1207
(3) m_-3,4_Dihydroxyphenylalanine (carboxyl-14C). Specific activity, 3.75 mc/mM. Commercial source, New England Corp. (4) DL-3,4_Dihydroxyphenylalanine (alanine-2-14C). Specific activity, 5.7 mc/mM. Commercial source, The Radiochemical Centre, Amersham. (5) 3,4-Dihydroxyphenylethylamine-1J4C hydrobromide. Specific activity, 6.4 mc/mM. Commercial source, New England Nuclear Corp. From O-3 to 3.1 PC (0*02-0*06 ml in aqueous solution) of each compound was injected into one prepupa. After the emergence of the butterflies, the wings were cut off, dried in a desiccator, and then brought into contact with Fuji, Medical KX X-ray film. The time of exposure was from 10 to 21 days. The autoradiograph of the two-dimensional chromatogram of the crude extract of the yellow pigments was taken with the same X-ray film as described above. The time of exposure was 50 to 70 days. The autoradiographof the one-dimensional chromatogram after decomposition of the yellow pigments by heating was also taken in the same way. RESULTS
Paper chromatography of the crude extract of yellow pigments
The yellow pigments were extracted by steeping the yellow scales in a small quantity of distilled water or 80% ethanol for a short time. The crude extract thus obtained was spotted on filter paper immediately after the extraction and twodimensionally chromatographed. The chromatogram is shown in Fig. 1, and the colour tests are given in Table 1. Spots Y-IIa, IIb, IIIa, IIIb, and V are the yellow pigments of this species. Spot K corresponds to authentic L-kynurenine in its chromatographic behaviour,
FIG. 1. Two-dimensional chromatogram of the crude extract of yellow pigments.
SIN MIL FEC PMA DNT
NNP EVA
7 8 9 10 11
12 13
Fluorescence Colour in visible rays Identity
0 P
Slightly 0 Y after first reagent tt
Pale Y Pale Y -
Slightly 0 Y after 8rs.t reagent, final Pi
Pale Yf$
Pale Y -
Neg Neg
WBu
Colourless Kynurenine
Immediate Pi
Neg
Br 0 P$ Neg
!r§ Dark Gn Bu II BrPT
F$ Neg Neg Immediate Pi
Br
$ Dark Br Bu II BrPT
Neg Neg Neg Neg Neg
Neg Neg Neg
RPt
K
Y-IIIa and Y-IIIb
I-COLOUR
?)
Br
Colourless uric acid ( ?)
?
Colourless -
None
None Colourless -
-
?
Bu Pale Y or BrlI -
-
-
-
-
-
-
None
-
Br
Bu Pale Y or Bry -
-
-
-
-
-
Bu Pale Y**
Br
Q
P
U
1
Pale Y
Slightly 0 Y after first reagent t t
Dark Gn Bu II BrPfl
Y§
Neg Neg Immediate Pi ( I) Br
f 0 f (or
Y-V
TESTS OF THE SPOTS IN FIG.
Colourless
None
-
-
RP
N
* Explanations of the abbreviations for colour tests are in the paragraph concerning Materials and Methods, pp. 1204, 1205. t Abbreviations for colours. These are common to Tables 1, 2, and 3. Br, brown; Bu, blue; Gn, green; 0, orange; P, purple; Pi, pink; R, red; Ro, rose; W, white; f, faint; Neg, negative. $ The purple colour is not so deep as in kynurenine and appears later than in kynurenine. 8 After a while, the yellow colour turns dark or yellowish grey. 11The blue colour appears shortly after spraying 2% phosphomolybdic acid and before exposing to ammonia vapour. 7 These spots show an orangeish yellow colour after spraying the first reagent. ** This spot does not show an orangeish yellow colour after spraying the first reagent. tt Whether or not these spots show a pink colour after spraying the second reagent was not clear because of their small quantities. $1 Papiliochrome IIa shows a bluish yellow fluorescence and Ilb, a yellow fluorescence.
NIN’ DAB TSU AHP BEE TTC
1 2 3 4 5 6
Test
Y-IIa and Y-IIb
TABLE
B
B
3
F
c
YELLOW PIGMENTS IN WINGS OF PAPILZO XUTHUS
1209
fluorescence, and colour tests and has been identified as such. As was reported in previous papers (UMEBACHI and TAKAHASHI, 1956; UMEBACHI and YAMADA,1964), the U.V.spectrum of spot K coincides with that of authentic kynurenine. Spot U corresponds to uric acid, not only in its position and colour reactions but also in the U.V. spectrum of its eluate. Uric acid and some o-diphenolic compounds, however, are similar in colour reactions and U.V.spectra. In addition, when old samples of butterflies were used as materials,spot U’ appeared instead of spot U. So, whether spot U is uric acid or not remains unsettled. Spots P, Q, and N also remain unidentified, though the colour tests suggest that the first two substances belong to phenolic compounds and the last one, to amino compounds. The more detailed properties of the spots U, U’, P, Q, and N were not investigated in the present paper, because they were present only in small quantities. The yellow pigments are pale yellow in visible rays and give a bluish yellow to pale yellow fluorescence under U.V.rays. Among these yellow pigments, Y-IIa and IIb are the main components. Y-V was too slight to be examined. Although Y-IIa, IIb, IIIa, and IIIb are positive to the tests for reducing sugars, such as silver nitrate and TTC, they are not sugars,because they are negativeto the aniline hydrogen phthalate and benzidine tests. The yellow pigments are positive to all the tests for phenols, that is silver nitrate, ferric chloride, Evans, diazotized p-nitroaniline, TTC, phosphomolybdic acid, Millon, and cl-nitroso-/3-naphthol. It is especially important to note that the yellow pigments (1) are positive to Evans’s test for o-diphenols, (2) give not a red but a slightly orange colour with the or-nitroso+naphthol test, and (3) show a yellow colour with Millon’s test. Moreover, the yellow pigments are also positive to the ninhydrin, p-diiethylaminobenzaldehyde, and Tsuda’s tests. All these resultsindicatethat the yellow pigments are o-diphenols which contain an amino group and moreover suggest that they have both aromatic and aliphatic amino groups. The Y-II group and Y-III group can be distinguished by the ferric chloride test. In the previous paper (UIVIEBACHI, 1961), these yellow pigments were named Papiliochrome II and III. In the present paper, they were found to contain two separatepigments respectively and are referred to as Papiliochrome IIa, IIb, IIIa, and IIIb. As mentioned above, Papiliochrome IIa and IIb are the main components. Papiliochrome IIIa and IIIb are rather small in quantity. Therefore, the chemical and physical properties of IIa and IIb were investigated in detail. Papiliochrome IIa and IIb are the same in all the colour tests, though they are a little different in fluorescence. The IIa gives a bluish yellow fluorescence, and the IIb, a yellow fluorescence. The elution of Papiliochrome IIa or IIb from the chromatogram and the rechromatography of the eluate did not give rise to the conversion of IIa into IIb or vice versa. Purajication of Papiliochrome
IIa and IIb
The wings of 150 to 200 butterflies were cut into small pieces and extracted with about 200 ml 80% ethanol. Two vol. of chloroform were added to the extract, 38
1210
Y. UMEBACHI AND K. YOSHIDA
and
after a while the water layer was taken. After one more repetition of the same treatment, the combined water layer was applied to the DEAE cellulose column (2 x 15 cm). The yellow pigments were not adsorbed and were washed down with water. Some brownish substances were adsorbed at the top of the column. The water effluents (yellow, 50-70 ml) were applied to the Dowex-50 X4 column (0.9 x 10 cm), on which the yellow pigments were adsorbed. The column was washed successively with 250 ml each of 0.1 M pyridine-formic acid buffer (pH 3-l), O-2 M pyridine-formic acid buffer (pH 3*1), O-2 M pyridineacetic acid buffer (pH 3-l), and 0.4 M pyridine-acetic acid buffer (pH 4.7) at a rate of 5 to 6 drops/min. Finally, the yellow pigments were eluted with 3-O M pyridine-acetic acid buffer (pH 5.5) at a rate of 2 to 3 drops/min. Only the Papiliochrome II group was eluted, while the Papiliochrome III group remained at the top of the column. The combined eluate (about 30 ml) of the Papiliochrome II group was concentrated to 2 ml by repeatedly shaking it with ethylacetate. Then, 1 to 2 ml of BAW solvent was added. The mixture was concentrated to a syrup (about 0.5 ml) under reduced pressure. Just enough of the cellulose powder, prepared as described above, was added to take up the yellow syrup and to give a dried yellow powder. The yellow cellulose powder was put on the top of the cellulose column (0.9 x 80 cm) and developed with BAW solvent at a rate of 2 drops/3 min. Papiliochrome IIa and IIb were separated. The fractions of IIa and IIb were collected separately, and about 5 ml of cold ethyl ether was added to each fraction (1.0 to 1.5 ml). The water layer was taken, and an equal volume of cold 99% ethanol was added. On top of it, ethyl ether was added dropwise until the solution became turbid. After keeping the mixture at 0°C for 1 hr, the yellow powder produced was gathered by centrifugation, washed with ethyl ether, and dried under reduced pressure. The powders of Papiliochrome IIa and IIb were respectively tested for their purity by paper chromatography and confirmed to give a single spot respectively, though in some cases a trace of kynurenine was produced by spotting the sample on the filter paper.
Ultra-violet absorption spectra of Papiliochrome IIa and IIb Purified Papiliochrome IIa and IIb were dissolved in the phosphate buffer, and their U.V. spectra were determined. Both pigments gave the same absorption peaks of 262 to 263, 280 to 284 (shoulder), and 380 to 381 nm as shown in Fig. 2. For the purpose of comparison, the U.V. spectra of authentic kynurenine are shown in Fig. 3, for, as will be shown later, Papiliochrome IIa and IIb are decomposed to some o-diphenol derivative and kynurenine by heating. The kynurenine obtained from Papiliochrome II showed the same spectral behaviour as that of authentic kynurenine (curve c of Fig. 7). It is known that the absorption peak at 360 nm of kynurenine in the phosphate buffer is due to the presence of the aromatic carbonyl group which is under the influence of the amino group in the o-position. This is supported by the shift of the peak to 369 to 371 nm in 99% methanol (Fig. 3). In order to examine the property of the absorption peak at 380 nm of
YELLOW PIGMENTS IN WINGS OF PAPILIO
1211
XUTHCJS
1-o
0.5
I
1
LENGTH
WAVE
Ultra-violet
300 WAVE
Ultra-violet
I
400
(mpl)
spectra of Papiliochrome IIa and IIb in phosphate buffer (pH 7-O). (a) IIa; (b) IIb.
250
FIG. 3.
350
300
250
FIG. 2.
I
I
350 LENGTH
400
(mr*)
spectra of authentic kynurenine. (a) In phosphate buffer (pH 7.0); (b) in 99% methanol.
