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Further studies on the dopamine derivative, SN-1 derived from the yellow pigments of Papilio xuthus
Insect Biochem., 1975, Vol. 5, pp. 73 to 92. Pergamon Press. Printed in Great Britain
F U R T H E R S T U D I E S ON T H E DOPAMINE DERIVATIVE, SN-1 DERIVED FROM T H E YELLOW P I G M E N T S OF PAPILIO X U T H U S Y. UMEBACHI Department of Biology, Faculty of Science, Kanazawa University, Kanazawa 920, Japan
(Received 24 June 1974) A b s t r a c t - - F o r the purpose of purifying the dopamine derivative SN-1 which
had been reported as a decomposition-product of the yellow pigments of Papilio xuthus, the isolation of the substance with column chromatography was tried. The crude extract from wings or yellow scales was heated in 10-' N HCI and chromatographed on the Amberlite CG-50 column. On this column, kynurenine and an unknown o-diphenolic substance were clearly separated. The latter coincided with the SN-1 and was re-confirmed to be a dopamine derivative using 14C-dopamine. On the basis of these observations, the purification of SN-1 was performed through the DEAE-Cellulose, Amberlite CG-50, and Biogel P-2 columns. The purified SN-1 was hydrolyzed in 1 N HC1, and the hydrolysates were again ehromatographed on the Biogel P-2 column, in which two main products were found. One has been identified as fl-alanine. The other was a dopamine derivative closely related to arterenol. It has been presumed that SN-1 is a dopamine derivative containing fl-alanine in the side chain. The contents of SN-1 and kynurenine in wings were estimated during the pharate adult stage. It was shown that both substances increase in parallel with each other during the yellow pigment formation. In addition to the above fl-alanine, this amino acid was found also in the scales remaining after the yellow pigments and other water-soluble substances were extracted. INTRODUCTION AmONQ wing pigments of butterflies, pterins of the Pieridae and ommatins of the Nymphalidae have been investigated in detail from the standpoints of organic chemistry and biochemistry. For other families, however, no such detailed investigation has been made. UMEBACm and NAKAMURA (1954) and UMrBACHI and TAKAHASHr (1956) found a large amount of kynurenine in the hot water extract of the yellow scales of Papilio xuthis (Papilionidae). Since then, physical and chemical properties of the yellow pigments of this species have been studied in detail (UMEBACm, 1958, 1959, 1961, 1962; UMEBACHI and YOSHIDA, 1970). Although the yellow pigments are derived from tryptophan, they do not belong to ommochrome. T h e y have been presumed to be the pigments which are formed from both tryptophan and tyrosine metabolites. W h e n the water extract of the yellow scales is two-dimensionally chromatographed, several yellow pigments are found. Among them, the main yellow 73
74
Y. UMEBACHI
pigments were named Papiliochrome 2a and 2b, and their chemical and spectral properties were reported by UMEBACHI and YOSHIDA (1970). Papiliochrome 2a and 2b are so unstable to heating that when their solution in 10 -a N HC1 is heated at 100°C for 30 min, the pigments readily decompose to L-kynurenine and a o-diphenol derivative. The latter substance was termed SN-1 and was proved to be a dopamine derivative (UM~BACHIand YOSHIDA, 1970). The present paper deals mainly with the purification of SN-1 and the identification of the hydrolysates. As SN-1 had been confirmed to be a decompositionproduct of the yellow pigments (UMEBACHIand YOSHIDA, 1970), the crude extract of the yellow pigments from wings or yellow scales was used as the starting material for SN-1 in the present paper. From such extract, SN-1 was purified through column chromatographies and hydrolyzed in 1 N HC1. Chromatographic examinations of the hydrolysates showed the presence of two main products. One has been identified as fl-alanine, and the other has been presumed to be a dopamine derivative which is very similar to arterenol. The SN-1 and kynurenine contents in wings during the pharate adult stage were also studied. Moreover, the presence of fl-alanine in the scales remaining after the pigment extraction was shown.
MATERIALS AND METHODS Materials As the materials for the extraction of the yellow pigments, the wings or yellow scales of the male adults of P. xuthus were used. Some of the butterflies were obtained through the Okura Biological Institute, and others were raised in our laboratory from the larval stage. For the injection-experiments of 14C-labelled compounds, the pharate pupae were used, and after the injection, were raised to adults at 25 :~ I°C.
Extraction of yellow pigments The wings or yellow scales were homogenized in 70% ethanol. After repeating centrifugation and extraction with 70% ethanol, the combined supernatant solution was evaporated to dryness under reduced pressure. The residue was washed with ethyl ether thrice and then dissolved in water. After centrifugation, the supernatant fluid is referred to below as a crude extract of the yellow pigments. In some cases, the yellow pigments were extracted by shaking the yellow scales in water and, without any pretreatment, were subjected to paper and thin-layer chromatography. All these crude extracts, of course, contain a large amount of Papiliochromes. Release of SN-1 .from the yellow pigments in the crude extract To the above-mentioned crude extract or to the yellow pigment solution which was obtained after the DEAE-Cellulose column mentioned below, hydrochloric acid was added to a final concentration of 10 -8 N, and the solution was heated in a
D O P A M I N E DERIVATIVE I N P A P I L I O X U T H U S
75
boiling water bath for 30 min. It had been confirmed in the previous paper (UMEBACm and YOSmDA, 1970) that the yellow pigments decompose to SN-1 and kynurenine by this procedure.
DEAE-Cellulose column After DEAE-Cellulose powder was washed with 0.2 N HCI and water, a 1 × 13 cm column was prepared. The yellow pigments are not adsorbed in this column. So, after the application of the crude extract to the column, the yellow pigments were washed down with water. After use, the column could be repeatedly used by being washed with 0.2 N HCI and water.
Amberlite CG-50 column After the powder of Amberlite CG-50 was washed successively with 2 N NaOH, water, 2 N HC1, and water, the resin was equilibrated with 0.2 M ammonium acetate buffer (pH 6.0) and adjusted to pH 6.0 with ammonia. A 1 × 30 cm column was prepared and washed with the same buffer. The elution was done with 0"4 M ammonium acetate buffer (pH 5.0) (KIRSHNERand GOODALL,1957). The rate of flow was about 0.12 ml per min.
Biogel P-2 column The powder of Biogel P-2 was suspended in water, packed to a 1.8 × 50 cm column, and washed with 0.2 N acetic acid. The elution was done with the same acetic acid at a rate of about 0.2 ml per min. This column was reported to be effective for the separation of o-diphenolic derivatives by ANDERSEN(1970).
Measurements of absorbance of the effluentsfrom columns The effluents from the Amberlite CG-50 and Biogel P-2 columns were fractionated using an automatic fraction collector. One fraction was 2.8 to 3.0 ml. The absorbance of each fraction was determined at both 280 and 360 nm with a Hitachi model 139 spectrophotometer.
Ninhydrin reaction in the effluent from the Biogel P-2 column A 1-ml aliquot was withdrawn from each fraction of the effluent, and the pH was adjusted to 5 4-0.2 by adding 0.5 ml of 0.3 N NaOH solution. To the mixture, 1.2 ml of Yemm and Cocking's KCN-ninhydrin solution was added (¥EMM and COCKmC, 1955). After shaking, the solution was heated in a boiling water bath for 15 min and then cooled with running water for 5 min. After the addition of 3 ml of 60% ethanol, the absorbance at 570 nm was measured.
Paper and thin-layer chromatographies For paper chromatography, Toyo No. 51A paper ( 3 0 × 3 0 c m ) was used. The solvents were (1) the upper layer of n-butanol-acetic acid-water (4 : 1 : 5) mixture (BAW), (2) methanol-water-pyridine ( 2 0 : 5 : 1 ) (MWP), and (3) nbutanol-water-pyridine (1 : 1 : 1) (BWP).
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Y. UMEBACHI
For thin-layer chromatography, the following two kinds of sheets and one kind of plate were used: (1) TLC aluminium sheet precoated with silica gel (20 × 20 cm, Merck No. 5553), (2) TLC aluminium sheet precoated with cellulose (20×20 cm, Merck No. 5552), and (3) glass plate precoated with Avicel SF (microcrystalline cellulose) (10 × 10 cm, Funakoshi Pharm. Co.). The solvents for silica gel sheet were phenol-water (75 : 25 g/g) (PhW) and methanol-chloroform17% ammonia aq. (2 : 2 : 1) (MCA). The solvents for cellulose sheet and Avicel plate were (1) BAW, (2) MWP, (3) phenol-ethanol-water-conc, ammonia aq. (15 : 4 : 1 : 0.1) (PhEA), (4) ethylmethylketone-acetone-formic acid-water (20 : 1 : 0.5 : 3) (EAF), and n-butanol saturated with 3 N HC1 (BHC1). After development, one of the following colour tests was made (UMEBACHI and YOSrItDA, 1970): (1) ninhydrin test, (2) phosphomolybdic acid-ammonia test, and (3) ethylenediamine-ammonia test.
