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5-thiolhistidine, a new amino acid from eggs of Octopus vulgaris
pp. 843-845, 1985
TIDINE, A NEW AMINC GS OF OCTOPUS VULG F. Rossi,* G. NARDI,* A. PALUMB( and G. PROTAf 2omunale 80121, Napoli, Italy; and tDi r 'Universitd, Via Mezzocannone 16, Napo
?ROM
Chimica Organica
(Received 29 June 1984) Abstraet--l. Eggs of Octopus vulgaris were found to contai contain a new an was isolated and characterized as the corresponding disulpl~hide. 2. Eggs of Loligo vulgaris contain a related metabolite, identified ider as 1-~ amino acid was also detected in urine of Octopus and Loli~~o. 3. The biochemical interest of these amino acids is briefly briefl discussed biogenesis of adenochrome, an unusual iron(III) chelating peptide l fro1
INTRODUCTION eviously we have shown that adenochrome the m(III)-containing pigment from Octopus is a mixre of unusual peptides derived from Gly and three vel isomeric catechol amino acids adenochromines novel A, B, C, responsible for the iron binding properties (Fig. 1) (Ito et al., 1979a). Degradative and model biosynthetic experiments owed that these characteristic components arise showed bioagenetically by addition of a hitherto unknown 5-thiol-L-histidine to dopaquinone formed by tysinase oxidation of dopa (Ito et al., 1979b). roslnase In further experiments with labelled precursors, evidence was obtained that, amon among the adenochrome containing organs of Octopuss vulgaris, only the white bodies are involved in the bios, ~synthesis of the pigment (Nardi et al., 1982). However,'r attempts to detect free 5-thiol-L-histidine(s) in these organs were unsuccessful. abolites (2 and 3) have Recently two related metabolites been isolated by Palumbo ett al. (1984) from several echinoderm species. These results prompted our reinvestigation of the of free occurrence 5-thiol-L-histidine(s) in the eggs, and physiological fluids of cephalopods, namel y Octopus vulgaris which rome and is able to synthesize adenochrome an Loligo vulgaris which is unable.
N
NH _ _
OH
listidine. The same the structure and
;arts.
METHODS
Biolog ical materi~ Spe~ ~ecimens of Octopus vulgaris and Loligo L vulgaris were collect in the Bay of Naples during the collected tl~ breeding season. flied with circulating were kept in aquaria suppli The animals al sea w~ water until required. Eggs were obtained obtai by shaking the ovarie in the sea water and were used fresh or frozen at ovaries 20°C. -
Analytical procedures Ultraviolet absorption spectra were recorded with a Ultz Perkin-Elmer model 552 spectrophototi ~hotometer. Optical rotaPerkin tions were determined with a PerkinPerkin-Elmer polarimeter model 141. NMR spectra were recorded recorde~ in D20 solutions with a Bruker WH-270 spectrometer. Chemical ( shifts are expressed in 6 values using as internal r~ reference the methyl signal of t-butyl alcohol at 6 1.23. Mass l~ spectra were obtained by direct insertion technique with wi an AEI MS-902 spectrometer (70 eV). Amino acid analys'ses were carried out with a Liquimat III (Kontron, Switzerla ;witzerland) using the standard program for physiological fluids. High performance liquid chromatography (HPLC) was carried out on a #-Bondapak C-18 column (3.9mm x 30cm) or a /~-Bondapak C-18 column (7.9 mm x 30 cm) with a Waters Associates Apparatus equipped with a u.v. absorbance detector (model 440). The columns were eluted with 0.05 M ammonium acetate, pH 4.5. Thin laye'er chromatography (TLC) was carried out on cellulose coated c glass plates (Merck, Darmstadt, West Germany) with wit n-butanol-acetic acid-water (60:20:20v/v/v) and/or n- propanol-1M t HCI (3/2 v/v). Low voltage electrophoresis was perfo ~erformed on Whatman No. 3mm paper in a pyridine--~ 'ridine-acetic acid-water (50:2:950v/v/v) buffer, pH 6.5. Chromatograms Chroml and electrophoretograms were examined under u.v. light (254nm) and then sprayed with ninhydrin and/or and/ Pauly reagents. Protein was determined by the method metho~ of Lowry et al. (1951) using crystalline bovine serum albl albumin as a standard.