1212
Y. UMEBACHIAND K. YOSHIDA
Papiliochrome IIa and IIb, their U.V. spectra were also determined in 99% methanol. As shown in Fig. 4, the peak at 380 to 381 nm shifted to 382 to 383 nm, and the peak at 262 to 263 nm to 260 to 261 nm. As Papiliochrome IIa and IIb seem to be a combination of o-diphenol derivative and kynurenine as will be shown later, these experimental results show that the peak at 380 to 381 nm is due to the 0.9
w 0.6 0 z a m E v) m a 0.3
300
250 WAVE FIG. 4.
Ultra-violet
350 LENGTH
400
(m&I
spectra of Papiliochrome IIa and IIb in 99% methanol. (a) IIa; (b) IIb.
aromatic carbonyl group of kynurenine contained as a component. It is conceivable that the peak at 360 nm of kynurenine shifts to 380 to 381 nm by a combination with o-diphenol derivative and gives a yellow colour to the compound. Infra-red absorption spectra of Papiliochrome IIa and Ilb
These curves are shown in Fig. 5. Papiliochrome IIa and IIb gave the same spectrum. As will be shown later, these yellow pigments contain both catecholamine derivative and kynurenine as their components. But it was difficult to assign exactly each absorption peak to any specific chemical group contained in the pigment, though the assignment of several peaks could be suggested. The broad absorption between 3000 and 3600 cm-l suggests the presence of an amino group, phenyl group, or hydroxyl group, or all of them. There is also the possibility that the broad absorption in this range is caused by water which is present in the sample, and judging from the fact that the yellow pigments contain kynurenine as a component, the absorption at 1638 cm-l seems to correspond to the aromatic carbonyl group which have an amino group in the o-position and/or to an amido or amino group. Moreover, the absorption at 757 cm-l suggests the presence of an
YELLOW PIGMENTS IN WINGS OF PAPILIO
1213
XUTHUS
o-substituted phenyl compound, and the absorption at 873 cm-l and 820 cm-’ may correspond to a 1,2,4+ubstituted phenyl compound. There is also the possibility that the absorption at 873 cm-l shows the presence of a 1,2,4,5substituted phenyl compound. It is clear that there is no ester bond. WAVE 100%
3 1
5 I
LENGTH
(,kL)
6 I
10 I
8 I
16 I-
50-
z z a I-
o-
_c loo-
I
I
I
I
I
I
I
I
t
l-
B cn z a E
50 -
O-
I
I
3200
2000 FREQUENCY
I
I
1600
1200
I 600
(cm-l)
FIG. 5. Infra-red spectra of Papiliochrome IIa and IIb.
ORD and CD spectra of Papiliochrome Ila and IIb
Aside from being able to separate Papiliochrome IIa and IIb by paper or cellulose column chromatography, they can also be distinguished by their fluorescence on the chromatogram, but both pigments are the same in their colour tests, U.V. spectra, ix, spectra, and decomposition products. Therefore, both yellow pigments were presumed to be optically isomeric, and their ORD and CD spectra were determined as shown in Fig. 6(A). Papiliochrome IIa showed a negative Cotten effect curve and a negative CD band at around 382 nm. On the other hand, Papiliochrome IIb gave the opposite curves. This indicates that Papiliochrome IIa and IIb are optically isomeric. As mentioned above, the U.V. absorption peak at 382 nm is due to the presence of an aromatic carbonyl group. This suggests that there is an asymmetric centre near the aromatic carbonyl group. As Papiliochrome IIa and IIb contain kynurenine as one of their components, the ORD and CD curves of kynurenine were determined for the purpose of comparison. As will be shown later, the kynurenine obtained from the yellow
1214
Y.
UMEBACHI
ANDK. YOSHIDA
pigments was L-isomer in both Papiliochrome IIa and IIb. D-Kynurenine was not found in P. xuthus. The ORD and CD spectra of authentic L-kynurenine are given in Fig. 6(B), which shows that the DL-isomerism of kynurenine hardly affects the ORD and CD curves at around 369 to 371 nm. That seems to be the reason why Papiliochrome IIa and IIb can show a mirror symmetry at least as far 3.0 -
B - 0.8
m 4
L
1
290
I
I
I
370
290
450 WAVE
LENGTH
370
450
(miu)
FIG. 6. ORD and CD spectra of Papiliochrome IIa and IIb and L-kynurenine. (A) (a), (b), and (c)-u.v., ORD, and CD spectra of Papiliochrome IIa; (d), (e), and (f ju.v., ORD, and CD spectra of Papiliochrome IIb. (B) (a), (b), and (c)u.v., ORD, and CD spectra of L-kynurenine. Solvent: 99% methanol in all the cases.
as the ORD and CD at around 382 nm are concerned. The u.v., ORD, and CD spectra of the yellow pigments and kynurenine indicate that a combination of kynurenine with o-diphenol derivative gives rise to a new asymmetric field near the aromatic carbonyl group of kynurenine and, moreover, that both components do not join together at the amino or carboxyl group of the side-chain of kynurenine. Decomposition of Pap&chrome IIa and Ilb in mild acid condition For this purpose, Papiliochrome IIa and IIb which were either eluted from the two-dimensional chromatogram or purified by column chromatographies were used. The yellow pigments from both of the separating methods gave the same results. Papiliochrome IIa and IIb were dissolved in 1O-3 N HCl and heated in a boiling water-bath for 30 to 60 min, and then the U.V. spectra of the solution were
YELLOWPIGMENTSIN WINGSOF PAPILIO
1215
XUTHUS
determined (curve (b) of Fig. 7). The absorption peak at 380 to 381 nm disappeared and shifted to 360 to 362 nm. An absorption peak at 279 to 281 nm appeared instead of the shoulder at 280 to 284 nm. The peak 262 to 263 nm shifted to 257 nm. Next, the same solution was one-dimensionally chromatographed with BAW
I
250
350
300 WAVE
LENGTH
400
(rnp)
FIG. 7. Ultra-violet spectra of the decomposition products of Papiliochrome IIa. (a) Papiliochrome IIa before decomposition; (b) after decomposition by heating in lo-* N HCl; (c) the kynurenine from Papiliochrome Ha; (d) the SN-1 substance. Solvent: lo-* N HCI. Papiliochrome IIb gave the same results.
solvent. After development, the filter paper was examined for fluorescence and then subjected to colour tests. Interestingly enough, Papiliochrome IIa and IIb gave the same chromatogram which is shown in Fig. 8(A). The results of the colour tests are given in Table 2. Judging from the colour reactions, fluorescence, Rtvalues, and the results of the previous papers (U-ACHI and TAKAHASHI, 1956; UMEBACHI, 1958, 1961; UMEBACHI and YAMADA, 1964), there is no doubt that the spot K is L-kynurenine. On the other hand, SN-1 is an unidentified substance, though the results of the colour tests indicate that the substance is o-diphenol which possibly contains amino nitrogen. Although the spot SN-1 is positive to the silver nitrate and TTC tests, it is not a sugar, because it is negative to the aniline hydrogen phthalate, benzidine, and resorcinol tests. The positive reactions to silver nitrate, TTC, phosphomolybdic acid, and diazotizedp-nitroaniline show that it belongs to the phenolic compounds, and, yet, as it is positive to the Evans, sodium molybdate, and ethylenediamine tests, and as it gives a yellow colour to Millon’s test and a faint orange colour to the ol-nitroso+-naphthol test, it is probable that SN-1 belongs to o-diphenol. Moreover, as it is negative or gives a
1216
Y. UMEBACHI AND K. YOSHIDA
light bluish colour to the ninhydrin test, the amino nitrogen contained seems to be neither the first amine nor cu-amino acid. The negativity to both Ehrhch’s and Tsuda’s tests shows that SN-1 does not have an aromatic amino group.
B
-S N-l’ -SN-2 -N-l -N-2
FIG. 8. (A) One-dimensional chromatogram of the decomposition products produced by heating Papiliochrome IIa in 10e9 N HCI. Solvent: BAW. (B) Onedimensional chromatogram of the degradation products produced by heating SN-1 in 1 N HCl. Solvent: BAW. In both (A) and (B), Papiliochrome IIb gave the same results as those of IIa.
The U.V. spectrum of SN-1 also suggests that it belongs to o-diphenol. The area of SN-1 on the chromatogram was cut out and eluted with 10-S N HCl, and the U.V. spectrum of the eluate was determined. The spectrum obtained is given in curve (d) of Fig. 7. Papiliochrome IIa and IIb gave the same result. The absorption peak was 279 to 281 nm. Fig. 7 shows that the solution of Papiliochrome IIa or IIb after heating in 10-s N HCl is a mixture of kynurenine and SN-1 substance. The U.V. spectrum of SN-1 is similar to those of DOPA, DOPAmine, and N-acetyl-DOPAmine as shown in Fig. 9. The absence of an R-band (n-n*) clearly shows that SN-1 does not contain an aromatic carbonyl group. For the purpose of comparison, the spectrum of protocatechuic acid is also shown in Fig. 9. On the other hand, it is evident from the paper chromatographic behaviour and colour tests that SN-1 is not DOPA, DOPAmine, or N-acetyl-DOPAmine. Moreover, as will be shown later, the injection experiments of 14C-labelled compounds indicate that SN-1 is a DOPAmine derivative. Although the result of Elson-Morgan’s test suggests the possibility that SN-1 is an N-acetyl compound, it remains to be proved. As mentioned above, there is no doubt that SN-1 is not N-acetyl-DOPAmine. The green colour reaction to 2,6-dichloroquinonechlorimide
YELLOWPIGMBNTSIN WINGS
OF PAPILIO
XUTHUS
1217
the possibility that SN-1 is a &substituted derivative of DOPAmine. But that also remains to be proved. At any rate, it is evident that SN-1 is a catecholamine derivative which has neither an aromatic carbonyl nor an aromatic amino group. Although the spot SN-2 of Fig. S(A) is also positive to the silver nitrate test, it is a substance which is derived from filter paper, because it appears only when
suggests
TABLB 2-Co~ot~~
TESTSOF THE SPOTSIN FIG. @A) spot K
Test
NIN*
1 2 3 4 5 6 7 8 9 10 11 12 13 14
DAB TSU AHP BEZ RES TTC SIN MIL FEC PMA DNT NNP EVA
15 16 17 18 19 20
SMO PFC EDA ELM DCQ BHC
Fluorescence Colour in visible rays Identity
SN-1
Neg Neg Neg 0 Neg Dark Bu
Sky Bu or Neg Neg Neg Neg Neg Neg Pi Br Y Gn or YGn Bu BrP f0 Y after first reagent, final Pi Y R: Pi (fluorescence) 0 fRoR 11 Slightly Gr PBuT
WBu
None
Colourless Kynurenine
Colourless
W
0 P Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
* Explanations of the abbreviations for colour tests are in the paragraph concerning Materials and Methods, pp. 1204, 1205. t Explanations of the abbreviations for colours are in the footnote of Table 1. $ Immediately after spraying the reagent, the spot becomes rose-red in visible rays. After several hours, the spot turns reddish purple in visible rays and gives a slightly purple fluorescence. 0 After several hours, the spot becomes orangeish yellow in visible rays and gives a pink fluorescence. After 1 to 3 days, the spot turns yellow in visible rays and gives an orangeish yellow fluorescence. (1After 1 to 2 days, the spot shows a faint rose-red colour. 7 The sensitivity is low.
1218
Y. UMEBACHIANDK. YOSHIDA
the eluate of Papiliochrome IIa and IIb from the paper chromatogram is heated and chromatographed again, and because it appears also in the chromatogram of the eluate of the blank filter paper which is treated in the same way. Spots N-l and N-2 of Fig. 8(A) are unidentified substances which are positive to the ninhydrin test and are only present in very small quantities. In some cases, these two spots were absent. They are probably secondary degradation products.
0.9
; 0.6 u m z fn m u 0.3
250 WAVE
LENGTH
(rnM,L)
FIG. 9. Ultra-violet spectra of authentic o&phenols. (a) Catechol; (b) DOPA; (c) DOPAmine; (d) IV-acetyl-DOPAmine; (e) protocatechuic acid.
The heating of Papiliochrome IIa and IIb in alkaline solution destroyed kynurenine. On the other hand, the heating of them in a strong acid solution broke down the SN-1 substance. Therefore, for the purpose of examining the primary decomposition products of Papiliochrome II, heating in a mild acid solution, for example 1O-3 N HCl, was most suitable.