Injection-experiments of 14C-labelled compounds The following two compounds were used: (1) Dopamine (ethylene-l-14C)hydrochloride obtained from the Radiochemical Centre. The specific activity was 56 mc/mM. The powder (50/zc) was dissolved in 2 ml of water. (2) Beta-alanine-l-14C obtained from the New England Nuclear. The specific activity was 4-29 mc/mM. The solution (50/lc) in 0"5 ml of 0.1 N HCI was neutralized with 0.033 N NaOH. Thirty to sixty/zl (0.2 to 3.0/zc) of these solutions was injected into one pharate pupa. After the emergence of butterflies, the wings were cut off and dried, and the autoradiographs of the wings were taken. The film was the Sakura, Medical Q, X-ray film, and the time of exposure was 14 to 22 days. After that, the crude extract of yellow pigments in 10 -8 N HCI was prepared from the wings, heated in a boiling water bath for 30 min, and submitted to the Amberlite CG-50 column. After the absorbance of each fraction of the effluent was measured at 280 and 360 nm, a 1-ml aliquot of each fraction was transferred to a stainless pan (diameter, 2.5 cm) for radioactivity measurement and evaporated to dryness under infra-red lamp. The radioactivity of each pan was measured with a Aloka gas flow counter. In the case of dopamine-l-14C, the autoradiograph of the wings was already reported in the previous paper (UMEBACHIand YOSHIDA,1970) and is not included in the present paper. RESULTS
Amberlite CG-50 column chromatographies of the crude extracts from wings and scales When the crude extract of yellow pigments from wings was, without any pretreatment, submitted to the Amberlite CG-50 column, the elution pattern as shown in Fig. la was obtained. As will be seen from the figure, a big peak was found from the fourth fraction to the eighth. But there was neither peak nor fraction corresponding to the yellow pigments (Papiliochromes). Although the
77
DOPAMINE DERIVATIVE I N PAPILIO X U T H U 8
fifth fraction was dirty yellow, it was evident that the substance was not Papiliochrome. Because, in the case of the extract from yellow scales, there was no yellow fraction. The yellow pigments were not eluted from this column with 0.4 M ammonium acetate.
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FIG. 1. Amberlite CG-50 column chromatographies of the crude extracts of wings. (a) Without any pretreatment; (b) after being heated in 10 -3 N HC1. Solid line, absorbance at 280 nm; dashed line, at 360 nm. The fraction numbers of the peaks of kynurenine and SN-1 were 10-13 and 22-26 respectively. When the crude extract from wings was acidified to a final concentration of 10 -3 N with 10 -= N HC1, heated at 100°C for 30 min, and then chromatographed on the Amberlite CG-50 column, the elution pattern changed to Fig. lb. As will be seen from the comparison between Figs. la and b, two new big peaks appeared after the heating. Of them, the first new peak was identified as kynurenine by paper chromatographies. From the results of the previous paper (UMEBACHI and YOSHIDA, 1970), there was no doubt about this identification. On the other hand, the second new peak was an unknown substance. After the fractions of the latter peak were combined and evaporated to dryness under reduced pressure, the residue was dissolved in water and one-dimensionally chromatographed on filter
78
Y. UMEBACHI
paper with BAW. On the chromatogram, a substance was found which was slightly brown or almost negative to the ninhydrin test and blue to the phosphomolybdic acid-NH8 test. To the ethylenediamine-NH~ test, the substance showed an orangish pink fluorescence which changed to a whitish yellow fluorescence after one to two days. The u.v. spectrum of the substance was measured in 0.4 M ammonium acetate (pH 5.0) as given in Fig. 2, which shows an absorption peak at 280 nm. From the colour tests, chromatographic behaviour, and absorption spectrum, it was evident that the substance was not other than the SN-1 substance which had been reported as a decomposition-product of the yellow pigments in the previous paper (UMEBACHIand YOSHIDA, 1970).
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Next, the crude extract from yellow scales was chromatographed on the Amberlite CG-50 column, without any pretreatment as in Fig. la or after being heated as in Fig. lb. The results are given in Figs. 3a and b, which clearly show that, only after heating, the big peaks of kynurenine and SN-1 appear. When the crude extract was chromatographed without being heated, SN-1 was not found, and kynurenine was present only in a very small amount (Fig. 3a). This indicates that there is no free SN-1 or, if any present, is negligible in the yellow scales, while free kynurenine is present in a small amount. Moreover, the crude extract from black scales was prepared in the same way as in the yellow scales, heated as in Fig. lb, and chromatographed on the Amberlite CG-50 column. The elution pattern is given in Fig. 3c, which shows that there is no SN-1. Kynurenine might be absent too.
79
D O P A M I N E DERIVATIVE I N PAPILIO XUTHUS
Injection-experiment of l~C-dopamine Dopamine-l-14C was injected at the pharate pupal stage as described in the paragraph of Methods. After the emergence of butterflies, the crude extract of yellow pigments from wings were prepared, heated as in Fig. lb, and submitted to the Amberlite CG-50 column. The absorbances of the effluent at 280 and 360 nm and the radioactivity were measured. The result is given in Fig. 4, which clearly shows that 14C-dopamine is incorporated into SN-1. 2.0
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Estimation of the relative contents of SN-1 during the pharate adult stage The crude extracts from wings during the pharate adult stage were treated in the same way as in Fig. lb and subjected to the Amberlite CG-50 column. The absorbance of each fraction was measured at 280 and 360 nm. The absorbances at 280 nm of the fractions of the SN-1 peak were summed. This sum gives the relative content of SN-1 in arbitrary unit. The result is given in Fig. 5a. For the purpose of comparison, the kynurenine contents were also determined. The fractions of the kynurenine-peak were combined and volumetrically diluted with water. The kynurenine content was estimated in the same way as in the previous papers
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FIG. 4. Amberlite CG-50 column chromatography of the crude extract from wings of the butterflies injected with dopamine-1-14C at the pharate pupal stage. The crude extract was treated in the same way as in Fig. lb. Solid line, absorbance at 280 n m ; dashed line, at 360 n m ; dotted line, radioactivity (counts/min).
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FIG. 5. Contents of (a) SN-1 and (b) kynurenine in wings during the pharate adult and emerged adult stages. Ordinate in (a) is an arbitrary unit and in (b),/~g kynurenine in the wings of one pharate adult or emerged adult. Abscissa shows the developmental stages. PE, Stage when the pigmentation of eye begins; BE, stage when eyes become black; RW, stage of the appearance of red spots in the hind-wings; E, the time of emergence. The triangle shows free kynurenine.
81
DERIVATIVE I N PAPILIO XUTHUS
DOPAMINE
(UMEBACHI and YAMADA, 1964; UMEBACHI and KATAYAMA, 1966). The result is given in Fig. 5b. From Figs. 5a and b, it will be seen that both SN-1 and kynurenine in wings increase almost in parallel with each other during the pharate adult stage.
Purification of SN-1 The wings of ten to twenty male adults were used as the material. First, the crude extract of yellow pigments from wings was submitted to the DEAE-Cellulose column without any pretreatment. Much substance was adsorbed in this column, and the top of the column became brown. The yellow pigments were not adsorbed
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FIG. 6. Amberlite CG-50 column chromatography of the wing-extract treated with DEAE-Cellulose and then heated in 10 -3 N HC1. Solid line, absorbance at 280 nm; dashed line, at 360 rim. in this column and could be washed down with water. The yellow effluents were combined, acidified to a final concentration of 10 -s N with 10 -2 N HCI, and heated at 100°C for 30 rain. After cooling, the solution was applied to the Amberlite CG-50 column and eluted with 0.4 M ammonium acetate buffer (pH 5.0). The elution pattern is given in Fig. 6, which shows the two big peaks of kynurenine and SN-1. As will be seen from the comparison between Figs. lb and 6, a big peak of the earlier effluents from the column (Fig. lb) is removed by using the DEAECeLlulose column. The fractions of SN-1 in Fig. 6 were combined and evaporated to dryness under reduced pressure. The residue was dissolved in a small amount of water. When a part of the solution was submitted to paper and thin-layer chromatographies, the spot of SN-1 was found which was slightly brown to the ninhydrin test and blue to the phosphomolybdic acid-NH8 test. Other substances were negligible.
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The remaining solution of SN-1 was, furthermore, applied to the Biogel P-2 column and eluted with 0.2 N acetic acid. The elution pattern is shown in Fig. 7. The fractions of SN-1 were combined and again evaporated to dryness under reduced pressure. The residue was dissolved in a small amount of water and chromatographed on filter paper and thin-layer plates. In the ninhydrin and phosphomolybdic acid-NH8 tests, a single spot of SN-1 was found, which was slightly brown to the former test and blue to the latter test. It was evident that the SN-1 thus obtained was chromatographically pure.
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FIG. 7. Biogel P-2 column chromatography of the S N - I which was isolated with the Amberlite CG-50 column. Solid line, absorbance at 280 n m ; dashed line, at 360 nm. SN-1 was'eluted in fractions 34-36.
From both the previous paper (UMEBACHIand YOSHIDA,1970) and Fig. 4 of the present paper, there can be no doubt that SN-1 is a dopamine derivative. But, SN-1 did not coincide in the chromatographic behaviour and colour reactions with any dopamine derivative which had been reported in animals and plants.
Hydrolysis of SN-1 Interestingly, SN-1 could be hydrolyzed in 1 N HCI at 100°C for 2 hr to yield two main substances. The SN-1 purified through the DEAE-Cellulose, Amberlite CG-50, and Biogel P-2 columns was evaporated to dryness under reduced pressure and dissolved in 1 N HC1. After being heated under reflux at 100°C for 2 hr, the solution was again evaporated to dryness under reduced pressure. The residue was dissolved in a small amount of water, applied again to the Biogel P-2 column, and eluted with 0-2 N acetic acid. The elution pattern is given in Fig. 8, which shows two new big peaks. They are referred to below as N-3 and SN-la respectively.