I~SH/~HOH2H~ OH
NO2C
M,~
iolhistidine, which
Isolation of 5-thiolhistidine (1)from Oct Octopus eggs Fresh collected eggs (1 kg wet wt) fro from Octopus vulgaris Adenochromine A Fig. 1. In adenochromines B and C, tln residues are in position 5,6 and 2,6
were homogenized in 3.01 of ethanol-1 M HC1 (80:20 v/v) np. After centrifugation at ttant was concentrated to a ~thyl ether to remove lipids
F. Rossi et al. as evaporated to dryr and left overnight at vert 5-thiolhistidine(s) ffter acidification, the amn (1.8 x 15cm) of esh). Successive wash21) removed homarine etabolite (2ma, 310nm ne (2~,, 262 nm, ~'max d HC1 gave a further and tryptophan. This :eparative HPLC C-18 ryptophan,
lation of l-methyl-5-thiolhistidine (2)from Loligo eggs Fresh eggs from Loligo vulgaris (700 g wet wt) were naogenized in 2.01 of ethanol 1 M HCI (80:20 v/v) and the ract worked up as described above• The fraction eluted h 4M HCI was further purified by semipreparative bLC chromatography to give, besides a large amount tryptophan (about 50mg), 2.0mg of l-methyl-5~lhistidine (2). This product was identified by comparison h an authentic sample. currence Of 5-thiolhistidine(s) in urine of Octopus and ligo Freshly collected urine (20 ml) was acidified to pH 2.0 to aove insoluble material and after centrifugation at 300g for 15min was passed down a column × 5.0cm) of Dowex. After prolonged washings with O and 0.5 M HC1, the fraction eluted with 4 M HC1 was COllectea lected anti and dried under vacuum. The residue, taken up in olume of water, was analysed by HPLC and TLC for the a volume presence of 5-thiolhistidine(s).
RESULTS
A reinvestigation by H P L C of the free amino acid fraction ~ction from white bodies of Octopus showed a peak at )'max 257 nm typical of 5-thiolhistidine(s). Attempts to characterize this metabolite te were unsuccessful owing to the extremely low concn :n as well as the relatively small size of white bodies. However -Iowever, the same cornpound was found to occur in somewhat greater amount in the ovaries of Octoopus vulgaris from which it could be isolated by combination 4nation of column chromatography on Dowex 50WV and H P L C on Bondapak (see Materials and Methods). This compound, prisms from •om a q / E t O H darkening at 198-200°C, M ÷ found 3722,4261.CIEH~6N604S2, requires M 372,4272; [~]2°0= : +60.26 ° (c = 1.1 HC1 0.1 M), )`max (0.1 M H C 1 ) = 257 nm, (log e = 9.200), was basic in character and, after reduction with •ction with Ellman's reKBH4, gave a positive reaction lm, taken in D20, shows agent. The 1H N M R spectrum, two signals in the sp 3 reg ion at 3.40 (2H x 2, d, J = 7.5 Hz, fl-CH2), 4.27 (1H × 2, t, J = 7.5 Hz, ~-CH) and a low field singlett (1H × 2) at 8.87, typical of H-2 protons of imidazole rings. Formulation of the co~aThese data allowed the formu pound as 5-thiol-L-histidine disulphide (1). / H02C ~/~----~ /S - H2N " N~/NH HNx,~.N 1
HO2C x H2N
)---/
• / CO2H S ~ N H 2 eN,,,,..~N
With this ba of Octopus w~ 5-thiol-L-histid analysis of the on Dowex 50 thiol. thiol-L-histidin )arison wi c°ml; al., 11984). It w~ in be detected d( eggs and uri to eg revealed the 1-me 1-methyl-5-thk gethe ether with lar
ailable, samples of urine ained for the presence of Led metabolites. H P L C Fraction partially purified presence of 1 methyl-5mg/20ml) identified by atic sample (Palumbo et I that neither 1 or 2 could xtension of these studies ~hich lacks adenochrome in both sources of '~2) (0.14mg/20ml) toof tryptophan.