Degradation of the SN-1 substance In order to examine further properties of SN-1, it was broken down by heating in a stronger acid solution. The area of SN-1 on the chromatogram was cut out and eluted in 1, 3, or 6 N HCl. The eluate was heated in a boiling water-bath for 30 min. After the solution was evaporated to dryness under reduced pressure, the residue was dissolved in a small quantity of water and chromatographed with BAW solvent again. Heating in 1, 3, and 6 N HCI gave the same result, which is shown in Fig. 8(B). SN-1 gave a spot which showed a little lower Rt value in BAW solvent and was different in colour tests. The spot was referred to as SN-1’. As seen in Table 3, SN-1’ shows a faint purple colour in the ninhydrin test. This
YELLOWPIGMENTSIN WINGSOF PAPILIO
XUTHUS
1219
fact suggests that the original substance SN-1 has an amino group in which the hydrogen is replaced by some unknown group. It is conceivable that this unknown group of the original substance SN-1 is removed by heating in 1 N HCl. The area TABLE 3-COLOUR TESTSOF SN-1’ IN FIG. S(B) 1 2 3 4 5 6 7 8
NIN* SIN PMA EVA SMO PFC EDA BHC
fW
Br Dark Bu Y after first reagent, final Pi BrY Pi: Y (fluorescence) f Dark Bu
* Explanations of the abbreviations for colour tests are in the paragraph concerning Materials and Methods, pp. 1204, 1205. t Explanations of the abbreviations for colours are in the footnote of Table 1. 3 Immediately after spraying the reagent, the spot becomes pink in visible rays. After several hours, the spot turns reddish purple in visible rays and gives a whitish blue fluorescence. $ After several hours, the spot becomes pink or orange in visible rays and gives a pink or whitish orange fluorescence.
of SN-1’ was cut out, and the U.V. spectra of the eluate was determined. As shown in Fig. 10, the peak was 280 run as for SN-1. It is probable from the colour tests and absorption spectrum that SN-1’ is a catecholamine derivative. The spot SN-2 of Fig. 8(B) is a reducing substance which is derived from filter paper as was already described in Fig. 8(A). Spots N-l and N-2 are ninhydrinpositive substances which are present in small quantities. They are also similar to those in Fig. 8(A), though this remains unsettled. The heating of the SN-1 solution in 1 N HCl at 100°C for 6 hr gave the same result as when heated for 30 min. But, in the heating of the SN-1 solution in 6 N HCl at 100°C for 6 hr, SN-1’ broke down further and gave several unidentified substances. Assuming that the conversion of SN-1 into SN-1’ might have resulted from heating in the air, the heating was carried out in nitrogen gas. The eluate of SN-1 was put into a glass ampoule and vacuumized, and nitrogen gas was introduced. After this procedure was repeated several times, the glass ampoule was sealed and heated. The heating of the SN-1 solution in 1 N HCl at 100°C for 30 or 60 min gave the same result as in Fig. 8(B). But the heating of the SN-1 solution in 6 N HCl at 100°C for 6 hr gave two substances, though the chemical properties of
1220
Y. UMEBACHIAND K. YOSHIDA
these two spots were not investigated in detail. At any rate, it was evident that further.degradation or changes of SN-1’ occurred in this procedure.
WAVE
LENGTH
(mr*.)
FIG. 10. Ultra-violet spectrum of SN-1’ substance.
Injection experiments of 1%~labelled tryptophan, DOPA, and DOPAmine The r4C of tryptophan (alanine-2-14C), DOPA-2-r4C, and DOPAmine-1-W was incorporated into the yellow pigments. The autoradiographs of these wings are shown in Figs. 11(A), (B), and (C). The crude extract from the yellow scales which incorporated the 1% of tryptophan (alanine-2J4C) was two-dimensionally chromatographed. The autoradiograph of the chromatogram obtained is shown in Fig. 12(A). The radioactivity is seen in spots T, K, Y-IIa, Y-IIb, Y-IIIa, and Y-IIIb, which are respectively tryptophan, kynurenine, Papiliochrome IIa, IIb, IIIa, and IIIb. Y-V is also radioactive. The areas of Papiliochrome IIa and IIb were cut out and separately eluted with lo4 N HCl. The eluatewas heated in a boiling water-bath for 30 min, concentrated under reduced pressure, and then one-dimensionally chromatographed. The autoradiograph of the chromatogram is shown in Fig. 13(A). Spot K is kynurenine. As had been expected, the 14C was incorporated into kynurenine and not into SN-1. The crude extracts from the yellow scales which incorporated the 14C of DOPA-2-W and DOPAmine-lJ4C were also treated in the same way as mentioned above. The results obtained from both compounds were the same concerning the incorporation of 14Cinto the yellow pigments, Fig. 12(B) shows the autoradiograph of the two-dimensional chromatogram in the case of DOPAmine-1-14C. Although the radioactivitywas weaker than in the case of tryptophan (alanine-2J4C), there was no doubt that the r%! was incorporated
1221
FIG. 11. Autoradiographs of the wings of the male adults, in the pharate pupal stage of which (A) tryptophan (alanine-2J4C), (B) DOPA-2J4C, and (C) DOPAmine-l -14C was injected respectively.
FIG. 12. Autoradiographs of the two-dimensional chromatograms of the crude extracts from the yellow scales which incorporated the 14C of (A) tryptophan (alanine-2-14C) and (B) DOPAmine-lJ4C.
1223
S N-l
Frc. 13. Autoradiographs of the one-dimensional chromatograms of the decomposition products of Papiliochrome IIa which incorporated the l*C of (A) tryptophan (alanine-2-14C) and (B) DOPAmine-lJ4C. Solvent: BAW. In the case of DOPA-2-14C, the result was the same as in DOPAmine-l-i4C. In both (A) and (B), Papiliochrome IIb gave the same results as those of IIa.
FIG. 14.
Autoradiographs
of the wings
of the male adult, (B) DOPA-l-14C
in the pharate pupal stage of which was injected respectively.
(A) tryptophan
(ring-2-l%)
and
YELLOW PIGMENTS IN WINGS OF PAPILIO
XUTHUS
1225
into Papiliochrome IIa, IIb, IIIa, and IIIb. The area including the startingpoint and Y-V was also radioactive. The autoradiographs of the one-dimensional chromatograms obtained after heating the eluate of Papiliochrome IIa and IIb are shown in Fig. 13(B). It is evident that the 14C is incorporated into SN-1 but not into kynurenine. The 14C of DOPA-1J4C and tryptophan (ring-2J4C) was not incorporated into the yellow pigments (Figs. 14A and B). But, in the case of the latter labelled compounds, there were some cases in which the yellow markingsof the wings were slightly radioactive. The reasonwhy the injection experimentof tryptophan (ring-214C)was carried out will be mentioned later. DISCUSSION
In the previous paper (UMEBACHI, 1961), it was reported that there were two kinds of yellow pigments in the wings of P. mthus, that is Y-II and Y-III. After that, it was found that Y-II and Y-III contain two separatepigments respectively, and in the present paper they were referred to as Papiliochrome IIa, IIb, IIIa, and IIIb. Among them, only IIa and IIb have been investigatedfor detailed physical and chemical properties, because Papiliochrome IIa and IIb are the main yellow pigments. Papiliochrome IIIa and IIIb are too small in quantity to be purified. Since UMEBACHI and NJXAMURA (1954) found that a largequantityof kynurenine was present in the extract of the wings of P. mthus, the following facts have been proved: (1) In the pupa, as the result of the increase of tryptophan pyrrolase activity during the pharate adult stage, the content of kynurenine increases. The kynurenine thus formed accumulates in the yellow scales as a bound form. The bound form of kynurenine means a compound which produces kynurenine by heating (UMEBACHI and KATAYAMA, 1966). (2) The yellow pigments of the yellow scales readily decompose to give a phenolic compound and kynurenine (UMEBACHI, 1961). In the present paper, Papiliochrome IIa and IIb were purified, and their physical and chemical properties were investigated. It was confirmed that Papiliochrome IIa and IIb can be readily decomposed to kynurenine and o-diphenol (SN-1) by heat’mg in a mild acid condition. The o-diphenol SN-1 is negative to the ninhydrin test or gives a faint light bluish colour, and is converted by heating in 1 N HCl into a substance (SN-1’) which gives a faint purple colour for the same test. From the colour tests and the injection experiments of i4C-labelled compounds, SN-1 seems to be an N-substituted derivative of DOPAmine. The Elson-Morgan test suggests the possibility that SN-1 is an N-ace@ derivative, though it does not correspond to N-acetyl DOPAmine. Moreover, judging from the negative result to 2,6-dichloroquinonechlorimide, the possibility exists that the hydrogen of the 6-position of SN-1 is also replaced by some group. In the previous paper (UMEBACHI and KATAYAMA, 1966), the possibility was suggested that the yellow pigments might be formed by a combination of odiphenol derivative and formylkynurenine, though the pigments are decomposed to o-diphenol derivative and kynurenine by heating. But, as the 1% of tryptophan
1226
Y. UMEESACHI AND K. YOSHIDA
(ring-2J4C) was not incorporated into the yellow pigments, this possibility was ruled out. The possibility is also excluded that after the decomposition of the yellow pigments to o-diphenol derivative and kynurenine, the formyl group of formylkynurenine keeps attaching to the o-diphenol derivative because it is clear from the U.V. spectrum that SN-1 does not have an aromatic carbonyl group. After all, Papiliochrome IIa and IIb seem to be formed by a combination of DOPAmine derivative and kynurenine (not formylkynurenine). The possibility exists that Papiliochrome IIa and IIb are formed by a combination of N-substituted DOPAmine and kynurenine and readily decompose to give kynurenine and a 6and N-substituted DOPAmine. Interestingly enough, Papiliochrome IIa and IIb gave the same decompositionproducts, U.V. spectra, and i.r. spectra. Before the present paper, Papiliochrome IIa and IIb could be distinguished only by their chromatographic behaviour and fluorescence. But, in the present paper, it was found that Papiliochrome IIa and IIb are different also in their ORD and CD curves, and, yet, both yellow pigments give the opposite Cotten effect. These results show that both yellow pigments are optically isomeric. It is almost certain that the absorption peak (360 nm) caused by the aromatic carbonyl group of kynurenine is shifted to 380 to 381 nm by the formation of the pigment, that is, by a combination with a DOPAmine derivative. In other words, the shift of 360 nm to 380 nm is the reason why the compounds turn yellow. It is very interesting that Papiliochrome IIa and IIb give just the opposite Cotten effect at around this peak (382 run in methanol). It must be kept in mind that the optical activity which is caused by the rn.-isomerism of the side-chain of kynurenine hardly affects the optical activity at around 370 nm (in methanol). The kynurenine contained in the pigments is L-kynurenine in both Papiliochrome IIa and IIb. From these spectral studies, the greatest possibility for the combination of DOPAmine derivative and kynurenine is that the former compound combines with the latter by way of the aromatic amino nitrogen of the latter, though further investigations are necessary to confirm it. For the tyrosine metabolism in insects, the following three main pathways have been known or suggested: The first is the pathway of melanin formation. It is generally thought to be similar to that in vertebrates, that is, melanin is formed by way of DOPA, DOPAquinone, and DOPAchrome. But detailed biochemical studies have not yet been made in insects. The second is the way of DOPAmine, noradrenaline, and adrenaline formation by way of DOPA. It is interesting that DOPAmine and noradrenaline have been reported to be present in large quantities in insects (BRUNET, 1963). N-Acetyl-DOPAmine is also formed in this way. The third is the way of phenolic acid formation, such as p-hydroxyphenylpyruvic acid and p-hydroxyphenyllactic acid. In the present paper, when DOPA-1J4C or DOPA-2-W was injected into the prepupa of P. xuthus, the 14C was not incorporated into the black scales. The reason remains unsolved, but one possibility is that when the injection is made at the pharate pupal stage, the metabolic transition of the injected compound and the development of black scales (or the stage of
YELLOWPIGMENTSIN WINGSOF PAPILIO
XUTHVS
1227
melanin formation) do not match well with each other. Studies on the metabolism of DOPA and DOPAmine are being made in our laboratory. In P. xuthus, the yellow and black scales are adjacent to each other in the border of the markings. In some papilionid butterflies, for example Ldhdorja japomka, in addition to the yellow and black scales, there are red scales which also incorporate the 14C of tryptophan (alanine-% W) (unpublished data). The black pigment is melanin which is probably derived from tyrosine. As for the red pigments, it is possible that they belong to the ommochrome group which is formed by way of On the other hand, as mentioned above, the yellow pig3-hydroxykynurenine. ments are not ommochrome but a combination of o-diphenol derivative and kynurenine. Thus, the papilionid butterflies present an interesting problem in the relationship between tryptophan and tyrosine metabolism. From the biological point of view, it presents a problem of the differentiation, regulation, and diversity of insect pigment metabolism, Acknowledgements-We wish to express our thanks to Miss Y. Ishizaki and Mr. T. Tanaka for their technical assistance. REFERENCES ACHER R. and CROCKERC. (1952) Reactions colorees spdcifiques de l’arginine et de la tyrosine real&es apres chromatographie sur papier. Biochim. biophys. Acta 9, 704-705. BRUNETP. C. J. (1963) Tyrosine metabolism in insects. Ann. N.Y. Acad. Sci. 100, 10201034. BUTENANDT A. and SCHXFERW. (1962) Ommochromes. In Recent Progress in the Chemistry of Natural and Synthetic Colouring Matters and Related Fields (Ed. by GORYT. S. et aZ.), pp. 13-33. Academic Press, New York. COULSONC. B. and EVANSW. C. (1958) Paper chromatography and paper electrophoresis of phenols and glycosides. J. Chronuatog. 1, 374-379. CUR F. (1954) Paper Chromatography. Macmillan, London. DALGLIE~HC. E. (1952) The relation between pyridoxin and tryptophan metabolism, studied in the rat. Bi0chem.J. 52, 3-14. FURNSAUXP. J. S. and MCFARLANEJ. E. (1965) Identification, estimation, and localization of catecholamines in eggs of the house cricket, Acheta domesticus (L.). J. Insect Physiol. 11, 591-600. GIBBS H. D. (1927) Phenol tests-III. The indophenol test. g. biol. Chem. 72, 649-664. Hoxnocxs R. H. (1949) Paper partition chromatography of reducing sugars with be&dine as a spraying reagent. Nature, Lond. 164,444. LEDBRERE. and LEDERERM. (1957) Chromatography, A Review of Principles and Applications. Elsevier, Amsterdam. MAZURR. H., ELLIS B. W., and CAMMARATA P. S. (1962) A new reagent for detection of peptides, nucleotides, and other N-H containing compounds on paper chromatograms. J. biol. Chenz. 237, 1619-1621. PARTRIDGES. M. (1948) Filter-paper partition chromatography of sugars-I. General description and application to the qualitative analysis of sugars in apple juice, egg white and foetal blood of sheep. Biochem. J. 42, 238-248. PITTARDA. J., GIBSON F., and DOY C. H. (1961) Phenolic compounds accumulated by washed cell suspensions of a tryptophan auxotroph of Aerobacter aerogenes. Biochim. biophys. Acta 49, 485-494.