83
D O P A M I N E DERIVATIVE I N PAPILIO XUTHUS
N-3 is a substance which is detected not by the absorption at 280 nm but by the ninhydrin reaction. On the other hand, $ N - l a is a substance which is detected by the absorption at 280 nm. The fractions of both peaks were respectively evaporated to dryness under reduced pressure. The residues were dissolved in a small amount of water and submitted to thin-layer chromatographies. N-3 was one-dimensionally chromatographed on the Avicel plate with BAW, MWP, or PhEA as solvent and on the silica gel sheet with PhW or MCA. In all the chromatograms, N-3 gave only a single spot which showed the same chromatographic behaviour and the
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]FIG. 8. Biogel P-2 column chromatography of the hydrolysates of SN-1. T h e hydrolysis was performed in 1 N HC1 at 100°C for 2 hr. Solid line, absorbance at 280 nm; dashed line, ninhydrin reaction. T h e fraction numbers of the peaks of N-3 and S N - l a were 26-28 and 40--42 respectively.
same purplish blue colour to the ninhydrin test as authentic fl-alanine. The result was very clear, and N-3 has been identified as fl-alanine. On the other hand, SN-la was chromatographed on the Avicel plate and cellulose sheet with BAW, BHC1, or EAF as solvent and was found to show almost the same chromatographic behaviour and the same yellow-brown colour to the ninhydrin test as arterenol. Moreover, the u.v. spectrum of SN-la was taken in water. The result is given in Fig. 9, which shows an absorption peak at 280 nm. The spectrum suggests that SN-la may be a dopamine derivative which is different from dopamine only in the side chain. The hydrolysis of SN-1 in 6 N HC1 was also carried out. After the solution of SN-1 in 6 N HCI was heated under reflux at 100°C for 2 hr and evaporated to dryness at 60°C with a rotary evaporator, the residue was dissolved in a small
84
Y. UMEBACHI
amount of water and submitted to paper chromatography. The production of p-alanine was the same as in the case of 1 N HC1. But, instead of SN-la, two phenolic, ninhydrin-negative substances were found.
Injection-experiment of 14C-fl-alanine Beta-alanine-l-~4C was injected at the pharate pupal stage as described in the paragraph of Methods. After the emergence of butterflies, the autoradiograph of wings was taken (Fig. 10). It was evident that ~4C-/~-alanine was incorporated into the yellow scales.
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Fro. 9. U.V. spectrum of SN-la substance in water. The crude extract from the wings was treated in the same way as in Fig. lb and submitted to the Amberlite CG-50 column. The absorbance of each fraction of the effluent was determined at 280 and 360 nm, and then the radioactivity of each fraction was measured. The resuk is given in Fig. 11, which clearly shows that 14C-fl-alanine is incorporated into SN-1. Moreover, the fractions of the SN-1 peak in Fig. 11 were combined and evaporated to dryness under reduced pressure. The residue was dissolved in 1 N HCI, heated under reflux at 100°C for 2 hr, and again evaporated to dryness under reduced pressure. The residue was dissolved in a small amount of water and submitted to thin-layer chromatographies of the Avicel plate and the silica gel sheet. The presence of p-alanine was confirmed by the ninhydrin test and by co-chromatography with authentic/~-alanine.
The production of fl-alaninefrom Papiliochrome 2 In the previous paper (UMEBACHI and YOSHIDA, 1970), it was reported that SN-1 is a decomposition-product of Papiliochrome 2a and 2b, and is a dopamine
85
Frc. 10. Autoradiograph of the wings of the male adult, in the pharate pupal stage of which fl-alanine-1-14C was injected.
DOPAMINE
DERIVATIVE
87
I N PAPILIO XUTHUS
derivative. From the experiments of the present paper, moreover, it has been shown that SN-1 is a dopamine derivative containing p-alanine. In the present paper, however, the source for SN-1 was not the pure Papiliochrome but the crude extract from wings or yellow scales. So, in order to make sure that fl-alanine is produced from Papiliochrome itself, the following experiment was carried out. The yellow scales were gathered, and the yellow pigments were extracted only by dipping the scales in water and shaking. The yellow extract thus obtained was immediately applied to the T L C cellulose sheets as a streak and 7" i
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FIG. 11. Amberlite CG-50 column chromatography of the crude extract from wings of the butterflies injected with fl-alanine-l-14C at the pharate pupal stage The crude extract was treated in the same way as in Fig. lb. Solid line, absorbance at 280 nm; dashed line, at 360 nm; dotted line, radioactivity (counts/min). developed with BAW. After development, the sheets were examined under u.v. rays. Papiliochrome 2a partly overlapped kynurenine. But Papiliochrome 2b could be separated from any other substance. So, from the area of Papiliochrome 2b, the cellulose powder was scraped, and the pigment was extracted with water. After centrifugation, the supernatant fluid contained neither fl-alanine nor SN-1 and was thin-layer chromatographically pure. After the solution was evaporated to dryness under reduced pressure, the residue was dissolved in 10 -3 N HC1, heated at 100°C for 30 min, and again evaporated to dryness under reduced pressure. The residue was dissolved in water, applied again to the T L C cellulose sheets as a streak and developed with BAW. After development, marker strips 2 cm from both edges of the sheets were cut off, and the ninhydrin and phosphomolybdic acid-NH~ tests were made on the strips. Kynurenine and SN-1 were found, and neither fl-alanine nor SN-la was present. From the areas of SN-1 of the sheets,
88
Y. UMEBAem
the cellulose powder was scraped and shaken with water. After centrifugation, the supernatant fluid was evaporated to dryness under reduced pressure. The residue was dissolved in 1 N HC1 and heated under reflux at 100°C for 2 hr, and the solution was evaporated to dryness under reduced pressure. The residue was dissolved in water, applied to the TLC cellulose sheets or Avicel plates, and developed with BAW, EAF, MWP or PhEA. After development, the ninhydrin and phosphomolybdic acid-NH8 tests were made. In all of these thin-layer chromatographies, the presence of •-alanine was confirmed. The presence of SN-la was proved in the chromatographies with BAW and EAF. MWP and PhEA were unsuitable for SN-la.
The presence of fl-alanine in the scales after the extraction of yellow pigments The crude extract from yellow scales was, without any pretreatment, submitted to one-dimensional paper chromatography w~th MWP. In some cases, the yellow scales were shaken in water, and the extract was one-dimensionally chromatographed on the TLC cellulose sheet with BAW. In all these chromatograms, no free fl-alanine was found by the ninhydrin test. Next, the yellow scales were treated successively and repeatedly with hot water (at 50°C) and 70% ethanol (at 45°C). After that, the scales were washed repeatedly with acetone and ethyl ether. The resulting scales, which did not contain the yellow pigments, were hydrolyzed under reflux in 6 N HC1 at 100°C for 24 hr. The hydrolysates were evaporated at 65°C with a rotary evaporator. The residue was dissolved in water and examined for/~-alanine with two-dimensional thinlayer chromatographies of silica gel and Avicel. The solvent for the first direction was MCA and MWP respectively. For the second direction, PhW and BAW were used respectively. Interestingly,/%alanine was detected by the ninhydrin test and confirmed by co-chromatography with authentic/~-alanine. DISCUSSION As described in the previous paper (UMEBACmand YOSmDA, 1970), the yellow pigments, Papilioehrome 2a and 2b, of the wings of P. xuthus, readily decompose to kynurenine and the dopamine derivative SN-1 by being heated in 10 -3 N HC1 at 100°C for 30 min. In fact, the decomposition does not necessitate heating in 10 -3 N HC1. Heating of the pigment solution in water is enough. But, in the latter case, kynurenine is destroyed to some extent. On the other hand, when the pigment solution in 6 N HC1 is heated, SN-1 is broken down. In order to minimize such secondary degradation, the heating in 10 -3 N HC1 was most suitable. Therefore, in the previous and present papers, this condition of decomposition was adopted. The ease of the decomposition suggests the possibility that Papiliochrome 2 may be a molecular complex of kynurenine and a dopamine derivative. In the previous paper (UMEBACHI and YOSaIDA, 1970), some chemical and physical properties of SN-1 were described. They included the colour tests on the paper chromatogram, u.v. spectrum, and the incorporation of l~C-dopamine.