HO2C~_ (Me)2N-leN ,,.~N
DISCUSSION
~rovide evidence for the Th results of this study provide The -thiol-L-histidine (1), the occurrence in Octopus of 5-thiol-L. OCCUl parer ~arent amino acid of the N-methyld derivatives 2 and 3 found fou Although in various species off echinoderms. echinc the isolated is c o m p o u n d corresponds to the characterdenochrome, no specific istic amino : acid found in adenoch relationship seems to exist between the occurrence of ,nthesis of pigment. No5-thiolhistidine and the biosynthesi tably, the l-methyl derivative (2) is i found in Loligo 'nthesize adenochrome. which lacks the ability to synthesi unusual structure of Indeed, it would appear that the un the adenochromines found in the adenochrome of Octopus results primarily from the wide distribution fiolhistidine(s) which can in marine organisms of 5-thiolhistfi in place act as chemical scavengers for dopaquinone dop~ of glutathione and/or cysteine as seen e.g. in the 3as in mammals biosynthesis of glutathionyldop~ ion it is (Prota, 1980). In this connection i noteworthy that glutathione and the ratio between reduced 5-thiol-histidine in sea urchin eggs is 1:60. ~termination was made of In the present study no determin~ the GSH/5-thiolhistidine ratio in adenochromesynthesizing organs, but the occurrence of a (2) in the significant amount of N-methyl derivative de of the parent amino urine suggests a wide distribution ot acid in Octopus as well as in Lolig"O.
CO2H
Acknowledgements--The authors wish to thank Mr Luigi
NH 2
De Martino for technical assistance. This work was supported in part by a research grant from CNR (P.F. Chimica ctral data were provided by assa del CNR e dell 'Univer-
stidine, a new amino acid from eggs of O
ta G. (i 979a) Isolation aae, a unique iron-(III)-
lgaris. J. Chem. Soc. Prota G. (1979b) A sis of adenochromines. r A. L. and Randall R. vith the Folin phenol 75.
Nardi G., Palum white bodies il
Biochem.Phy~
Palumbo A., Mi Prota G. ( 1-methyl-5-thi, metabolite in Physiol.78B, I Prota G. (1980 vertebrates. In by Scheuer), I~
ta G. (1982) The role of the ,'sis of adenochrome. Comp. 300. Eschia M., Donaudy F. and an and distribution of disulphide and a related dnoderms. Comp. Biochem. pigments in marine in-
al Products, Vol. III (edited cademic Press, New York.
TIDINE, A NEW AMINC GS OF OCTOPUS VULG F. Rossi,* G. NARDI,* A. PALUMB( and G. PROTAf 2omunale 80121, Napoli, Italy; and tDi r 'Universitd, Via Mezzocannone 16, Napo
?ROM
Chimica Organica
(Received 29 June 1984) Abstraet--l. Eggs of Octopus vulgaris were found to contai contain a new an was isolated and characterized as the corresponding disulpl~hide. 2. Eggs of Loligo vulgaris contain a related metabolite, identified ider as 1-~ amino acid was also detected in urine of Octopus and Loli~~o. 3. The biochemical interest of these amino acids is briefly briefl discussed biogenesis of adenochrome, an unusual iron(III) chelating peptide l fro1
INTRODUCTION eviously we have shown that adenochrome the m(III)-containing pigment from Octopus is a mixre of unusual peptides derived from Gly and three vel isomeric catechol amino acids adenochromines novel A, B, C, responsible for the iron binding properties (Fig. 1) (Ito et al., 1979a). Degradative and model biosynthetic experiments owed that these characteristic components arise showed bioagenetically by addition of a hitherto unknown 5-thiol-L-histidine to dopaquinone formed by tysinase oxidation of dopa (Ito et al., 1979b). roslnase In further experiments with labelled precursors, evidence was obtained that, amon among the adenochrome containing organs of Octopuss vulgaris, only the white bodies are involved in the bios, ~synthesis of the pigment (Nardi et al., 1982). However,'r attempts to detect free 5-thiol-L-histidine(s) in these organs were unsuccessful. abolites (2 and 3) have Recently two related metabolites been isolated by Palumbo ett al. (1984) from several echinoderm species. These results prompted our reinvestigation of the of free occurrence 5-thiol-L-histidine(s) in the eggs, and physiological fluids of cephalopods, namel y Octopus vulgaris which rome and is able to synthesize adenochrome an Loligo vulgaris which is unable.
N
NH _ _
OH
listidine. The same the structure and
;arts.