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Y. UMIXBACHI ANDK. YOSHIDA
PRIDHAMJ. B. (1959) Paper electrophoresis and paper chromatography of phenolic compounds. g. ChromatogY 2,605-611. RILEYR. F. (1950) Paper partition chromatography of some simple phenols. J. Am. them. Sac. 72. 5782-5783. SCHtiPF C: (1964) Die Anfringe der Pterin-Chemie. In Pteridine Chemistry (Ed. by PFLEIDHRBR W. and TAYLOR E. C.), pp. 3-14. Pergamon Press, Oxford. SBNOHS., CRBVBLWGC. R., UDENFRIEND S., and WITKOPB. (1959) Chemical, enzymatic and metabolic studies on the mechanism of oxidation of DOPAmine. J. Am. them. Sot. 81,6236-6240. SENOH S. and WITKOP B. (1959) Non-enzymatic conversions of DOPAmine to norepinephrine and trihydroxyphenethylamines. J. Am. them. Sot. 81, 6222-6231. Sounxxs T. L., DENTONR. L., MURPHY G. F., CHAVJXZ B., and SAINT CYR S. (1963) The excretion of dihydroxyphenylalanine, DOPAmine, and dihydroxyphenylacetic acid in neuroblastoma. Pediatrics 31, 660-668. SWAIN T. (1953) The identification of coumarins and related compounds by filter-paper chromatography. Biochem. J. 53, 20&208. )T(R&VBLYAN W. E., PROCTERD. P., and HARRISONJ. S. (1950) Detection of sugars on paper chromatograms. Nature, Lond. 166, 444445. UM~BACHIY. (1958) Yellow pigments in the wings of the papilionid butterflies-I. The relation between kynurenine and the yellow pigments of Papilio xuthus. Sci. Rep. Kanazawa Univ. 6, 45-55. UME~ACHIY. (1959a) Yellow pigments in the wings of the papilionid butterflies-II. The presence of a pale blue fluorescent substance supposed to be kynurenine in the wings of the Zerynthiinae. Sci. Rep. Kanazawa Univ. 6, 69-75. U~VIEBACHI Y. (1959b) Yellow pigments in the wings of the papilionid butterflies-III. The radioautographs of the wings of five species of Papilio injected with 14C-labeled tryptophan. Antwt. Zool.~apan 32,112-116. UMEBACHIY. (1960) Yellow pigments in the wings of the papilionid butterflies-IV. The presence or absence of kynurenine in the wings of the genus Graph&m. Sci. Rep. Kanazawa Univ. 7,107-112. U~ACHI Y. (1961) Yellow pigments in the wings of the papilionid butterflies-V. Some chemical properties of the yellow pigments of Papilio xuthus. Sci. Rep. Kunaxawa Univ. 7,139-150. UM~BACHIY. (1962) Yellow pigments in the wings of the papilionid butterflies-VII. Consideration of the nature and distribution of the wing pigments of the papilionid butterflies. Sn’. Rep. Kunazawa Univ. 8, 135-142. UMEBACHIY. and KATAYAMA M. (1966) Tryptophan and tyrosine metabolism in the pupa of papilionid butterflies-II. The general pattern of tryptophan metabolism during the pupal stage of Papilio xuthus. J. Insect Physiol. 12, 1539-l 547. A. (1954) The presence of kynurenine in the wings of the UM~BACHIY. and NAKAMURA papilionid butterflies. Zool. Mug. Tokyo 63, 57-61. (In Japanese, English summary.) UMEBACHIY. and TAKAHASHI H. (1956) Kynurenine in the wings of the papilionid butterflies. 3. Biochem. Tokyo 43,73-81. UMEEIACHI Y. and TSUCHITANIK. (1955) The presence of xanthurenic acid in the fruit-fly, Drosophila melanogaster. J. Biochem. Tokyo 42, 817-824. UMEEIACHI Y. and YAMADAM. (1964) Tryptophan and tyrosine metabolism in the pupa of papilionid butter!Iies-I. Accumulation of the bound form of kynurenine in Pupilio xuthus. Annot. Zool. Japon. 37, 51-57.
SOME CHEMICAL AND PHYSICAL PROPERTIES PAPILIOCHROME II IN THE WINGS OF
PAPILIO Y. UMEBACHI
OF
XUTHUS and K. YOSHIDA
Department of Biology, Faculty of Science, Kanazawa University, Kanazawa, Japan (Received 8 September 1969)
Abstract-The yellow pigments in the wings of Papilio xuthus were extracted and two-dimensionally chromatographed. Two major and two minor yellow pigments were found and named Papiliochrome IIa, IIb, IIIa, and IIIb respectively. Some chemical properties of these pigments were examined by colour tests. After being purified using DEAE cellulose, Dowex SO-W, and cellulose columns, IIa and IIb were examined for u.v., i.r., optical rotatory dispersion, and circular dichroism spectra. The decomposition products of these pigments were also examined for their chemical and physical properties. Moreover, ‘%Xabelled tryptophan, DOPA, and DOPAmine were injected into the prepupa and examined for their incorporation into the yellow pigments. It was inferred from the results of these experiments that Papiliochrome IIa and IIb are formed by a combination of DOPAmine derivative and kynurenine and are optically isomeric to each other. INTRODUCTION
AMONG the wing pigments
of butterflies, the pterins of the Pieridae (SCHBPF, 1964) of the Nymphalidae (BUTENANDT and SCHAFER, 1962) have been investigated in detail from the chemical and biochemical points of view. However, little work has been done on the pigments of other families. It was discovered by UMEBACHIand NAKAMUEW (1954) that the hot-water extract of the yellow scales of the papilionid butterflies contained a large quantity of kynurenine, an intermediate of tryptophan metabolism. Since then we have investigated the chemical properties and formation of the yellow pigments of this family (UMEBACHIand TAKAHASHI, 1956; UMEBACHI,1958,1959a, b, 1960,1961,1962; UMEBACHIand YAMADA,1964; UMEBACHI and KATAYAMA,1966). The yellow pigments of Pupilio mthus in particular have been used as the material for our chemical studies. In the previous paper (UMEBACHI,1961), two groups of yellow pigments were found in the yellow scales of this species and were named Papiliochrome II and III respectively. Both groups of pigments were readily decomposed by heating and yielded kynurenine and a phenolic compound. The present paper deals mainly with the more detailed chemical and physical properties of Papiliochrome II. It was proved that Papiliochrome II and III respectively contain two kinds of yellow pigments. They were named Papiliochrome IIa and IIb and IIIa and IIIb. and the ommatins
1203
1204
Y. UMEBACHI AND K. YOSHIDA
Papiliochrome IIa and IIb were separated, purified, and examined for their chemical and physical properties. From these experiments and the incorporation tests of 14C-labelled compounds, it was inferred that Papiliochrome IIa and IIb are formed by a combination of DOPAmine derivative and kynurenine. Interestingly enough, Papiliochrome IIa and IIb gave the same results regarding colour tests, decomposition products, U.V. and i.r. spectra, and the incorporation of i4C-labelled compounds. But, as to optical rotatory dispersion and circular dichroism, both pigments showed opposite curves, and thus it was suggested that Papiliochrome IIa and IIb are optically isomeric. MATERIALS
AND METHODS
Materials The male adults of Pap&o xuthus were used in the experiments other than the incorporation tests of 14C-labelled compounds. Most of them were obtained through the Okura Biological Institute. Some of them were collected during their larval stage in Kanazawa and raised to the adult stage in the laboratory. For the incorporation tests of i*C-labelled compounds, they were injected during their pharate pupal stage with the compounds which will be mentioned later and then raised to the adult stage. Extraction of yellow pigments The yellow pigments of the wings are soluble in water and 80% ethanol and are insoluble in ethyl ether and chloroform (UMEBACHI, 1961). Therefore, the yellow pigments were extracted from the wings or yellow scales with water or 80% ethanol. After centrifugation, the supernatant fluid, which is referred to below as a crude extract of the yellow pigments, was used as the material for the chromatographic separation and purification. Paper chromatography Toyo No. 51A filter paper was used. The chromatogram was run by the ascending method with 80% methanol or the upper layer of n-butanol-glacial acetic acid-water (4 : 1 : 5) mixture (BAW). For two-dimensional separation, the former solvent was used for the first direction, and the latter for the second direction. After development, the filter paper was inspected under U.V. rays and then subjected to one of the following colour tests: (1) Ninhydrin test (NIN). (2) Ehrhch’s test for indole structure and aromatic ammo compounds usingp-dimethylaminobenzaldehyde (DAB) (DALCLIESH, 1952). After the reagent was sprayed, the filter paper was warmed. (3) The test for aromatic amines using Tsuda’s reagent (fi-diethylaminoethyl-anaphthylamine oxalate) (TSU) (UMEBACHIand TSUCHITANI,1955). (4) Aniline hydrogen phthalate test for reducing sugars (AHP) (CRAMER,1954). (5) Be&dine test for reducing sugars (BEZ) (HORROCKS,1949). (6) Resorcinol test for sugars (RES) (LBDERERand LEDERBR,1957).