DOPAMINEDEmVATIV~IN p A P ~ u o
xvTnvs
89
From those results, SN-1 was proved to be a dopamine derivative. In the present paper, the purification of SN-1 was made with the DEAE-Cellulose, Amberlite CG-50, and Biogel P-2 columns. From the colour tests, chromatographic behaviour, and u.v. spectrum, it was evident that the SN-1 prepared through these columns was identical with the SN-1 of the previous paper, though the result of the ninhydrin test was a little different, that is, light bluish or negative in the previous paper and slightly brownish or almost negative in the present paper. And Figs. 2a-c indicate that SN-1 is derived from the yellow scales. Moreover, the chromatographic examination of the decomposition-products of the thin-layer chromatographically pure Papiliochrome 2b re-confirmed the previous result that SN-1 was derived from Papiliochrome. From the results of both previous (UMEBACHIand YOSHIDA, 1970) and present papers, there is now no doubt at all that SN-1 is a dopamine derivative. But, SN-1 is not identical with any dopamine derivative which has been reported up to the present. In other words, SN-1 is an unknown derivative of dopamine. The fact that the absorption peak is at 280 nm as in dopa, dopamine, arterenol, and adrenaline suggests that SN-1 may be different from dopamine only in the side chain. The fact that SN-1 is hydrolyzed by being heated in 1 N HC1 at 100°C for 2 hr to yield fl-alanine and the ninhydrin yellow-brown o-diphenolic substance (SN-la) is important. In this regard, the result of the previous paper (UMEBACHI and YOSrlIDA, 1970) must be corrected. In the previous paper, it was reported that, by the hydrolysis in 1 N HC1, SN-1 was changed to an o-diphenol derivative which was purple to the ninhydrin test on the paper chromatogram. This erroneous observation resulted from the fact that, when the hydrolysates in 1 N HC1 were one-dimensionally chromatographed on paper with BAW, SN-la and fl-alanine had almost the same R~ values. The positions of SN-la and fl-alanine overlap on the chromatogram, though the position of the former is a little higher than that~of the latter. But, when the paper chromatogram is run with MWP, SN-la and p-alanine can be clearly separated, because this solvent is not suited for SN-la. In addition, SN-la is yellowish brown to the ninhydrin test on the chromatogram, while fl-alanine is purplish blue to the test. The latter fact is, in the present paper, one of the evidences which support that the N-3 substance obtained after the hydrolysis of SN-1 is fl-alanine, because authentic fl-alanine is neither reddish purple nor purple but purplish blue to the ninhydrin test. From these results, there was no doubt that SN-1 decomposed to fl-alanine and the ninhydrinyellowish brown o-diphenolic substance (SN-la). From the u.v. spectruin, it is probable that SN-la is different from dopamine only in the side chain. The fact that SN-la is yellowish brown to the ninhydrin test suggests that the substance still contains the nitrogen of the side chain of dopamine. In the thin-layer chromatographies with BAW, BHC1, and EAF and the colour reactions, SN-la resembled arterenol very closely. It is probable that SN-la is a dopamine derivative which is very similar to arterenol. But it was evident that SN-la was not adrenaline. In this connection, the report of ANDERSEN(1971) that arterenol
90
Y. UMEBACHZ
can be incorporated into the cuticle, though it is presumably not natural tanning agent is very interesting. LEVENBOOKet al. (1969) reported the presence of/?-alanyl-tyrosine (Sarcophagine) in the larvae of the fleshfly Sarcophaga bullata. This dipeptide seems to be a reservoir of tyrosine in this fly group (BODNARYKand LEVENBOOK,1969 ; BODNARYK, 1972). It seems that, just at the pupation, /~-alanyl-tyrosine is cleaved and is incorporated into the cuticle (BoDNARYK, 1970, 1971a). BOD~ARYK (1971b) reported that, in Sareophagine,/~-alanine is N-terminal and that, in general, when /%alanine is present as a N-terminal amino acid of peptide bond, the bond is hydrolyzed much easier than other peptide bonds. According to his report, in the peptide in which/Lalanine is N-terminal,/%alanine is released by hydrolysis in 2 N HC1 for 2 hr. In this regard, the fact that SN-1 is hydrolyzed in 1 N HC1 at 100°C for 2 hr is interesting. This suggests the possibility that SN-1 may be N-(p-alanyl)-dopamine derivative. The absorption spectrum of SN-1 also is not in conflict with this possibility, because the spectrum suggests that SN-1 may be different from dopamine only in the side chain. The SN-1 contents in wings have been proved to be parallel to the kynurenine contents during the yellow pigment formation. In fact, this result was what was expected. Because, both SN-1 and kynurenine had been presumed to be decomposition-products of the yellow pigments (UMEBACHI and YOSmDA, 1970). And yet, no free SN-1 was found, and free kynurenine was present only in a small quantity. Therefore, the quantitative parallelism of SN-1 and kynurenine during the yellow pigment formation was to be expected. But, anyway, this result is further evidence which indicates that Papiliochrome 2 contains both dopamine derivative and kynurenine as constituents. As to the difference between Papiliochrome 2a and 2b, the possibility that they were a kind of isomer was suggested in the previous paper (UMEBACHIand YOSHIDA,1970). It is interesting that fl-alanine was found in the hydrolysates of the scales from which water-soluble substances including the yellow pigments had been removed. This suggests the presence of protein-bound fl-alanine. Anyway, the relation between such fl-alanine and the fl-alanine as a constituent of the yellow pigments remains to be studied. Up to the present, there have been a considerable number of reports on the presence of fl-alanine in insects. For example, AGRELL (1949) reported the presence of fl-alanine as a free-amino acid in the extracts of whole pupae and pharate adults of Calliphora erythrocephala. CHEN (1959) found flalanine in the hydrolysates of haemolymph protein of Culex pipiens. CHiN (1958) and DUFFY (1964) reported the sexual differences in free fl-alanine content in mosquitoes. Moreover, DENNELL (1958) reported the presence of fl-alanine in the hydrolysates of puparium cuticle of Calliphora vomitoria and mentioned the possibility that this amino acid may be related to the hardening of cuticle. In this regard, Seki and his collaborators' reports on black mutant strains of several species are very interesting. SEKI (1962), FUKUSHIand SEKI (1965), FUKUSHI(1967), and NAKAI (1971) reported that the pupal sheaths of the wild strains of Bombyx mori, Musca domestica, and Drosophila viliris contain fl-alanine and that the black
DOPAMINE DERIVATIVE I N PAPILIO XUTHUS
91
mutants, for example, so of B. mori, bp of M . domestica, and eb of D. virilis lack this amino acid in their pupal sheaths. After or in parallel with these reports, several reports dealing with fl-alanine in the cuticle of insects were published (JAcoBs and BRUBAKER, 1963; JACOBS, 1966; BODNARYK, 1971a; HODGETTS, 1972; ROSS and MONROE, 1972). F r o m these reports, it is probable that fl-alanine is incorporated into cuticle at the time of hardening. Screlotization of cuticle may be related to the following three factors: the amino groups of protein, o-diphanol derivative (for example, N-acetyldopamine), and fl-alanine, though the function of fl-alanine in the hardening process remains to be studied. Now, in the present paper, the yellow pigments of P. xuthus have proved to be related to kynurenine, dopamine derivative, and fl-alanine. So, it can be said that the formation of yellow pigments in P. xuthus bears some resemblance to the hardening of cuticle in insects, at least as far as their constituents concern. I n this regard, the yellow pigments of wings in the Papilionidae are not only a new field of insect pigment but also an interesting subject which may give a clue for elucidating the function of fl-alanine in insects.
REFERENCES
AGRELL I. (1949) Occurrence and metabolism of free amino acids during insect metamorphosis. Acta Physiol. Scan& 18, 247-258. ANDEaS~r S. O. (1970) Isolation of arterenone (2-amino-3',4'-dihydroxyacetophenone) from hydrolysates of sclerotized insect cuticle. J. Insect Physiol. 16, 1951-1959. ANDERSEN S. O. (1971) Phenolic compounds isolated from insect hard cuticle and their relationship to the sclerotization process. Insect Biochem. 1, 157-170. BODNARYK R. P. (1970) Effect of dopa-decarboxylase inhibition on the metabolism of fl-alanyl-L-tyrosine during puparium formation in the fleshfly Sarcophaga bullata Parker. Comp. Biochem. Physiol. 35, 221-227. BODNARYKR. P. (1971a) Studies of the incorporation of fl-alanine into the puparium of the fly, Sarcophaga bullata. J. Insect Physiol. 17, 1201-1210. BODNARYK R. P. (1971b) N-Terminal fl-alanine in the puparium of the fly Sarcophaga bullata: Evidence from kinetic studies of its release by partial acid hydrolysis. Insect Biochem. 1, 228-236. BODNARYKR. P. (1972) A survey of the occurrence of fl-alanyl-tyrosine, 7-glutamyl-phenylalanine, and tyrosine-0-phosphate in the larval stage of flies (Diptera). Comp. Biochem. Physiol. 43B~ 587-592. BODNAR'/K R. P. and LBV~BOOK L. (1969) The role of/~-alanyl-L-tyrosine (Sarcophagine) in puparium formation in the fleshfly Sarcophaga bullata. Comp. Biochem. Physiol. 30, 909-921. CHm~ P. S. (1958) Studies on the protein metabolism of Culex pipiens L.--II. Quantitative differences in free amino acids between male and female adult mosquitoes. J. Insect Physiol. 2, 128-136. CHEN P. S. (1959) Studies on the protein metabolism of Culex pipiens L.--III. A comparative analysis of the protein contents in the larval haemolymph of autogenous and anautogenous forms. J. Insect Physiol. 3, 335-344. DF~NZLL R. (1958) The amino acid metabolism of a developing insect cuticle: the larval cuticle and puparium of Calliphora vomitoria--I. Changes in amino acid composition during development. Proc. R. Soc. (B) 148, 270-279. DUFFY J. P. (1964) Sex differences in the free amino acids of three colonized mosquito species. Ann. ent. Soc. Am. 57, 24-28.
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FUKUSHIY. (1967) Genetic and biochemical studies on amino acid compositions and color manifestation in pupal sheaths of insects. Jap. ft. Genetics 42, 11-21. FUKUSHI Y. and SEKI T. (1965) Differences in amino acid compositions of pupal sheaths between wild and black pupa strains in some species of insects. Jap. J. Genetics 40, 203-208. HODGETT8 R. B. (1972) Biochemical characterization of mutants affecting the metabolism of fl-alanine in Drosophila. J. Insect Physiol. 18, 937-947. JACOBSM. E. (1966) Deposition of labelled beta-alanine in ebony and non-ebony Drosophila raelanogaster with notes on other amino acids. Genetics 53, 777-784. JACOBSM. E. and BRUBAKERK. K. (1963) Beta-alanine utilization of ebony and non-ebony Drosophila melanogaster. Science, Wash. 139, 1282-1283. KIRSHNER N. and GOODALLMcC. (1957) Separation of adrenaline, noradrenaline, and hydroxytyramine by ion exchange chromatography, ft. biol. Chem. 226, 207-212. LEVENBOOKL., BODNARYKR. P., and SPANDET. F. (1969) fl-Alanyl-L-tyrosine. Chemical synthesis, properties, and occurrence in larvae of the fleshfly Sarcophaga bullata Parker. Biochem. ft. 113, 837-841. NAKAI S. (1971) Genetic and biochemical studies on the fl-alanine metabolism in the housefly, Musca domestica L. flap. ft. Genetics 46, 53-60. Ross R. H. and MONROER.E. (1972) fl-Alanine metabolism in the housefly, Musca domestica: Concentrations in the larvae, pupae, and adults, ft. Insect Physiol. 18~ 791-796. SEKI T. (1962) Absence of beta-alanine in hydrolyzate of the pupal sheaths of ebony mutant of Drosophila virilis. Drosophila Inf. Serv. 36, 115. UMEBACHIY. (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. UMEBACHIY. (1959) Yellow pigments in the wings of the Papilionid butterfiies--III. The radioautographs of the wings of five species of Papilio injected with 14C-labelled tryptophan. Annot. Zool.Jap. 32, 112-116. UMEBACHIY. (1961) Yellow pigments in the wings of the Papilionid butterflies--V. Some chemical properties of the yellow pigments of Papilio xuthus. Sci. Rep. Kanazawa Univ. 7~ 139-150. UMEBACm Y. (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. Sci. Rep. Kanazawa Univ. 8~ 135-142. UMEBACHXY. and KATAYAMAM. (1966). Tryptophan and tyrosine metabolism in the pupa of Papilionid butterflies--II. The general pattern of tryptophan metabolism during the pupal stage of PapiIio xuthus. J. Insect Physiol. 12, 1539-1547. UMEI3ACHIY. and NAKAMURAA. (1954) The presence of kynurenine in the wings of the Papilionid butterflies. Zool. Mag. Tokyo 63~ 57-61. (In Japanese, English summary.) UMEBACHIY. and TAKAHASHIn . (1956) Kynurenine in the wings of the Papilionid butterflies. J. Biochem. Tokyo 43~ 73-81. UMEBACHIY. and YAMADAM. (1964) Tryptophan and tyrosine metabolism in the pupa of Papilionid butterflies--I. Accumulation of the bound form of kynurenine in Papilio xuthus. Annot. Zool. Jap. 37, 51-57. UMEBACHIY. and YOSHIDAK. (1970) Some chemical and physical properties of Papiliochrome 2 in the wings of Papilio xuthus. J. Insect Physiol. 16, 1203-1228. YEMM E. W. and COCKINGE. C. (1955) The determination of amino-acids with ninhydrin. Biochem. J. 80~ 209-213.