METHODS
Biolog ical materi~ Spe~ ~ecimens of Octopus vulgaris and Loligo L vulgaris were collect in the Bay of Naples during the collected tl~ breeding season. flied with circulating were kept in aquaria suppli The animals al sea w~ water until required. Eggs were obtained obtai by shaking the ovarie in the sea water and were used fresh or frozen at ovaries 20°C. -
Analytical procedures Ultraviolet absorption spectra were recorded with a Ultz Perkin-Elmer model 552 spectrophototi ~hotometer. Optical rotaPerkin tions were determined with a PerkinPerkin-Elmer polarimeter model 141. NMR spectra were recorded recorde~ in D20 solutions with a Bruker WH-270 spectrometer. Chemical ( shifts are expressed in 6 values using as internal r~ reference the methyl signal of t-butyl alcohol at 6 1.23. Mass l~ spectra were obtained by direct insertion technique with wi an AEI MS-902 spectrometer (70 eV). Amino acid analys'ses were carried out with a Liquimat III (Kontron, Switzerla ;witzerland) using the standard program for physiological fluids. High performance liquid chromatography (HPLC) was carried out on a #-Bondapak C-18 column (3.9mm x 30cm) or a /~-Bondapak C-18 column (7.9 mm x 30 cm) with a Waters Associates Apparatus equipped with a u.v. absorbance detector (model 440). The columns were eluted with 0.05 M ammonium acetate, pH 4.5. Thin laye'er chromatography (TLC) was carried out on cellulose coated c glass plates (Merck, Darmstadt, West Germany) with wit n-butanol-acetic acid-water (60:20:20v/v/v) and/or n- propanol-1M t HCI (3/2 v/v). Low voltage electrophoresis was perfo ~erformed on Whatman No. 3mm paper in a pyridine--~ 'ridine-acetic acid-water (50:2:950v/v/v) buffer, pH 6.5. Chromatograms Chroml and electrophoretograms were examined under u.v. light (254nm) and then sprayed with ninhydrin and/or and/ Pauly reagents. Protein was determined by the method metho~ of Lowry et al. (1951) using crystalline bovine serum albl albumin as a standard.
I~SH/~HOH2H~ OH
NO2C
M,~
iolhistidine, which
Isolation of 5-thiolhistidine (1)from Oct Octopus eggs Fresh collected eggs (1 kg wet wt) fro from Octopus vulgaris Adenochromine A Fig. 1. In adenochromines B and C, tln residues are in position 5,6 and 2,6
were homogenized in 3.01 of ethanol-1 M HC1 (80:20 v/v) np. After centrifugation at ttant was concentrated to a ~thyl ether to remove lipids
F. Rossi et al. as evaporated to dryr and left overnight at vert 5-thiolhistidine(s) ffter acidification, the amn (1.8 x 15cm) of esh). Successive wash21) removed homarine etabolite (2ma, 310nm ne (2~,, 262 nm, ~'max d HC1 gave a further and tryptophan. This :eparative HPLC C-18 ryptophan,
lation of l-methyl-5-thiolhistidine (2)from Loligo eggs Fresh eggs from Loligo vulgaris (700 g wet wt) were naogenized in 2.01 of ethanol 1 M HCI (80:20 v/v) and the ract worked up as described above• The fraction eluted h 4M HCI was further purified by semipreparative bLC chromatography to give, besides a large amount tryptophan (about 50mg), 2.0mg of l-methyl-5~lhistidine (2). This product was identified by comparison h an authentic sample. currence Of 5-thiolhistidine(s) in urine of Octopus and ligo Freshly collected urine (20 ml) was acidified to pH 2.0 to aove insoluble material and after centrifugation at 300g for 15min was passed down a column × 5.0cm) of Dowex. After prolonged washings with O and 0.5 M HC1, the fraction eluted with 4 M HC1 was COllectea lected anti and dried under vacuum. The residue, taken up in olume of water, was analysed by HPLC and TLC for the a volume presence of 5-thiolhistidine(s).