YELLOW
PIGMBN’ISIN
WINGS OF PAPILIO
XUTHUS
1205
(7) 2,3,5-Triphenyltetrszoliumchloride test for reducing sugars, o-diphenols, and other reducing substancea (TTC) (TREVELYAN et OZ., 1950: PITTARD et uZ., 1961). (8) Silver nitrate test for reducing sugars, o-diphenols, and other reducing substances (SIN) (SWAIN, 1953). (9) Millon’s test for phenolic compounds (MIL) (CRAMJXR,1954). (10) Ferric chloride test for phenolic compounds (FEC) (CRAMER, 1954). After the reagent was sprayed, the filter paper was warmed. (11) Phosphomolybdic acid-ammonia test for phenolic compounds (PMA) (RILEY, 1950). (12) Diazotized p-nitroaniline test for phenolic compounds (DNT) (SWAIN, 1953; COULSONand EVANS,1958). (13) or-Nitroso-fi-naphthol test for p-substituted phenolic compounds (NNP) (A&RR and CROCKER,1952). (14) Evans’s test for o-diphenols (EVA) (COULSONand EVANS,1958). (15) Sodium molybdate test for o-diphenols (SMO) (PRIDHAM,1959). (16) Potassium ferricyanide test for adrenaline and related compounds (PFC) (SENOH and WITKOP, 1959; SENOHet al., 1959). (17) Ethylenediamine-ammonia test for o-diphenols (EDA) (Soet al., 1963; FURNEAUXand MCFARLANE, 1965). DOPA and DOPAmine give a whitish yellow and yellow fluorescence respectively. Noradrenaline and adrenaline show an orsngeish yellow fluorescence. (18) Elson-Morgan’s test for glucosamine (ELM). Of the reagents a, b, and c used for detecting glucosamine, only the last two reagents were used. These two reagents were reported to be useful as a test for the presence of iV-acetylated derivative (PARTRIDGE,1948). Although this test is not suitable for o-diphenols, the following results were obtained for DOPA derivatives by us. DOPA and DOPAmine did not give a red but rather, an orangeish brown colour, while N-acetyl-DOPAmine gave a red colour. (19) 2,6-Dichloroquinone chlorimide test for phenols (DCQ). This is generally said to be a test for phenols in which the position puru to the hydroxyl group is unsubstituted (GIBBS, 1927). But, according to SENOH et al. (1959), 6-hydroxy-DOPAmine is also positive. (20) The test for peptides, amines, amides, and other N-H-containing compounds using tert-butylhypochlorite and potassium iodide (BHC) (MAZUR et al., 1962). A 1 : 1 mixture of saturated o-tolidine in 2% acetic acid and 0.05 N KI was used instead of the 1 y0 KI and 1 y0 starch solution. Tyrosine and DOPA are negative, while DOPAmine and kynurenine give a dark blue colour.
Preparation
of DEAE
cellulose column
One hundred ml of DEAE cellulose was suspended in about 500 ml of distilled water, and the fine and coarse powders were repeatedly removed by decantation. For the purpose of the present experiment, other pretreatment was not necessary. A 2 x 15 cm column of the DEAE cellulose was prepared, washed with about 175 ml of 0.2 N HCl and finally washed with about 750 ml of distilled water. Preparation of Dowex-50 W column Dowex-50 W X4 (200-400 mesh) was used. One hundred ml of dry resin was suspended in distilled water, and the particle size was made uniform by repeating decantation. The resin suspension in water was filtered through a glass filter and washed with 2 N NaOH until the filtrate turned alkaline. After washing with distilled water until neutralization, the resin was washed with 4 N HCl until the
1206
Y. UMJIBACHI AND K. YOSHIDA
resin turned pale in colour. It was washed again with distilled water and then with 2 N NaOH until the filtrate became colourless. These washing procedures were carried out in the same glass filter. Next, the resin was suspended in 3 vol. of 1 N NaOH in a beaker and heated in a water-bath at 60 to 70°C for about 3 hr. After standing at room temperature for 1 hr, the supernatant fluid was removed by decantation and hot 1 N NaOH was again added. This procedure was repeated five times. Next the resin was washed with distilled water, about 100 ml of 4 N HCl, and about 100 ml of 2 N HCl successively. After washing with distilled water until neutralization, the resin was suspended in 2 vol. of 2 M pyridine and stirred at room temperature for 1 to 2 hr. After that, the resin was filtered through a glass filter and washed with distilled water until the filtrate became neutral. Finally, the resin was suspended in 2 vol. of O-1 M pyridine-formic acid buffer (pH 3.1) and equilibrated by storing in the cold for at least 2 weeks. A O-9 x 10 cm column of the resin was prepared and washed with 500 ml of the above-mentioned buffer. Preparation
of cellulose column
About 100 ml of cellulose powder (Toyo, 100-200 mesh) was suspended in about 1 1. of 0.1 N HCl in 99% ethanol and boiled in a water-bath under reflux for 20 hr. After that, the powder was filtered through a glass filter and dried. Then the powder was suspended in BAW solvent and a 0.9 x 80 cm column was prepared. Ultra-violet
absorption spectra
These were determined in the range from 240 to 400 nm with a Hitachi model The solvent was 0.067 or O-1 M Sorensen’s phosphate 139 spectrophotometer. buffer (pH 7.0) 99% methanol, or 1O-3 N HCl. Infra-red These Bunko DS was mixed a pressure
absorption spectra were determined in the range from 4000 to 650 cm-l with a Nihon 301 i.r. spectrophotometer. The powder of the purified yellow pigment with KBr powder, and a micro-pellet (diameter, 5 mm) was made under of 4 to 6 tons.
Optical rotatory
dispersion (ORD)
and circular dichroism (CD) spectra
These measurements were made in the range from 450 to 250 nm with a Nihon Bunko ORD/UV-5 spectropolarimeter equipped with a CD attachment. The solvent was 99% methanol. Injection experiments
of 14C-labelled compounds
The labelled compounds used were as follows: (1) nL-Tryptophan (alanine-2- 1%). Specific activity, 0.45 mc/mM. Commercial source, Tracerlab. (2) nL-Tryptophan (ring-Z i4C). Specific activity, 21 mc/mM. Commercial source, Commissariat a l’$nergie Atomique Livraison de Molecules Marquees.
YELLOW
PIGMENTS
IN WINGS
OF PAPILIO XVTHVS
1207
(3) m_-3,4_Dihydroxyphenylalanine (carboxyl-14C). Specific activity, 3.75 mc/mM. Commercial source, New England Corp. (4) DL-3,4_Dihydroxyphenylalanine (alanine-2-14C). Specific activity, 5.7 mc/mM. Commercial source, The Radiochemical Centre, Amersham. (5) 3,4-Dihydroxyphenylethylamine-1J4C hydrobromide. Specific activity, 6.4 mc/mM. Commercial source, New England Nuclear Corp. From O-3 to 3.1 PC (0*02-0*06 ml in aqueous solution) of each compound was injected into one prepupa. After the emergence of the butterflies, the wings were cut off, dried in a desiccator, and then brought into contact with Fuji, Medical KX X-ray film. The time of exposure was from 10 to 21 days. The autoradiograph of the two-dimensional chromatogram of the crude extract of the yellow pigments was taken with the same X-ray film as described above. The time of exposure was 50 to 70 days. The autoradiographof the one-dimensional chromatogram after decomposition of the yellow pigments by heating was also taken in the same way. RESULTS
Paper chromatography of the crude extract of yellow pigments
The yellow pigments were extracted by steeping the yellow scales in a small quantity of distilled water or 80% ethanol for a short time. The crude extract thus obtained was spotted on filter paper immediately after the extraction and twodimensionally chromatographed. The chromatogram is shown in Fig. 1, and the colour tests are given in Table 1. Spots Y-IIa, IIb, IIIa, IIIb, and V are the yellow pigments of this species. Spot K corresponds to authentic L-kynurenine in its chromatographic behaviour,
FIG. 1. Two-dimensional chromatogram of the crude extract of yellow pigments.
SIN MIL FEC PMA DNT
NNP EVA
7 8 9 10 11
12 13
Fluorescence Colour in visible rays Identity
0 P
Slightly 0 Y after first reagent tt
Pale Y Pale Y -
Slightly 0 Y after 8rs.t reagent, final Pi
Pale Yf$
Pale Y -
Neg Neg
WBu
Colourless Kynurenine
Immediate Pi
Neg
Br 0 P$ Neg
!r§ Dark Gn Bu II BrPT
F$ Neg Neg Immediate Pi
Br
$ Dark Br Bu II BrPT
Neg Neg Neg Neg Neg
Neg Neg Neg
RPt
K
Y-IIIa and Y-IIIb
I-COLOUR
?)
Br
Colourless uric acid ( ?)
?
Colourless -
None
None Colourless -
-
?
Bu Pale Y or BrlI -
-
-
-
-
-
-
None
-
Br
Bu Pale Y or Bry -
-
-
-
-
-
Bu Pale Y**
Br
Q
P
U
1
Pale Y
Slightly 0 Y after first reagent t t
Dark Gn Bu II BrPfl
Y§
Neg Neg Immediate Pi ( I) Br
f 0 f (or
Y-V
TESTS OF THE SPOTS IN FIG.
Colourless
None
-
-
RP
N
* Explanations of the abbreviations for colour tests are in the paragraph concerning Materials and Methods, pp. 1204, 1205. t Abbreviations for colours. These are common to Tables 1, 2, and 3. Br, brown; Bu, blue; Gn, green; 0, orange; P, purple; Pi, pink; R, red; Ro, rose; W, white; f, faint; Neg, negative. $ The purple colour is not so deep as in kynurenine and appears later than in kynurenine. 8 After a while, the yellow colour turns dark or yellowish grey. 11The blue colour appears shortly after spraying 2% phosphomolybdic acid and before exposing to ammonia vapour. 7 These spots show an orangeish yellow colour after spraying the first reagent. ** This spot does not show an orangeish yellow colour after spraying the first reagent. tt Whether or not these spots show a pink colour after spraying the second reagent was not clear because of their small quantities. $1 Papiliochrome IIa shows a bluish yellow fluorescence and Ilb, a yellow fluorescence.