F U R T H E R S T U D I E S ON T H E DOPAMINE DERIVATIVE, SN-1 DERIVED FROM T H E YELLOW P I G M E N T S OF PAPILIO X U T H U S Y. UMEBACHI Department of Biology, Faculty of Science, Kanazawa University, Kanazawa 920, Japan
(Received 24 June 1974) A b s t r a c t - - F o r the purpose of purifying the dopamine derivative SN-1 which
had been reported as a decomposition-product of the yellow pigments of Papilio xuthus, the isolation of the substance with column chromatography was tried. The crude extract from wings or yellow scales was heated in 10-' N HCI and chromatographed on the Amberlite CG-50 column. On this column, kynurenine and an unknown o-diphenolic substance were clearly separated. The latter coincided with the SN-1 and was re-confirmed to be a dopamine derivative using 14C-dopamine. On the basis of these observations, the purification of SN-1 was performed through the DEAE-Cellulose, Amberlite CG-50, and Biogel P-2 columns. The purified SN-1 was hydrolyzed in 1 N HC1, and the hydrolysates were again ehromatographed on the Biogel P-2 column, in which two main products were found. One has been identified as fl-alanine. The other was a dopamine derivative closely related to arterenol. It has been presumed that SN-1 is a dopamine derivative containing fl-alanine in the side chain. The contents of SN-1 and kynurenine in wings were estimated during the pharate adult stage. It was shown that both substances increase in parallel with each other during the yellow pigment formation. In addition to the above fl-alanine, this amino acid was found also in the scales remaining after the yellow pigments and other water-soluble substances were extracted. INTRODUCTION AmONQ wing pigments of butterflies, pterins of the Pieridae and ommatins of the Nymphalidae have been investigated in detail from the standpoints of organic chemistry and biochemistry. For other families, however, no such detailed investigation has been made. UMEBACm and NAKAMURA (1954) and UMrBACHI and TAKAHASHr (1956) found a large amount of kynurenine in the hot water extract of the yellow scales of Papilio xuthis (Papilionidae). Since then, physical and chemical properties of the yellow pigments of this species have been studied in detail (UMEBACm, 1958, 1959, 1961, 1962; UMEBACHI and YOSHIDA, 1970). Although the yellow pigments are derived from tryptophan, they do not belong to ommochrome. T h e y have been presumed to be the pigments which are formed from both tryptophan and tyrosine metabolites. W h e n the water extract of the yellow scales is two-dimensionally chromatographed, several yellow pigments are found. Among them, the main yellow 73
74
Y. UMEBACHI
pigments were named Papiliochrome 2a and 2b, and their chemical and spectral properties were reported by UMEBACHI and YOSHIDA (1970). Papiliochrome 2a and 2b are so unstable to heating that when their solution in 10 -a N HC1 is heated at 100°C for 30 min, the pigments readily decompose to L-kynurenine and a o-diphenol derivative. The latter substance was termed SN-1 and was proved to be a dopamine derivative (UM~BACHIand YOSHIDA, 1970). The present paper deals mainly with the purification of SN-1 and the identification of the hydrolysates. As SN-1 had been confirmed to be a decompositionproduct of the yellow pigments (UMEBACHIand YOSHIDA, 1970), the crude extract of the yellow pigments from wings or yellow scales was used as the starting material for SN-1 in the present paper. From such extract, SN-1 was purified through column chromatographies and hydrolyzed in 1 N HC1. Chromatographic examinations of the hydrolysates showed the presence of two main products. One has been identified as fl-alanine, and the other has been presumed to be a dopamine derivative which is very similar to arterenol. The SN-1 and kynurenine contents in wings during the pharate adult stage were also studied. Moreover, the presence of fl-alanine in the scales remaining after the pigment extraction was shown.
MATERIALS AND METHODS Materials As the materials for the extraction of the yellow pigments, the wings or yellow scales of the male adults of P. xuthus were used. Some of the butterflies were obtained through the Okura Biological Institute, and others were raised in our laboratory from the larval stage. For the injection-experiments of 14C-labelled compounds, the pharate pupae were used, and after the injection, were raised to adults at 25 :~ I°C.
Extraction of yellow pigments The wings or yellow scales were homogenized in 70% ethanol. After repeating centrifugation and extraction with 70% ethanol, the combined supernatant solution was evaporated to dryness under reduced pressure. The residue was washed with ethyl ether thrice and then dissolved in water. After centrifugation, the supernatant fluid is referred to below as a crude extract of the yellow pigments. In some cases, the yellow pigments were extracted by shaking the yellow scales in water and, without any pretreatment, were subjected to paper and thin-layer chromatography. All these crude extracts, of course, contain a large amount of Papiliochromes. Release of SN-1 .from the yellow pigments in the crude extract To the above-mentioned crude extract or to the yellow pigment solution which was obtained after the DEAE-Cellulose column mentioned below, hydrochloric acid was added to a final concentration of 10 -8 N, and the solution was heated in a
D O P A M I N E DERIVATIVE I N P A P I L I O X U T H U S
75
boiling water bath for 30 min. It had been confirmed in the previous paper (UMEBACm and YOSmDA, 1970) that the yellow pigments decompose to SN-1 and kynurenine by this procedure.
DEAE-Cellulose column After DEAE-Cellulose powder was washed with 0.2 N HCI and water, a 1 × 13 cm column was prepared. The yellow pigments are not adsorbed in this column. So, after the application of the crude extract to the column, the yellow pigments were washed down with water. After use, the column could be repeatedly used by being washed with 0.2 N HCI and water.
Amberlite CG-50 column After the powder of Amberlite CG-50 was washed successively with 2 N NaOH, water, 2 N HC1, and water, the resin was equilibrated with 0.2 M ammonium acetate buffer (pH 6.0) and adjusted to pH 6.0 with ammonia. A 1 × 30 cm column was prepared and washed with the same buffer. The elution was done with 0"4 M ammonium acetate buffer (pH 5.0) (KIRSHNERand GOODALL,1957). The rate of flow was about 0.12 ml per min.
Biogel P-2 column The powder of Biogel P-2 was suspended in water, packed to a 1.8 × 50 cm column, and washed with 0.2 N acetic acid. The elution was done with the same acetic acid at a rate of about 0.2 ml per min. This column was reported to be effective for the separation of o-diphenolic derivatives by ANDERSEN(1970).
Measurements of absorbance of the effluentsfrom columns The effluents from the Amberlite CG-50 and Biogel P-2 columns were fractionated using an automatic fraction collector. One fraction was 2.8 to 3.0 ml. The absorbance of each fraction was determined at both 280 and 360 nm with a Hitachi model 139 spectrophotometer.
Ninhydrin reaction in the effluent from the Biogel P-2 column A 1-ml aliquot was withdrawn from each fraction of the effluent, and the pH was adjusted to 5 4-0.2 by adding 0.5 ml of 0.3 N NaOH solution. To the mixture, 1.2 ml of Yemm and Cocking's KCN-ninhydrin solution was added (¥EMM and COCKmC, 1955). After shaking, the solution was heated in a boiling water bath for 15 min and then cooled with running water for 5 min. After the addition of 3 ml of 60% ethanol, the absorbance at 570 nm was measured.
Paper and thin-layer chromatographies For paper chromatography, Toyo No. 51A paper ( 3 0 × 3 0 c m ) was used. The solvents were (1) the upper layer of n-butanol-acetic acid-water (4 : 1 : 5) mixture (BAW), (2) methanol-water-pyridine ( 2 0 : 5 : 1 ) (MWP), and (3) nbutanol-water-pyridine (1 : 1 : 1) (BWP).
76
Y. UMEBACHI
For thin-layer chromatography, the following two kinds of sheets and one kind of plate were used: (1) TLC aluminium sheet precoated with silica gel (20 × 20 cm, Merck No. 5553), (2) TLC aluminium sheet precoated with cellulose (20×20 cm, Merck No. 5552), and (3) glass plate precoated with Avicel SF (microcrystalline cellulose) (10 × 10 cm, Funakoshi Pharm. Co.). The solvents for silica gel sheet were phenol-water (75 : 25 g/g) (PhW) and methanol-chloroform17% ammonia aq. (2 : 2 : 1) (MCA). The solvents for cellulose sheet and Avicel plate were (1) BAW, (2) MWP, (3) phenol-ethanol-water-conc, ammonia aq. (15 : 4 : 1 : 0.1) (PhEA), (4) ethylmethylketone-acetone-formic acid-water (20 : 1 : 0.5 : 3) (EAF), and n-butanol saturated with 3 N HC1 (BHC1). After development, one of the following colour tests was made (UMEBACHI and YOSrItDA, 1970): (1) ninhydrin test, (2) phosphomolybdic acid-ammonia test, and (3) ethylenediamine-ammonia test.