RESULTS
A reinvestigation by H P L C of the free amino acid fraction ~ction from white bodies of Octopus showed a peak at )'max 257 nm typical of 5-thiolhistidine(s). Attempts to characterize this metabolite te were unsuccessful owing to the extremely low concn :n as well as the relatively small size of white bodies. However -Iowever, the same cornpound was found to occur in somewhat greater amount in the ovaries of Octoopus vulgaris from which it could be isolated by combination 4nation of column chromatography on Dowex 50WV and H P L C on Bondapak (see Materials and Methods). This compound, prisms from •om a q / E t O H darkening at 198-200°C, M ÷ found 3722,4261.CIEH~6N604S2, requires M 372,4272; [~]2°0= : +60.26 ° (c = 1.1 HC1 0.1 M), )`max (0.1 M H C 1 ) = 257 nm, (log e = 9.200), was basic in character and, after reduction with •ction with Ellman's reKBH4, gave a positive reaction lm, taken in D20, shows agent. The 1H N M R spectrum, two signals in the sp 3 reg ion at 3.40 (2H x 2, d, J = 7.5 Hz, fl-CH2), 4.27 (1H × 2, t, J = 7.5 Hz, ~-CH) and a low field singlett (1H × 2) at 8.87, typical of H-2 protons of imidazole rings. Formulation of the co~aThese data allowed the formu pound as 5-thiol-L-histidine disulphide (1). / H02C ~/~----~ /S - H2N " N~/NH HNx,~.N 1
HO2C x H2N
)---/
• / CO2H S ~ N H 2 eN,,,,..~N
With this ba of Octopus w~ 5-thiol-L-histid analysis of the on Dowex 50 thiol. thiol-L-histidin )arison wi c°ml; al., 11984). It w~ in be detected d( eggs and uri to eg revealed the 1-me 1-methyl-5-thk gethe ether with lar
ailable, samples of urine ained for the presence of Led metabolites. H P L C Fraction partially purified presence of 1 methyl-5mg/20ml) identified by atic sample (Palumbo et I that neither 1 or 2 could xtension of these studies ~hich lacks adenochrome in both sources of '~2) (0.14mg/20ml) toof tryptophan.
HO2C~_ (Me)2N-leN ,,.~N
DISCUSSION
~rovide evidence for the Th results of this study provide The -thiol-L-histidine (1), the occurrence in Octopus of 5-thiol-L. OCCUl parer ~arent amino acid of the N-methyld derivatives 2 and 3 found fou Although in various species off echinoderms. echinc the isolated is c o m p o u n d corresponds to the characterdenochrome, no specific istic amino : acid found in adenoch relationship seems to exist between the occurrence of ,nthesis of pigment. No5-thiolhistidine and the biosynthesi tably, the l-methyl derivative (2) is i found in Loligo 'nthesize adenochrome. which lacks the ability to synthesi unusual structure of Indeed, it would appear that the un the adenochromines found in the adenochrome of Octopus results primarily from the wide distribution fiolhistidine(s) which can in marine organisms of 5-thiolhistfi in place act as chemical scavengers for dopaquinone dop~ of glutathione and/or cysteine as seen e.g. in the 3as in mammals biosynthesis of glutathionyldop~ ion it is (Prota, 1980). In this connection i noteworthy that glutathione and the ratio between reduced 5-thiol-histidine in sea urchin eggs is 1:60. ~termination was made of In the present study no determin~ the GSH/5-thiolhistidine ratio in adenochromesynthesizing organs, but the occurrence of a (2) in the significant amount of N-methyl derivative de of the parent amino urine suggests a wide distribution ot acid in Octopus as well as in Lolig"O.
CO2H
Acknowledgements--The authors wish to thank Mr Luigi
NH 2
De Martino for technical assistance. This work was supported in part by a research grant from CNR (P.F. Chimica ctral data were provided by assa del CNR e dell 'Univer-
stidine, a new amino acid from eggs of O
ta G. (i 979a) Isolation aae, a unique iron-(III)-
lgaris. J. Chem. Soc. Prota G. (1979b) A sis of adenochromines. r A. L. and Randall R. vith the Folin phenol 75.
Nardi G., Palum white bodies il
Biochem.Phy~
Palumbo A., Mi Prota G. ( 1-methyl-5-thi, metabolite in Physiol.78B, I Prota G. (1980 vertebrates. In by Scheuer), I~
ta G. (1982) The role of the ,'sis of adenochrome. Comp. 300. Eschia M., Donaudy F. and an and distribution of disulphide and a related dnoderms. Comp. Biochem. pigments in marine in-
al Products, Vol. III (edited cademic Press, New York.