NIN’ DAB TSU AHP BEE TTC
1 2 3 4 5 6
Test
Y-IIa and Y-IIb
TABLE
B
B
3
F
c
YELLOW PIGMENTS IN WINGS OF PAPILZO XUTHUS
1209
fluorescence, and colour tests and has been identified as such. As was reported in previous papers (UMEBACHI and TAKAHASHI, 1956; UMEBACHI and YAMADA,1964), the U.V.spectrum of spot K coincides with that of authentic kynurenine. Spot U corresponds to uric acid, not only in its position and colour reactions but also in the U.V. spectrum of its eluate. Uric acid and some o-diphenolic compounds, however, are similar in colour reactions and U.V.spectra. In addition, when old samples of butterflies were used as materials,spot U’ appeared instead of spot U. So, whether spot U is uric acid or not remains unsettled. Spots P, Q, and N also remain unidentified, though the colour tests suggest that the first two substances belong to phenolic compounds and the last one, to amino compounds. The more detailed properties of the spots U, U’, P, Q, and N were not investigated in the present paper, because they were present only in small quantities. The yellow pigments are pale yellow in visible rays and give a bluish yellow to pale yellow fluorescence under U.V.rays. Among these yellow pigments, Y-IIa and IIb are the main components. Y-V was too slight to be examined. Although Y-IIa, IIb, IIIa, and IIIb are positive to the tests for reducing sugars, such as silver nitrate and TTC, they are not sugars,because they are negativeto the aniline hydrogen phthalate and benzidine tests. The yellow pigments are positive to all the tests for phenols, that is silver nitrate, ferric chloride, Evans, diazotized p-nitroaniline, TTC, phosphomolybdic acid, Millon, and cl-nitroso-/3-naphthol. It is especially important to note that the yellow pigments (1) are positive to Evans’s test for o-diphenols, (2) give not a red but a slightly orange colour with the or-nitroso+naphthol test, and (3) show a yellow colour with Millon’s test. Moreover, the yellow pigments are also positive to the ninhydrin, p-diiethylaminobenzaldehyde, and Tsuda’s tests. All these resultsindicatethat the yellow pigments are o-diphenols which contain an amino group and moreover suggest that they have both aromatic and aliphatic amino groups. The Y-II group and Y-III group can be distinguished by the ferric chloride test. In the previous paper (UIVIEBACHI, 1961), these yellow pigments were named Papiliochrome II and III. In the present paper, they were found to contain two separatepigments respectively and are referred to as Papiliochrome IIa, IIb, IIIa, and IIIb. As mentioned above, Papiliochrome IIa and IIb are the main components. Papiliochrome IIIa and IIIb are rather small in quantity. Therefore, the chemical and physical properties of IIa and IIb were investigated in detail. Papiliochrome IIa and IIb are the same in all the colour tests, though they are a little different in fluorescence. The IIa gives a bluish yellow fluorescence, and the IIb, a yellow fluorescence. The elution of Papiliochrome IIa or IIb from the chromatogram and the rechromatography of the eluate did not give rise to the conversion of IIa into IIb or vice versa. Purajication of Papiliochrome
IIa and IIb
The wings of 150 to 200 butterflies were cut into small pieces and extracted with about 200 ml 80% ethanol. Two vol. of chloroform were added to the extract, 38
1210
Y. UMEBACHI AND K. YOSHIDA
and
after a while the water layer was taken. After one more repetition of the same treatment, the combined water layer was applied to the DEAE cellulose column (2 x 15 cm). The yellow pigments were not adsorbed and were washed down with water. Some brownish substances were adsorbed at the top of the column. The water effluents (yellow, 50-70 ml) were applied to the Dowex-50 X4 column (0.9 x 10 cm), on which the yellow pigments were adsorbed. The column was washed successively with 250 ml each of 0.1 M pyridine-formic acid buffer (pH 3-l), O-2 M pyridine-formic acid buffer (pH 3*1), O-2 M pyridineacetic acid buffer (pH 3-l), and 0.4 M pyridine-acetic acid buffer (pH 4.7) at a rate of 5 to 6 drops/min. Finally, the yellow pigments were eluted with 3-O M pyridine-acetic acid buffer (pH 5.5) at a rate of 2 to 3 drops/min. Only the Papiliochrome II group was eluted, while the Papiliochrome III group remained at the top of the column. The combined eluate (about 30 ml) of the Papiliochrome II group was concentrated to 2 ml by repeatedly shaking it with ethylacetate. Then, 1 to 2 ml of BAW solvent was added. The mixture was concentrated to a syrup (about 0.5 ml) under reduced pressure. Just enough of the cellulose powder, prepared as described above, was added to take up the yellow syrup and to give a dried yellow powder. The yellow cellulose powder was put on the top of the cellulose column (0.9 x 80 cm) and developed with BAW solvent at a rate of 2 drops/3 min. Papiliochrome IIa and IIb were separated. The fractions of IIa and IIb were collected separately, and about 5 ml of cold ethyl ether was added to each fraction (1.0 to 1.5 ml). The water layer was taken, and an equal volume of cold 99% ethanol was added. On top of it, ethyl ether was added dropwise until the solution became turbid. After keeping the mixture at 0°C for 1 hr, the yellow powder produced was gathered by centrifugation, washed with ethyl ether, and dried under reduced pressure. The powders of Papiliochrome IIa and IIb were respectively tested for their purity by paper chromatography and confirmed to give a single spot respectively, though in some cases a trace of kynurenine was produced by spotting the sample on the filter paper.
Ultra-violet absorption spectra of Papiliochrome IIa and IIb Purified Papiliochrome IIa and IIb were dissolved in the phosphate buffer, and their U.V. spectra were determined. Both pigments gave the same absorption peaks of 262 to 263, 280 to 284 (shoulder), and 380 to 381 nm as shown in Fig. 2. For the purpose of comparison, the U.V. spectra of authentic kynurenine are shown in Fig. 3, for, as will be shown later, Papiliochrome IIa and IIb are decomposed to some o-diphenol derivative and kynurenine by heating. The kynurenine obtained from Papiliochrome II showed the same spectral behaviour as that of authentic kynurenine (curve c of Fig. 7). It is known that the absorption peak at 360 nm of kynurenine in the phosphate buffer is due to the presence of the aromatic carbonyl group which is under the influence of the amino group in the o-position. This is supported by the shift of the peak to 369 to 371 nm in 99% methanol (Fig. 3). In order to examine the property of the absorption peak at 380 nm of
YELLOW PIGMENTS IN WINGS OF PAPILIO
1211
XUTHCJS
1-o
0.5
I
1
LENGTH
WAVE
Ultra-violet
300 WAVE
Ultra-violet
I
400
(mpl)
spectra of Papiliochrome IIa and IIb in phosphate buffer (pH 7-O). (a) IIa; (b) IIb.
250
FIG. 3.
350
300
250
FIG. 2.
I
I
350 LENGTH
400
(mr*)
spectra of authentic kynurenine. (a) In phosphate buffer (pH 7.0); (b) in 99% methanol.
1212
Y. UMEBACHIAND K. YOSHIDA
Papiliochrome IIa and IIb, their U.V. spectra were also determined in 99% methanol. As shown in Fig. 4, the peak at 380 to 381 nm shifted to 382 to 383 nm, and the peak at 262 to 263 nm to 260 to 261 nm. As Papiliochrome IIa and IIb seem to be a combination of o-diphenol derivative and kynurenine as will be shown later, these experimental results show that the peak at 380 to 381 nm is due to the 0.9
w 0.6 0 z a m E v) m a 0.3
300
250 WAVE FIG. 4.
Ultra-violet
350 LENGTH
400
(m&I
spectra of Papiliochrome IIa and IIb in 99% methanol. (a) IIa; (b) IIb.
aromatic carbonyl group of kynurenine contained as a component. It is conceivable that the peak at 360 nm of kynurenine shifts to 380 to 381 nm by a combination with o-diphenol derivative and gives a yellow colour to the compound. Infra-red absorption spectra of Papiliochrome IIa and Ilb
These curves are shown in Fig. 5. Papiliochrome IIa and IIb gave the same spectrum. As will be shown later, these yellow pigments contain both catecholamine derivative and kynurenine as their components. But it was difficult to assign exactly each absorption peak to any specific chemical group contained in the pigment, though the assignment of several peaks could be suggested. The broad absorption between 3000 and 3600 cm-l suggests the presence of an amino group, phenyl group, or hydroxyl group, or all of them. There is also the possibility that the broad absorption in this range is caused by water which is present in the sample, and judging from the fact that the yellow pigments contain kynurenine as a component, the absorption at 1638 cm-l seems to correspond to the aromatic carbonyl group which have an amino group in the o-position and/or to an amido or amino group. Moreover, the absorption at 757 cm-l suggests the presence of an
YELLOW PIGMENTS IN WINGS OF PAPILIO
1213
XUTHUS
o-substituted phenyl compound, and the absorption at 873 cm-l and 820 cm-’ may correspond to a 1,2,4+ubstituted phenyl compound. There is also the possibility that the absorption at 873 cm-l shows the presence of a 1,2,4,5substituted phenyl compound. It is clear that there is no ester bond. WAVE 100%
3 1
5 I
LENGTH
(,kL)
6 I
10 I
8 I
16 I-
50-
z z a I-
o-
_c loo-
I
I
I
I
I
I
I
I
t
l-
B cn z a E
50 -
O-
I
I
3200
2000 FREQUENCY
I
I
1600
1200
I 600
(cm-l)
FIG. 5. Infra-red spectra of Papiliochrome IIa and IIb.
ORD and CD spectra of Papiliochrome Ila and IIb
Aside from being able to separate Papiliochrome IIa and IIb by paper or cellulose column chromatography, they can also be distinguished by their fluorescence on the chromatogram, but both pigments are the same in their colour tests, U.V. spectra, ix, spectra, and decomposition products. Therefore, both yellow pigments were presumed to be optically isomeric, and their ORD and CD spectra were determined as shown in Fig. 6(A). Papiliochrome IIa showed a negative Cotten effect curve and a negative CD band at around 382 nm. On the other hand, Papiliochrome IIb gave the opposite curves. This indicates that Papiliochrome IIa and IIb are optically isomeric. As mentioned above, the U.V. absorption peak at 382 nm is due to the presence of an aromatic carbonyl group. This suggests that there is an asymmetric centre near the aromatic carbonyl group. As Papiliochrome IIa and IIb contain kynurenine as one of their components, the ORD and CD curves of kynurenine were determined for the purpose of comparison. As will be shown later, the kynurenine obtained from the yellow
1214
Y.
UMEBACHI
ANDK. YOSHIDA
pigments was L-isomer in both Papiliochrome IIa and IIb. D-Kynurenine was not found in P. xuthus. The ORD and CD spectra of authentic L-kynurenine are given in Fig. 6(B), which shows that the DL-isomerism of kynurenine hardly affects the ORD and CD curves at around 369 to 371 nm. That seems to be the reason why Papiliochrome IIa and IIb can show a mirror symmetry at least as far 3.0 -
B - 0.8
m 4
L
1
290
I
I
I
370
290
450 WAVE
LENGTH
370
450
(miu)
FIG. 6. ORD and CD spectra of Papiliochrome IIa and IIb and L-kynurenine. (A) (a), (b), and (c)-u.v., ORD, and CD spectra of Papiliochrome IIa; (d), (e), and (f ju.v., ORD, and CD spectra of Papiliochrome IIb. (B) (a), (b), and (c)u.v., ORD, and CD spectra of L-kynurenine. Solvent: 99% methanol in all the cases.
as the ORD and CD at around 382 nm are concerned. The u.v., ORD, and CD spectra of the yellow pigments and kynurenine indicate that a combination of kynurenine with o-diphenol derivative gives rise to a new asymmetric field near the aromatic carbonyl group of kynurenine and, moreover, that both components do not join together at the amino or carboxyl group of the side-chain of kynurenine. Decomposition of Pap&chrome IIa and Ilb in mild acid condition For this purpose, Papiliochrome IIa and IIb which were either eluted from the two-dimensional chromatogram or purified by column chromatographies were used. The yellow pigments from both of the separating methods gave the same results. Papiliochrome IIa and IIb were dissolved in 1O-3 N HCl and heated in a boiling water-bath for 30 to 60 min, and then the U.V. spectra of the solution were
YELLOWPIGMENTSIN WINGSOF PAPILIO
1215
XUTHUS
determined (curve (b) of Fig. 7). The absorption peak at 380 to 381 nm disappeared and shifted to 360 to 362 nm. An absorption peak at 279 to 281 nm appeared instead of the shoulder at 280 to 284 nm. The peak 262 to 263 nm shifted to 257 nm. Next, the same solution was one-dimensionally chromatographed with BAW
I
250
350
300 WAVE
LENGTH
400
(rnp)
FIG. 7. Ultra-violet spectra of the decomposition products of Papiliochrome IIa. (a) Papiliochrome IIa before decomposition; (b) after decomposition by heating in lo-* N HCl; (c) the kynurenine from Papiliochrome Ha; (d) the SN-1 substance. Solvent: lo-* N HCI. Papiliochrome IIb gave the same results.
solvent. After development, the filter paper was examined for fluorescence and then subjected to colour tests. Interestingly enough, Papiliochrome IIa and IIb gave the same chromatogram which is shown in Fig. 8(A). The results of the colour tests are given in Table 2. Judging from the colour reactions, fluorescence, Rtvalues, and the results of the previous papers (U-ACHI and TAKAHASHI, 1956; UMEBACHI, 1958, 1961; UMEBACHI and YAMADA, 1964), there is no doubt that the spot K is L-kynurenine. On the other hand, SN-1 is an unidentified substance, though the results of the colour tests indicate that the substance is o-diphenol which possibly contains amino nitrogen. Although the spot SN-1 is positive to the silver nitrate and TTC tests, it is not a sugar, because it is negative to the aniline hydrogen phthalate, benzidine, and resorcinol tests. The positive reactions to silver nitrate, TTC, phosphomolybdic acid, and diazotizedp-nitroaniline show that it belongs to the phenolic compounds, and, yet, as it is positive to the Evans, sodium molybdate, and ethylenediamine tests, and as it gives a yellow colour to Millon’s test and a faint orange colour to the ol-nitroso+-naphthol test, it is probable that SN-1 belongs to o-diphenol. Moreover, as it is negative or gives a
1216
Y. UMEBACHI AND K. YOSHIDA
light bluish colour to the ninhydrin test, the amino nitrogen contained seems to be neither the first amine nor cu-amino acid. The negativity to both Ehrhch’s and Tsuda’s tests shows that SN-1 does not have an aromatic amino group.