Injection-experiments of 14C-labelled compounds The following two compounds were used: (1) Dopamine (ethylene-l-14C)hydrochloride obtained from the Radiochemical Centre. The specific activity was 56 mc/mM. The powder (50/zc) was dissolved in 2 ml of water. (2) Beta-alanine-l-14C obtained from the New England Nuclear. The specific activity was 4-29 mc/mM. The solution (50/lc) in 0"5 ml of 0.1 N HCI was neutralized with 0.033 N NaOH. Thirty to sixty/zl (0.2 to 3.0/zc) of these solutions was injected into one pharate pupa. After the emergence of butterflies, the wings were cut off and dried, and the autoradiographs of the wings were taken. The film was the Sakura, Medical Q, X-ray film, and the time of exposure was 14 to 22 days. After that, the crude extract of yellow pigments in 10 -8 N HCI was prepared from the wings, heated in a boiling water bath for 30 min, and submitted to the Amberlite CG-50 column. After the absorbance of each fraction of the effluent was measured at 280 and 360 nm, a 1-ml aliquot of each fraction was transferred to a stainless pan (diameter, 2.5 cm) for radioactivity measurement and evaporated to dryness under infra-red lamp. The radioactivity of each pan was measured with a Aloka gas flow counter. In the case of dopamine-l-14C, the autoradiograph of the wings was already reported in the previous paper (UMEBACHIand YOSHIDA,1970) and is not included in the present paper. RESULTS
Amberlite CG-50 column chromatographies of the crude extracts from wings and scales When the crude extract of yellow pigments from wings was, without any pretreatment, submitted to the Amberlite CG-50 column, the elution pattern as shown in Fig. la was obtained. As will be seen from the figure, a big peak was found from the fourth fraction to the eighth. But there was neither peak nor fraction corresponding to the yellow pigments (Papiliochromes). Although the
77
DOPAMINE DERIVATIVE I N PAPILIO X U T H U 8
fifth fraction was dirty yellow, it was evident that the substance was not Papiliochrome. Because, in the case of the extract from yellow scales, there was no yellow fraction. The yellow pigments were not eluted from this column with 0.4 M ammonium acetate.
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FIG. 1. Amberlite CG-50 column chromatographies of the crude extracts of wings. (a) Without any pretreatment; (b) after being heated in 10 -3 N HC1. Solid line, absorbance at 280 nm; dashed line, at 360 nm. The fraction numbers of the peaks of kynurenine and SN-1 were 10-13 and 22-26 respectively. When the crude extract from wings was acidified to a final concentration of 10 -3 N with 10 -= N HC1, heated at 100°C for 30 min, and then chromatographed on the Amberlite CG-50 column, the elution pattern changed to Fig. lb. As will be seen from the comparison between Figs. la and b, two new big peaks appeared after the heating. Of them, the first new peak was identified as kynurenine by paper chromatographies. From the results of the previous paper (UMEBACHI and YOSHIDA, 1970), there was no doubt about this identification. On the other hand, the second new peak was an unknown substance. After the fractions of the latter peak were combined and evaporated to dryness under reduced pressure, the residue was dissolved in water and one-dimensionally chromatographed on filter
78
Y. UMEBACHI
paper with BAW. On the chromatogram, a substance was found which was slightly brown or almost negative to the ninhydrin test and blue to the phosphomolybdic acid-NH8 test. To the ethylenediamine-NH~ test, the substance showed an orangish pink fluorescence which changed to a whitish yellow fluorescence after one to two days. The u.v. spectrum of the substance was measured in 0.4 M ammonium acetate (pH 5.0) as given in Fig. 2, which shows an absorption peak at 280 nm. From the colour tests, chromatographic behaviour, and absorption spectrum, it was evident that the substance was not other than the SN-1 substance which had been reported as a decomposition-product of the yellow pigments in the previous paper (UMEBACHIand YOSHIDA, 1970).
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F[o. 2. U.V. spectrum of SN-1 substance in 0"4 M ammonium acetate buffer (pH 5"0).
Next, the crude extract from yellow scales was chromatographed on the Amberlite CG-50 column, without any pretreatment as in Fig. la or after being heated as in Fig. lb. The results are given in Figs. 3a and b, which clearly show that, only after heating, the big peaks of kynurenine and SN-1 appear. When the crude extract was chromatographed without being heated, SN-1 was not found, and kynurenine was present only in a very small amount (Fig. 3a). This indicates that there is no free SN-1 or, if any present, is negligible in the yellow scales, while free kynurenine is present in a small amount. Moreover, the crude extract from black scales was prepared in the same way as in the yellow scales, heated as in Fig. lb, and chromatographed on the Amberlite CG-50 column. The elution pattern is given in Fig. 3c, which shows that there is no SN-1. Kynurenine might be absent too.
79
D O P A M I N E DERIVATIVE I N PAPILIO XUTHUS
Injection-experiment of l~C-dopamine Dopamine-l-14C was injected at the pharate pupal stage as described in the paragraph of Methods. After the emergence of butterflies, the crude extract of yellow pigments from wings were prepared, heated as in Fig. lb, and submitted to the Amberlite CG-50 column. The absorbances of the effluent at 280 and 360 nm and the radioactivity were measured. The result is given in Fig. 4, which clearly shows that 14C-dopamine is incorporated into SN-1. 2.0
a
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NUMBER FIG. 3. Amberlite CG-50 column chromatographies of the crude extracts of scales. (a) Extract from yellow scales, without any pretreatment; (b) extract from yeUow scales, after being heated in 10 -8 N HCI; (c) extract from black scales, after being heated in 10 -~ N HCI. Solid line, absorbance at 280 nm; dashed line, at 360 nm. FRACTION
Estimation of the relative contents of SN-1 during the pharate adult stage The crude extracts from wings during the pharate adult stage were treated in the same way as in Fig. lb and subjected to the Amberlite CG-50 column. The absorbance of each fraction was measured at 280 and 360 nm. The absorbances at 280 nm of the fractions of the SN-1 peak were summed. This sum gives the relative content of SN-1 in arbitrary unit. The result is given in Fig. 5a. For the purpose of comparison, the kynurenine contents were also determined. The fractions of the kynurenine-peak were combined and volumetrically diluted with water. The kynurenine content was estimated in the same way as in the previous papers
80
Y . UMEBACHI i "~
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FIG. 4. Amberlite CG-50 column chromatography of the crude extract from wings of the butterflies injected with dopamine-1-14C at the pharate pupal stage. The crude extract was treated in the same way as in Fig. lb. Solid line, absorbance at 280 n m ; dashed line, at 360 n m ; dotted line, radioactivity (counts/min).
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FIG. 5. Contents of (a) SN-1 and (b) kynurenine in wings during the pharate adult and emerged adult stages. Ordinate in (a) is an arbitrary unit and in (b),/~g kynurenine in the wings of one pharate adult or emerged adult. Abscissa shows the developmental stages. PE, Stage when the pigmentation of eye begins; BE, stage when eyes become black; RW, stage of the appearance of red spots in the hind-wings; E, the time of emergence. The triangle shows free kynurenine.
81
DERIVATIVE I N PAPILIO XUTHUS
DOPAMINE
(UMEBACHI and YAMADA, 1964; UMEBACHI and KATAYAMA, 1966). The result is given in Fig. 5b. From Figs. 5a and b, it will be seen that both SN-1 and kynurenine in wings increase almost in parallel with each other during the pharate adult stage.
Purification of SN-1 The wings of ten to twenty male adults were used as the material. First, the crude extract of yellow pigments from wings was submitted to the DEAE-Cellulose column without any pretreatment. Much substance was adsorbed in this column, and the top of the column became brown. The yellow pigments were not adsorbed
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FIG. 6. Amberlite CG-50 column chromatography of the wing-extract treated with DEAE-Cellulose and then heated in 10 -3 N HC1. Solid line, absorbance at 280 nm; dashed line, at 360 rim. in this column and could be washed down with water. The yellow effluents were combined, acidified to a final concentration of 10 -s N with 10 -2 N HCI, and heated at 100°C for 30 rain. After cooling, the solution was applied to the Amberlite CG-50 column and eluted with 0.4 M ammonium acetate buffer (pH 5.0). The elution pattern is given in Fig. 6, which shows the two big peaks of kynurenine and SN-1. As will be seen from the comparison between Figs. lb and 6, a big peak of the earlier effluents from the column (Fig. lb) is removed by using the DEAECeLlulose column. The fractions of SN-1 in Fig. 6 were combined and evaporated to dryness under reduced pressure. The residue was dissolved in a small amount of water. When a part of the solution was submitted to paper and thin-layer chromatographies, the spot of SN-1 was found which was slightly brown to the ninhydrin test and blue to the phosphomolybdic acid-NH8 test. Other substances were negligible.
82
Y. UMEBACHI
The remaining solution of SN-1 was, furthermore, applied to the Biogel P-2 column and eluted with 0.2 N acetic acid. The elution pattern is shown in Fig. 7. The fractions of SN-1 were combined and again evaporated to dryness under reduced pressure. The residue was dissolved in a small amount of water and chromatographed on filter paper and thin-layer plates. In the ninhydrin and phosphomolybdic acid-NH8 tests, a single spot of SN-1 was found, which was slightly brown to the former test and blue to the latter test. It was evident that the SN-1 thus obtained was chromatographically pure.
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10
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FIG. 7. Biogel P-2 column chromatography of the S N - I which was isolated with the Amberlite CG-50 column. Solid line, absorbance at 280 n m ; dashed line, at 360 nm. SN-1 was'eluted in fractions 34-36.
From both the previous paper (UMEBACHIand YOSHIDA,1970) and Fig. 4 of the present paper, there can be no doubt that SN-1 is a dopamine derivative. But, SN-1 did not coincide in the chromatographic behaviour and colour reactions with any dopamine derivative which had been reported in animals and plants.
Hydrolysis of SN-1 Interestingly, SN-1 could be hydrolyzed in 1 N HCI at 100°C for 2 hr to yield two main substances. The SN-1 purified through the DEAE-Cellulose, Amberlite CG-50, and Biogel P-2 columns was evaporated to dryness under reduced pressure and dissolved in 1 N HC1. After being heated under reflux at 100°C for 2 hr, the solution was again evaporated to dryness under reduced pressure. The residue was dissolved in a small amount of water, applied again to the Biogel P-2 column, and eluted with 0-2 N acetic acid. The elution pattern is given in Fig. 8, which shows two new big peaks. They are referred to below as N-3 and SN-la respectively.
83
D O P A M I N E DERIVATIVE I N PAPILIO XUTHUS
N-3 is a substance which is detected not by the absorption at 280 nm but by the ninhydrin reaction. On the other hand, $ N - l a is a substance which is detected by the absorption at 280 nm. The fractions of both peaks were respectively evaporated to dryness under reduced pressure. The residues were dissolved in a small amount of water and submitted to thin-layer chromatographies. N-3 was one-dimensionally chromatographed on the Avicel plate with BAW, MWP, or PhEA as solvent and on the silica gel sheet with PhW or MCA. In all the chromatograms, N-3 gave only a single spot which showed the same chromatographic behaviour and the
i
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]FIG. 8. Biogel P-2 column chromatography of the hydrolysates of SN-1. T h e hydrolysis was performed in 1 N HC1 at 100°C for 2 hr. Solid line, absorbance at 280 nm; dashed line, ninhydrin reaction. T h e fraction numbers of the peaks of N-3 and S N - l a were 26-28 and 40--42 respectively.
same purplish blue colour to the ninhydrin test as authentic fl-alanine. The result was very clear, and N-3 has been identified as fl-alanine. On the other hand, SN-la was chromatographed on the Avicel plate and cellulose sheet with BAW, BHC1, or EAF as solvent and was found to show almost the same chromatographic behaviour and the same yellow-brown colour to the ninhydrin test as arterenol. Moreover, the u.v. spectrum of SN-la was taken in water. The result is given in Fig. 9, which shows an absorption peak at 280 nm. The spectrum suggests that SN-la may be a dopamine derivative which is different from dopamine only in the side chain. The hydrolysis of SN-1 in 6 N HC1 was also carried out. After the solution of SN-1 in 6 N HCI was heated under reflux at 100°C for 2 hr and evaporated to dryness at 60°C with a rotary evaporator, the residue was dissolved in a small
84
Y. UMEBACHI
amount of water and submitted to paper chromatography. The production of p-alanine was the same as in the case of 1 N HC1. But, instead of SN-la, two phenolic, ninhydrin-negative substances were found.
Injection-experiment of 14C-fl-alanine Beta-alanine-l-~4C was injected at the pharate pupal stage as described in the paragraph of Methods. After the emergence of butterflies, the autoradiograph of wings was taken (Fig. 10). It was evident that ~4C-/~-alanine was incorporated into the yellow scales.
0.6
l.d o Z 0.4
0 al 0.2
I
I
i
240
280
320
WAVE
LENGTH
360
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Fro. 9. U.V. spectrum of SN-la substance in water. The crude extract from the wings was treated in the same way as in Fig. lb and submitted to the Amberlite CG-50 column. The absorbance of each fraction of the effluent was determined at 280 and 360 nm, and then the radioactivity of each fraction was measured. The resuk is given in Fig. 11, which clearly shows that 14C-fl-alanine is incorporated into SN-1. Moreover, the fractions of the SN-1 peak in Fig. 11 were combined and evaporated to dryness under reduced pressure. The residue was dissolved in 1 N HCI, heated under reflux at 100°C for 2 hr, and again evaporated to dryness under reduced pressure. The residue was dissolved in a small amount of water and submitted to thin-layer chromatographies of the Avicel plate and the silica gel sheet. The presence of p-alanine was confirmed by the ninhydrin test and by co-chromatography with authentic/~-alanine.
The production of fl-alaninefrom Papiliochrome 2 In the previous paper (UMEBACHI and YOSHIDA, 1970), it was reported that SN-1 is a decomposition-product of Papiliochrome 2a and 2b, and is a dopamine
85
Frc. 10. Autoradiograph of the wings of the male adult, in the pharate pupal stage of which fl-alanine-1-14C was injected.
DOPAMINE
DERIVATIVE
87
I N PAPILIO XUTHUS
derivative. From the experiments of the present paper, moreover, it has been shown that SN-1 is a dopamine derivative containing p-alanine. In the present paper, however, the source for SN-1 was not the pure Papiliochrome but the crude extract from wings or yellow scales. So, in order to make sure that fl-alanine is produced from Papiliochrome itself, the following experiment was carried out. The yellow scales were gathered, and the yellow pigments were extracted only by dipping the scales in water and shaking. The yellow extract thus obtained was immediately applied to the T L C cellulose sheets as a streak and 7" i
3,0 2.0.
e c
400
~
7~
~
z
O Z
O
u) m <~
0.5. E c 0 co o,I i 10
20 FRACTION
30
40
50
60
NUMBER
FIG. 11. Amberlite CG-50 column chromatography of the crude extract from wings of the butterflies injected with fl-alanine-l-14C at the pharate pupal stage The crude extract was treated in the same way as in Fig. lb. Solid line, absorbance at 280 nm; dashed line, at 360 nm; dotted line, radioactivity (counts/min). developed with BAW. After development, the sheets were examined under u.v. rays. Papiliochrome 2a partly overlapped kynurenine. But Papiliochrome 2b could be separated from any other substance. So, from the area of Papiliochrome 2b, the cellulose powder was scraped, and the pigment was extracted with water. After centrifugation, the supernatant fluid contained neither fl-alanine nor SN-1 and was thin-layer chromatographically pure. After the solution was evaporated to dryness under reduced pressure, the residue was dissolved in 10 -3 N HC1, heated at 100°C for 30 min, and again evaporated to dryness under reduced pressure. The residue was dissolved in water, applied again to the T L C cellulose sheets as a streak and developed with BAW. After development, marker strips 2 cm from both edges of the sheets were cut off, and the ninhydrin and phosphomolybdic acid-NH~ tests were made on the strips. Kynurenine and SN-1 were found, and neither fl-alanine nor SN-la was present. From the areas of SN-1 of the sheets,
88
Y. UMEBAem
the cellulose powder was scraped and shaken with water. After centrifugation, the supernatant fluid was evaporated to dryness under reduced pressure. The residue was dissolved in 1 N HC1 and heated under reflux at 100°C for 2 hr, and the solution was evaporated to dryness under reduced pressure. The residue was dissolved in water, applied to the TLC cellulose sheets or Avicel plates, and developed with BAW, EAF, MWP or PhEA. After development, the ninhydrin and phosphomolybdic acid-NH8 tests were made. In all of these thin-layer chromatographies, the presence of •-alanine was confirmed. The presence of SN-la was proved in the chromatographies with BAW and EAF. MWP and PhEA were unsuitable for SN-la.
The presence of fl-alanine in the scales after the extraction of yellow pigments The crude extract from yellow scales was, without any pretreatment, submitted to one-dimensional paper chromatography w~th MWP. In some cases, the yellow scales were shaken in water, and the extract was one-dimensionally chromatographed on the TLC cellulose sheet with BAW. In all these chromatograms, no free fl-alanine was found by the ninhydrin test. Next, the yellow scales were treated successively and repeatedly with hot water (at 50°C) and 70% ethanol (at 45°C). After that, the scales were washed repeatedly with acetone and ethyl ether. The resulting scales, which did not contain the yellow pigments, were hydrolyzed under reflux in 6 N HC1 at 100°C for 24 hr. The hydrolysates were evaporated at 65°C with a rotary evaporator. The residue was dissolved in water and examined for/~-alanine with two-dimensional thinlayer chromatographies of silica gel and Avicel. The solvent for the first direction was MCA and MWP respectively. For the second direction, PhW and BAW were used respectively. Interestingly,/%alanine was detected by the ninhydrin test and confirmed by co-chromatography with authentic/~-alanine. DISCUSSION As described in the previous paper (UMEBACmand YOSmDA, 1970), the yellow pigments, Papilioehrome 2a and 2b, of the wings of P. xuthus, readily decompose to kynurenine and the dopamine derivative SN-1 by being heated in 10 -3 N HC1 at 100°C for 30 min. In fact, the decomposition does not necessitate heating in 10 -3 N HC1. Heating of the pigment solution in water is enough. But, in the latter case, kynurenine is destroyed to some extent. On the other hand, when the pigment solution in 6 N HC1 is heated, SN-1 is broken down. In order to minimize such secondary degradation, the heating in 10 -3 N HC1 was most suitable. Therefore, in the previous and present papers, this condition of decomposition was adopted. The ease of the decomposition suggests the possibility that Papiliochrome 2 may be a molecular complex of kynurenine and a dopamine derivative. In the previous paper (UMEBACHI and YOSaIDA, 1970), some chemical and physical properties of SN-1 were described. They included the colour tests on the paper chromatogram, u.v. spectrum, and the incorporation of l~C-dopamine.