B
-S N-l’ -SN-2 -N-l -N-2
FIG. 8. (A) One-dimensional chromatogram of the decomposition products produced by heating Papiliochrome IIa in 10e9 N HCI. Solvent: BAW. (B) Onedimensional chromatogram of the degradation products produced by heating SN-1 in 1 N HCl. Solvent: BAW. In both (A) and (B), Papiliochrome IIb gave the same results as those of IIa.
The U.V. spectrum of SN-1 also suggests that it belongs to o-diphenol. The area of SN-1 on the chromatogram was cut out and eluted with 10-S N HCl, and the U.V. spectrum of the eluate was determined. The spectrum obtained is given in curve (d) of Fig. 7. Papiliochrome IIa and IIb gave the same result. The absorption peak was 279 to 281 nm. Fig. 7 shows that the solution of Papiliochrome IIa or IIb after heating in 10-s N HCl is a mixture of kynurenine and SN-1 substance. The U.V. spectrum of SN-1 is similar to those of DOPA, DOPAmine, and N-acetyl-DOPAmine as shown in Fig. 9. The absence of an R-band (n-n*) clearly shows that SN-1 does not contain an aromatic carbonyl group. For the purpose of comparison, the spectrum of protocatechuic acid is also shown in Fig. 9. On the other hand, it is evident from the paper chromatographic behaviour and colour tests that SN-1 is not DOPA, DOPAmine, or N-acetyl-DOPAmine. Moreover, as will be shown later, the injection experiments of 14C-labelled compounds indicate that SN-1 is a DOPAmine derivative. Although the result of Elson-Morgan’s test suggests the possibility that SN-1 is an N-acetyl compound, it remains to be proved. As mentioned above, there is no doubt that SN-1 is not N-acetyl-DOPAmine. The green colour reaction to 2,6-dichloroquinonechlorimide
YELLOWPIGMBNTSIN WINGS
OF PAPILIO
XUTHUS
1217
the possibility that SN-1 is a &substituted derivative of DOPAmine. But that also remains to be proved. At any rate, it is evident that SN-1 is a catecholamine derivative which has neither an aromatic carbonyl nor an aromatic amino group. Although the spot SN-2 of Fig. S(A) is also positive to the silver nitrate test, it is a substance which is derived from filter paper, because it appears only when
suggests
TABLB 2-Co~ot~~
TESTSOF THE SPOTSIN FIG. @A) spot K
Test
NIN*
1 2 3 4 5 6 7 8 9 10 11 12 13 14
DAB TSU AHP BEZ RES TTC SIN MIL FEC PMA DNT NNP EVA
15 16 17 18 19 20
SMO PFC EDA ELM DCQ BHC
Fluorescence Colour in visible rays Identity
SN-1
Neg Neg Neg 0 Neg Dark Bu
Sky Bu or Neg Neg Neg Neg Neg Neg Pi Br Y Gn or YGn Bu BrP f0 Y after first reagent, final Pi Y R: Pi (fluorescence) 0 fRoR 11 Slightly Gr PBuT
WBu
None
Colourless Kynurenine
Colourless
W
0 P Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
* Explanations of the abbreviations for colour tests are in the paragraph concerning Materials and Methods, pp. 1204, 1205. t Explanations of the abbreviations for colours are in the footnote of Table 1. $ Immediately after spraying the reagent, the spot becomes rose-red in visible rays. After several hours, the spot turns reddish purple in visible rays and gives a slightly purple fluorescence. 0 After several hours, the spot becomes orangeish yellow in visible rays and gives a pink fluorescence. After 1 to 3 days, the spot turns yellow in visible rays and gives an orangeish yellow fluorescence. (1After 1 to 2 days, the spot shows a faint rose-red colour. 7 The sensitivity is low.
1218
Y. UMEBACHIANDK. YOSHIDA
the eluate of Papiliochrome IIa and IIb from the paper chromatogram is heated and chromatographed again, and because it appears also in the chromatogram of the eluate of the blank filter paper which is treated in the same way. Spots N-l and N-2 of Fig. 8(A) are unidentified substances which are positive to the ninhydrin test and are only present in very small quantities. In some cases, these two spots were absent. They are probably secondary degradation products.
0.9
; 0.6 u m z fn m u 0.3
250 WAVE
LENGTH
(rnM,L)
FIG. 9. Ultra-violet spectra of authentic o&phenols. (a) Catechol; (b) DOPA; (c) DOPAmine; (d) IV-acetyl-DOPAmine; (e) protocatechuic acid.
The heating of Papiliochrome IIa and IIb in alkaline solution destroyed kynurenine. On the other hand, the heating of them in a strong acid solution broke down the SN-1 substance. Therefore, for the purpose of examining the primary decomposition products of Papiliochrome II, heating in a mild acid solution, for example 1O-3 N HCl, was most suitable.
Degradation of the SN-1 substance In order to examine further properties of SN-1, it was broken down by heating in a stronger acid solution. The area of SN-1 on the chromatogram was cut out and eluted in 1, 3, or 6 N HCl. The eluate was heated in a boiling water-bath for 30 min. After the solution was evaporated to dryness under reduced pressure, the residue was dissolved in a small quantity of water and chromatographed with BAW solvent again. Heating in 1, 3, and 6 N HCI gave the same result, which is shown in Fig. 8(B). SN-1 gave a spot which showed a little lower Rt value in BAW solvent and was different in colour tests. The spot was referred to as SN-1’. As seen in Table 3, SN-1’ shows a faint purple colour in the ninhydrin test. This
YELLOWPIGMENTSIN WINGSOF PAPILIO
XUTHUS
1219
fact suggests that the original substance SN-1 has an amino group in which the hydrogen is replaced by some unknown group. It is conceivable that this unknown group of the original substance SN-1 is removed by heating in 1 N HCl. The area TABLE 3-COLOUR TESTSOF SN-1’ IN FIG. S(B) 1 2 3 4 5 6 7 8
NIN* SIN PMA EVA SMO PFC EDA BHC
fW
Br Dark Bu Y after first reagent, final Pi BrY Pi: Y (fluorescence) f Dark Bu
* Explanations of the abbreviations for colour tests are in the paragraph concerning Materials and Methods, pp. 1204, 1205. t Explanations of the abbreviations for colours are in the footnote of Table 1. 3 Immediately after spraying the reagent, the spot becomes pink in visible rays. After several hours, the spot turns reddish purple in visible rays and gives a whitish blue fluorescence. $ After several hours, the spot becomes pink or orange in visible rays and gives a pink or whitish orange fluorescence.
of SN-1’ was cut out, and the U.V. spectra of the eluate was determined. As shown in Fig. 10, the peak was 280 run as for SN-1. It is probable from the colour tests and absorption spectrum that SN-1’ is a catecholamine derivative. The spot SN-2 of Fig. 8(B) is a reducing substance which is derived from filter paper as was already described in Fig. 8(A). Spots N-l and N-2 are ninhydrinpositive substances which are present in small quantities. They are also similar to those in Fig. 8(A), though this remains unsettled. The heating of the SN-1 solution in 1 N HCl at 100°C for 6 hr gave the same result as when heated for 30 min. But, in the heating of the SN-1 solution in 6 N HCl at 100°C for 6 hr, SN-1’ broke down further and gave several unidentified substances. Assuming that the conversion of SN-1 into SN-1’ might have resulted from heating in the air, the heating was carried out in nitrogen gas. The eluate of SN-1 was put into a glass ampoule and vacuumized, and nitrogen gas was introduced. After this procedure was repeated several times, the glass ampoule was sealed and heated. The heating of the SN-1 solution in 1 N HCl at 100°C for 30 or 60 min gave the same result as in Fig. 8(B). But the heating of the SN-1 solution in 6 N HCl at 100°C for 6 hr gave two substances, though the chemical properties of
1220
Y. UMEBACHIAND K. YOSHIDA
these two spots were not investigated in detail. At any rate, it was evident that further.degradation or changes of SN-1’ occurred in this procedure.
WAVE
LENGTH
(mr*.)
FIG. 10. Ultra-violet spectrum of SN-1’ substance.
Injection experiments of 1%~labelled tryptophan, DOPA, and DOPAmine The r4C of tryptophan (alanine-2-14C), DOPA-2-r4C, and DOPAmine-1-W was incorporated into the yellow pigments. The autoradiographs of these wings are shown in Figs. 11(A), (B), and (C). The crude extract from the yellow scales which incorporated the 1% of tryptophan (alanine-2J4C) was two-dimensionally chromatographed. The autoradiograph of the chromatogram obtained is shown in Fig. 12(A). The radioactivity is seen in spots T, K, Y-IIa, Y-IIb, Y-IIIa, and Y-IIIb, which are respectively tryptophan, kynurenine, Papiliochrome IIa, IIb, IIIa, and IIIb. Y-V is also radioactive. The areas of Papiliochrome IIa and IIb were cut out and separately eluted with lo4 N HCl. The eluatewas heated in a boiling water-bath for 30 min, concentrated under reduced pressure, and then one-dimensionally chromatographed. The autoradiograph of the chromatogram is shown in Fig. 13(A). Spot K is kynurenine. As had been expected, the 14C was incorporated into kynurenine and not into SN-1. The crude extracts from the yellow scales which incorporated the 14C of DOPA-2-W and DOPAmine-lJ4C were also treated in the same way as mentioned above. The results obtained from both compounds were the same concerning the incorporation of 14Cinto the yellow pigments, Fig. 12(B) shows the autoradiograph of the two-dimensional chromatogram in the case of DOPAmine-1-14C. Although the radioactivitywas weaker than in the case of tryptophan (alanine-2J4C), there was no doubt that the r%! was incorporated
1221
FIG. 11. Autoradiographs of the wings of the male adults, in the pharate pupal stage of which (A) tryptophan (alanine-2J4C), (B) DOPA-2J4C, and (C) DOPAmine-l -14C was injected respectively.
FIG. 12. Autoradiographs of the two-dimensional chromatograms of the crude extracts from the yellow scales which incorporated the 14C of (A) tryptophan (alanine-2-14C) and (B) DOPAmine-lJ4C.
1223
S N-l
Frc. 13. Autoradiographs of the one-dimensional chromatograms of the decomposition products of Papiliochrome IIa which incorporated the l*C of (A) tryptophan (alanine-2-14C) and (B) DOPAmine-lJ4C. Solvent: BAW. In the case of DOPA-2-14C, the result was the same as in DOPAmine-l-i4C. In both (A) and (B), Papiliochrome IIb gave the same results as those of IIa.
FIG. 14.
Autoradiographs
of the wings
of the male adult, (B) DOPA-l-14C
in the pharate pupal stage of which was injected respectively.