DOPAMINEDEmVATIV~IN p A P ~ u o
xvTnvs
89
From those results, SN-1 was proved to be a dopamine derivative. In the present paper, the purification of SN-1 was made with the DEAE-Cellulose, Amberlite CG-50, and Biogel P-2 columns. From the colour tests, chromatographic behaviour, and u.v. spectrum, it was evident that the SN-1 prepared through these columns was identical with the SN-1 of the previous paper, though the result of the ninhydrin test was a little different, that is, light bluish or negative in the previous paper and slightly brownish or almost negative in the present paper. And Figs. 2a-c indicate that SN-1 is derived from the yellow scales. Moreover, the chromatographic examination of the decomposition-products of the thin-layer chromatographically pure Papiliochrome 2b re-confirmed the previous result that SN-1 was derived from Papiliochrome. From the results of both previous (UMEBACHIand YOSHIDA, 1970) and present papers, there is now no doubt at all that SN-1 is a dopamine derivative. But, SN-1 is not identical with any dopamine derivative which has been reported up to the present. In other words, SN-1 is an unknown derivative of dopamine. The fact that the absorption peak is at 280 nm as in dopa, dopamine, arterenol, and adrenaline suggests that SN-1 may be different from dopamine only in the side chain. The fact that SN-1 is hydrolyzed by being heated in 1 N HC1 at 100°C for 2 hr to yield fl-alanine and the ninhydrin yellow-brown o-diphenolic substance (SN-la) is important. In this regard, the result of the previous paper (UMEBACHI and YOSrlIDA, 1970) must be corrected. In the previous paper, it was reported that, by the hydrolysis in 1 N HC1, SN-1 was changed to an o-diphenol derivative which was purple to the ninhydrin test on the paper chromatogram. This erroneous observation resulted from the fact that, when the hydrolysates in 1 N HC1 were one-dimensionally chromatographed on paper with BAW, SN-la and fl-alanine had almost the same R~ values. The positions of SN-la and fl-alanine overlap on the chromatogram, though the position of the former is a little higher than that~of the latter. But, when the paper chromatogram is run with MWP, SN-la and p-alanine can be clearly separated, because this solvent is not suited for SN-la. In addition, SN-la is yellowish brown to the ninhydrin test on the chromatogram, while fl-alanine is purplish blue to the test. The latter fact is, in the present paper, one of the evidences which support that the N-3 substance obtained after the hydrolysis of SN-1 is fl-alanine, because authentic fl-alanine is neither reddish purple nor purple but purplish blue to the ninhydrin test. From these results, there was no doubt that SN-1 decomposed to fl-alanine and the ninhydrinyellowish brown o-diphenolic substance (SN-la). From the u.v. spectruin, it is probable that SN-la is different from dopamine only in the side chain. The fact that SN-la is yellowish brown to the ninhydrin test suggests that the substance still contains the nitrogen of the side chain of dopamine. In the thin-layer chromatographies with BAW, BHC1, and EAF and the colour reactions, SN-la resembled arterenol very closely. It is probable that SN-la is a dopamine derivative which is very similar to arterenol. But it was evident that SN-la was not adrenaline. In this connection, the report of ANDERSEN(1971) that arterenol
90
Y. UMEBACHZ
can be incorporated into the cuticle, though it is presumably not natural tanning agent is very interesting. LEVENBOOKet al. (1969) reported the presence of/?-alanyl-tyrosine (Sarcophagine) in the larvae of the fleshfly Sarcophaga bullata. This dipeptide seems to be a reservoir of tyrosine in this fly group (BODNARYKand LEVENBOOK,1969 ; BODNARYK, 1972). It seems that, just at the pupation, /~-alanyl-tyrosine is cleaved and is incorporated into the cuticle (BoDNARYK, 1970, 1971a). BOD~ARYK (1971b) reported that, in Sareophagine,/~-alanine is N-terminal and that, in general, when /%alanine is present as a N-terminal amino acid of peptide bond, the bond is hydrolyzed much easier than other peptide bonds. According to his report, in the peptide in which/Lalanine is N-terminal,/%alanine is released by hydrolysis in 2 N HC1 for 2 hr. In this regard, the fact that SN-1 is hydrolyzed in 1 N HC1 at 100°C for 2 hr is interesting. This suggests the possibility that SN-1 may be N-(p-alanyl)-dopamine derivative. The absorption spectrum of SN-1 also is not in conflict with this possibility, because the spectrum suggests that SN-1 may be different from dopamine only in the side chain. The SN-1 contents in wings have been proved to be parallel to the kynurenine contents during the yellow pigment formation. In fact, this result was what was expected. Because, both SN-1 and kynurenine had been presumed to be decomposition-products of the yellow pigments (UMEBACHI and YOSmDA, 1970). And yet, no free SN-1 was found, and free kynurenine was present only in a small quantity. Therefore, the quantitative parallelism of SN-1 and kynurenine during the yellow pigment formation was to be expected. But, anyway, this result is further evidence which indicates that Papiliochrome 2 contains both dopamine derivative and kynurenine as constituents. As to the difference between Papiliochrome 2a and 2b, the possibility that they were a kind of isomer was suggested in the previous paper (UMEBACHIand YOSHIDA,1970). It is interesting that fl-alanine was found in the hydrolysates of the scales from which water-soluble substances including the yellow pigments had been removed. This suggests the presence of protein-bound fl-alanine. Anyway, the relation between such fl-alanine and the fl-alanine as a constituent of the yellow pigments remains to be studied. Up to the present, there have been a considerable number of reports on the presence of fl-alanine in insects. For example, AGRELL (1949) reported the presence of fl-alanine as a free-amino acid in the extracts of whole pupae and pharate adults of Calliphora erythrocephala. CHEN (1959) found flalanine in the hydrolysates of haemolymph protein of Culex pipiens. CHiN (1958) and DUFFY (1964) reported the sexual differences in free fl-alanine content in mosquitoes. Moreover, DENNELL (1958) reported the presence of fl-alanine in the hydrolysates of puparium cuticle of Calliphora vomitoria and mentioned the possibility that this amino acid may be related to the hardening of cuticle. In this regard, Seki and his collaborators' reports on black mutant strains of several species are very interesting. SEKI (1962), FUKUSHIand SEKI (1965), FUKUSHI(1967), and NAKAI (1971) reported that the pupal sheaths of the wild strains of Bombyx mori, Musca domestica, and Drosophila viliris contain fl-alanine and that the black
DOPAMINE DERIVATIVE I N PAPILIO XUTHUS
91
mutants, for example, so of B. mori, bp of M . domestica, and eb of D. virilis lack this amino acid in their pupal sheaths. After or in parallel with these reports, several reports dealing with fl-alanine in the cuticle of insects were published (JAcoBs and BRUBAKER, 1963; JACOBS, 1966; BODNARYK, 1971a; HODGETTS, 1972; ROSS and MONROE, 1972). F r o m these reports, it is probable that fl-alanine is incorporated into cuticle at the time of hardening. Screlotization of cuticle may be related to the following three factors: the amino groups of protein, o-diphanol derivative (for example, N-acetyldopamine), and fl-alanine, though the function of fl-alanine in the hardening process remains to be studied. Now, in the present paper, the yellow pigments of P. xuthus have proved to be related to kynurenine, dopamine derivative, and fl-alanine. So, it can be said that the formation of yellow pigments in P. xuthus bears some resemblance to the hardening of cuticle in insects, at least as far as their constituents concern. I n this regard, the yellow pigments of wings in the Papilionidae are not only a new field of insect pigment but also an interesting subject which may give a clue for elucidating the function of fl-alanine in insects.
REFERENCES
AGRELL I. (1949) Occurrence and metabolism of free amino acids during insect metamorphosis. Acta Physiol. Scan& 18, 247-258. ANDEaS~r S. O. (1970) Isolation of arterenone (2-amino-3',4'-dihydroxyacetophenone) from hydrolysates of sclerotized insect cuticle. J. Insect Physiol. 16, 1951-1959. ANDERSEN S. O. (1971) Phenolic compounds isolated from insect hard cuticle and their relationship to the sclerotization process. Insect Biochem. 1, 157-170. BODNARYK R. P. (1970) Effect of dopa-decarboxylase inhibition on the metabolism of fl-alanyl-L-tyrosine during puparium formation in the fleshfly Sarcophaga bullata Parker. Comp. Biochem. Physiol. 35, 221-227. BODNARYKR. P. (1971a) Studies of the incorporation of fl-alanine into the puparium of the fly, Sarcophaga bullata. J. Insect Physiol. 17, 1201-1210. BODNARYK R. P. (1971b) N-Terminal fl-alanine in the puparium of the fly Sarcophaga bullata: Evidence from kinetic studies of its release by partial acid hydrolysis. Insect Biochem. 1, 228-236. BODNARYKR. P. (1972) A survey of the occurrence of fl-alanyl-tyrosine, 7-glutamyl-phenylalanine, and tyrosine-0-phosphate in the larval stage of flies (Diptera). Comp. Biochem. Physiol. 43B~ 587-592. BODNAR'/K R. P. and LBV~BOOK L. (1969) The role of/~-alanyl-L-tyrosine (Sarcophagine) in puparium formation in the fleshfly Sarcophaga bullata. Comp. Biochem. Physiol. 30, 909-921. CHm~ P. S. (1958) Studies on the protein metabolism of Culex pipiens L.--II. Quantitative differences in free amino acids between male and female adult mosquitoes. J. Insect Physiol. 2, 128-136. CHEN P. S. (1959) Studies on the protein metabolism of Culex pipiens L.--III. A comparative analysis of the protein contents in the larval haemolymph of autogenous and anautogenous forms. J. Insect Physiol. 3, 335-344. DF~NZLL R. (1958) The amino acid metabolism of a developing insect cuticle: the larval cuticle and puparium of Calliphora vomitoria--I. Changes in amino acid composition during development. Proc. R. Soc. (B) 148, 270-279. DUFFY J. P. (1964) Sex differences in the free amino acids of three colonized mosquito species. Ann. ent. Soc. Am. 57, 24-28.
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