(A) tryptophan
(ring-2-l%)
and
YELLOW PIGMENTS IN WINGS OF PAPILIO
XUTHUS
1225
into Papiliochrome IIa, IIb, IIIa, and IIIb. The area including the startingpoint and Y-V was also radioactive. The autoradiographs of the one-dimensional chromatograms obtained after heating the eluate of Papiliochrome IIa and IIb are shown in Fig. 13(B). It is evident that the 14C is incorporated into SN-1 but not into kynurenine. The 14C of DOPA-1J4C and tryptophan (ring-2J4C) was not incorporated into the yellow pigments (Figs. 14A and B). But, in the case of the latter labelled compounds, there were some cases in which the yellow markingsof the wings were slightly radioactive. The reasonwhy the injection experimentof tryptophan (ring-214C)was carried out will be mentioned later. DISCUSSION
In the previous paper (UMEBACHI, 1961), it was reported that there were two kinds of yellow pigments in the wings of P. mthus, that is Y-II and Y-III. After that, it was found that Y-II and Y-III contain two separatepigments respectively, and in the present paper they were referred to as Papiliochrome IIa, IIb, IIIa, and IIIb. Among them, only IIa and IIb have been investigatedfor detailed physical and chemical properties, because Papiliochrome IIa and IIb are the main yellow pigments. Papiliochrome IIIa and IIIb are too small in quantity to be purified. Since UMEBACHI and NJXAMURA (1954) found that a largequantityof kynurenine was present in the extract of the wings of P. mthus, the following facts have been proved: (1) In the pupa, as the result of the increase of tryptophan pyrrolase activity during the pharate adult stage, the content of kynurenine increases. The kynurenine thus formed accumulates in the yellow scales as a bound form. The bound form of kynurenine means a compound which produces kynurenine by heating (UMEBACHI and KATAYAMA, 1966). (2) The yellow pigments of the yellow scales readily decompose to give a phenolic compound and kynurenine (UMEBACHI, 1961). In the present paper, Papiliochrome IIa and IIb were purified, and their physical and chemical properties were investigated. It was confirmed that Papiliochrome IIa and IIb can be readily decomposed to kynurenine and o-diphenol (SN-1) by heat’mg in a mild acid condition. The o-diphenol SN-1 is negative to the ninhydrin test or gives a faint light bluish colour, and is converted by heating in 1 N HCl into a substance (SN-1’) which gives a faint purple colour for the same test. From the colour tests and the injection experiments of i4C-labelled compounds, SN-1 seems to be an N-substituted derivative of DOPAmine. The Elson-Morgan test suggests the possibility that SN-1 is an N-ace@ derivative, though it does not correspond to N-acetyl DOPAmine. Moreover, judging from the negative result to 2,6-dichloroquinonechlorimide, the possibility exists that the hydrogen of the 6-position of SN-1 is also replaced by some group. In the previous paper (UMEBACHI and KATAYAMA, 1966), the possibility was suggested that the yellow pigments might be formed by a combination of odiphenol derivative and formylkynurenine, though the pigments are decomposed to o-diphenol derivative and kynurenine by heating. But, as the 1% of tryptophan
1226
Y. UMEESACHI AND K. YOSHIDA
(ring-2J4C) was not incorporated into the yellow pigments, this possibility was ruled out. The possibility is also excluded that after the decomposition of the yellow pigments to o-diphenol derivative and kynurenine, the formyl group of formylkynurenine keeps attaching to the o-diphenol derivative because it is clear from the U.V. spectrum that SN-1 does not have an aromatic carbonyl group. After all, Papiliochrome IIa and IIb seem to be formed by a combination of DOPAmine derivative and kynurenine (not formylkynurenine). The possibility exists that Papiliochrome IIa and IIb are formed by a combination of N-substituted DOPAmine and kynurenine and readily decompose to give kynurenine and a 6and N-substituted DOPAmine. Interestingly enough, Papiliochrome IIa and IIb gave the same decompositionproducts, U.V. spectra, and i.r. spectra. Before the present paper, Papiliochrome IIa and IIb could be distinguished only by their chromatographic behaviour and fluorescence. But, in the present paper, it was found that Papiliochrome IIa and IIb are different also in their ORD and CD curves, and, yet, both yellow pigments give the opposite Cotten effect. These results show that both yellow pigments are optically isomeric. It is almost certain that the absorption peak (360 nm) caused by the aromatic carbonyl group of kynurenine is shifted to 380 to 381 nm by the formation of the pigment, that is, by a combination with a DOPAmine derivative. In other words, the shift of 360 nm to 380 nm is the reason why the compounds turn yellow. It is very interesting that Papiliochrome IIa and IIb give just the opposite Cotten effect at around this peak (382 run in methanol). It must be kept in mind that the optical activity which is caused by the rn.-isomerism of the side-chain of kynurenine hardly affects the optical activity at around 370 nm (in methanol). The kynurenine contained in the pigments is L-kynurenine in both Papiliochrome IIa and IIb. From these spectral studies, the greatest possibility for the combination of DOPAmine derivative and kynurenine is that the former compound combines with the latter by way of the aromatic amino nitrogen of the latter, though further investigations are necessary to confirm it. For the tyrosine metabolism in insects, the following three main pathways have been known or suggested: The first is the pathway of melanin formation. It is generally thought to be similar to that in vertebrates, that is, melanin is formed by way of DOPA, DOPAquinone, and DOPAchrome. But detailed biochemical studies have not yet been made in insects. The second is the way of DOPAmine, noradrenaline, and adrenaline formation by way of DOPA. It is interesting that DOPAmine and noradrenaline have been reported to be present in large quantities in insects (BRUNET, 1963). N-Acetyl-DOPAmine is also formed in this way. The third is the way of phenolic acid formation, such as p-hydroxyphenylpyruvic acid and p-hydroxyphenyllactic acid. In the present paper, when DOPA-1J4C or DOPA-2-W was injected into the prepupa of P. xuthus, the 14C was not incorporated into the black scales. The reason remains unsolved, but one possibility is that when the injection is made at the pharate pupal stage, the metabolic transition of the injected compound and the development of black scales (or the stage of
YELLOWPIGMENTSIN WINGSOF PAPILIO
XUTHVS
1227
melanin formation) do not match well with each other. Studies on the metabolism of DOPA and DOPAmine are being made in our laboratory. In P. xuthus, the yellow and black scales are adjacent to each other in the border of the markings. In some papilionid butterflies, for example Ldhdorja japomka, in addition to the yellow and black scales, there are red scales which also incorporate the 14C of tryptophan (alanine-% W) (unpublished data). The black pigment is melanin which is probably derived from tyrosine. As for the red pigments, it is possible that they belong to the ommochrome group which is formed by way of On the other hand, as mentioned above, the yellow pig3-hydroxykynurenine. ments are not ommochrome but a combination of o-diphenol derivative and kynurenine. Thus, the papilionid butterflies present an interesting problem in the relationship between tryptophan and tyrosine metabolism. From the biological point of view, it presents a problem of the differentiation, regulation, and diversity of insect pigment metabolism, Acknowledgements-We wish to express our thanks to Miss Y. Ishizaki and Mr. T. Tanaka for their technical assistance. REFERENCES ACHER R. and CROCKERC. (1952) Reactions colorees spdcifiques de l’arginine et de la tyrosine real&es apres chromatographie sur papier. Biochim. biophys. Acta 9, 704-705. BRUNETP. C. J. (1963) Tyrosine metabolism in insects. Ann. N.Y. Acad. Sci. 100, 10201034. BUTENANDT A. and SCHXFERW. (1962) Ommochromes. In Recent Progress in the Chemistry of Natural and Synthetic Colouring Matters and Related Fields (Ed. by GORYT. S. et aZ.), pp. 13-33. Academic Press, New York. COULSONC. B. and EVANSW. C. (1958) Paper chromatography and paper electrophoresis of phenols and glycosides. J. Chronuatog. 1, 374-379. CUR F. (1954) Paper Chromatography. Macmillan, London. DALGLIE~HC. E. (1952) The relation between pyridoxin and tryptophan metabolism, studied in the rat. Bi0chem.J. 52, 3-14. FURNSAUXP. J. S. and MCFARLANEJ. E. (1965) Identification, estimation, and localization of catecholamines in eggs of the house cricket, Acheta domesticus (L.). J. Insect Physiol. 11, 591-600. GIBBS H. D. (1927) Phenol tests-III. The indophenol test. g. biol. Chem. 72, 649-664. Hoxnocxs R. H. (1949) Paper partition chromatography of reducing sugars with be&dine as a spraying reagent. Nature, Lond. 164,444. LEDBRERE. and LEDERERM. (1957) Chromatography, A Review of Principles and Applications. Elsevier, Amsterdam. MAZURR. H., ELLIS B. W., and CAMMARATA P. S. (1962) A new reagent for detection of peptides, nucleotides, and other N-H containing compounds on paper chromatograms. J. biol. Chenz. 237, 1619-1621. PARTRIDGES. M. (1948) Filter-paper partition chromatography of sugars-I. General description and application to the qualitative analysis of sugars in apple juice, egg white and foetal blood of sheep. Biochem. J. 42, 238-248. PITTARDA. J., GIBSON F., and DOY C. H. (1961) Phenolic compounds accumulated by washed cell suspensions of a tryptophan auxotroph of Aerobacter aerogenes. Biochim. biophys. Acta 49, 485-494.
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PRIDHAMJ. B. (1959) Paper electrophoresis and paper chromatography of phenolic compounds. g. ChromatogY 2,605-611. RILEYR. F. (1950) Paper partition chromatography of some simple phenols. J. Am. them. Sac. 72. 5782-5783. SCHtiPF C: (1964) Die Anfringe der Pterin-Chemie. In Pteridine Chemistry (Ed. by PFLEIDHRBR W. and TAYLOR E. C.), pp. 3-14. Pergamon Press, Oxford. SBNOHS., CRBVBLWGC. R., UDENFRIEND S., and WITKOPB. (1959) Chemical, enzymatic and metabolic studies on the mechanism of oxidation of DOPAmine. J. Am. them. Sot. 81,6236-6240. SENOH S. and WITKOP B. (1959) Non-enzymatic conversions of DOPAmine to norepinephrine and trihydroxyphenethylamines. J. Am. them. Sot. 81, 6222-6231. Sounxxs T. L., DENTONR. L., MURPHY G. F., CHAVJXZ B., and SAINT CYR S. (1963) The excretion of dihydroxyphenylalanine, DOPAmine, and dihydroxyphenylacetic acid in neuroblastoma. Pediatrics 31, 660-668. SWAIN T. (1953) The identification of coumarins and related compounds by filter-paper chromatography. Biochem. J. 53, 20&208. )T(R&VBLYAN W. E., PROCTERD. P., and HARRISONJ. S. (1950) Detection of sugars on paper chromatograms. Nature, Lond. 166, 444445. UM~BACHIY. (1958) Yellow pigments in the wings of the papilionid butterflies-I. The relation between kynurenine and the yellow pigments of Papilio xuthus. Sci. Rep. Kanazawa Univ. 6, 45-55. UME~ACHIY. (1959a) Yellow pigments in the wings of the papilionid butterflies-II. The presence of a pale blue fluorescent substance supposed to be kynurenine in the wings of the Zerynthiinae. Sci. Rep. Kanazawa Univ. 6, 69-75. U~VIEBACHI Y. (1959b) Yellow pigments in the wings of the papilionid butterflies-III. The radioautographs of the wings of five species of Papilio injected with 14C-labeled tryptophan. Antwt. Zool.~apan 32,112-116. UMEBACHIY. (1960) Yellow pigments in the wings of the papilionid butterflies-IV. The presence or absence of kynurenine in the wings of the genus Graph&m. Sci. Rep. Kanazawa Univ. 7,107-112. U~ACHI Y. (1961) Yellow pigments in the wings of the papilionid butterflies-V. Some chemical properties of the yellow pigments of Papilio xuthus. Sci. Rep. Kunaxawa Univ. 7,139-150. UM~BACHIY. (1962) Yellow pigments in the wings of the papilionid butterflies-VII. Consideration of the nature and distribution of the wing pigments of the papilionid butterflies. Sn’. Rep. Kunazawa Univ. 8, 135-142. UMEBACHIY. and KATAYAMA M. (1966) Tryptophan and tyrosine metabolism in the pupa of papilionid butterflies-II. The general pattern of tryptophan metabolism during the pupal stage of Papilio xuthus. J. Insect Physiol. 12, 1539-l 547. A. (1954) The presence of kynurenine in the wings of the UM~BACHIY. and NAKAMURA papilionid butterflies. Zool. Mug. Tokyo 63, 57-61. (In Japanese, English summary.) UMEBACHIY. and TAKAHASHI H. (1956) Kynurenine in the wings of the papilionid butterflies. 3. Biochem. Tokyo 43,73-81. UMEEIACHI Y. and TSUCHITANIK. (1955) The presence of xanthurenic acid in the fruit-fly, Drosophila melanogaster. J. Biochem. Tokyo 42, 817-824. UMEEIACHI Y. and YAMADAM. (1964) Tryptophan and tyrosine metabolism in the pupa of papilionid butter!Iies-I. Accumulation of the bound form of kynurenine in Pupilio xuthus. Annot. Zool. Japon. 37, 51-57.