Isolation of a possible biosynthetic precursor of adenochrome from the white bodies of Octopus vulgaris

Comp. Biochem. Physiol., 1977. Vol. 58B. pp. 353 to 356. Pergamon Press. Printed in Great Britain

ISOLATION OF A POSSIBLE BIOSYNTHETIC PRECURSOR OF ADENOCHROME FROM THE WHITE BODIES OF OCTOPUS VULGARIS ANNA PALUMBO, SBOSUKE ITO, GIOVANNANARDI AND GIUSEPPE PROTA Stazione Zoologica di Napoli, 80121 Napoli, and Istituto di Chimica delrUniversit~ di Napoli, Via Mezzocannone 16, Napoli, Italy

(Received 21 February 1977) Abstract--1. Adenochrome, the red iron-containing pigment found originally in the branchial hearts of Octopus vulgaris, was isolated in the form of desferriadenochrome (DFA) from gills, hepatopancreas, ovaries after egg deposition, and amoebocytes of the animal. 2. Methods for identification of DFA included u.v. spectroscopy, paper electrophoresis and acid degradation. 3. The white bodies were found to contain" a more acid DFA-analogue (DFA-WB), identified as DFA-Asp2, which, by partial hydrolysis, was readily converted to authentic DFA by loss of the two aspartic acid residues. 4. A possible role of white bodies and amoebocytes in the biosynthesis and circulation of adenochrome in Octopus trulgaris is discussed.

and chromatographic properties (Ito et al., 1976; Prota et al., 1976). Extensive degradative studies have revealed that DFA is an inseparable mixture of closely related peptides which consist of 2 moles of glycine and 1 mole of one of three novel isomeric amino acids, the adenochromines A, B and C (Fig. 1), differing in the positions of the two 5-thiolhistidine residues on the dopa moiety (Ito et al., 1976; Prota et al., 1976). Adenochromines are the amino acids responsible for the iron-binding properties of DFA and probably arise biogeneticaUy by addition of 5-thiolhistidine to dopaquinone derived from tyrosinase oxidation of tyrosine. With this background available, a study was undertaken to establish the structural relationship between the adenochrome from the branchial hearts and similar pigments previously found in other organs of Octopus vuh3aris (Nardi & Steinberg, 1974). The possible chemical identity of all these adenochromes would imply the existence of a biological mechanism of transport from a site of biosynthesis throughout the body.

INTRODUCTION

A considerable variety of low molecular weight ironbinding metabolites, known as siderochromes, are produced by micro-organisms, in which they play an important role in the transport of iron (Neilands, 1972). A rare example of such substances in the animal kingdom is represented by adenochrome, a red iron (III)-containing pigment of unknown physiological significance, occurring as intracellular granules in the branchial hearts of some Octopus species (Curnot et al., 1908; Turchini, 1923; Bacq & Leiner, 1935; Fox & Uppdegraff, 1943; Nardi & Steinberg, 1974). Distinguishing features of this pigment include an amphoteric character, insolubility near neutrality, high instability in alkaline media and a broad absorption band around 505 nm. On account of its intractable nature, chemical investigation on adenochrome has proceeded very slowly, and only recently it has been isolated from branchial hearts of Octopus vulgaris as the colorless iron-free form, desferriadenochrome (DFA), displaying more favourable solubility

N,~'~,NR HIN / ' ~ S . . . ~ .OH

H,N C0,H

F~0H RN._._.~

"",,T.NH2 COz H

H02C NHz

" ' T " NH2 COzH

Adenochromine A

Secoadenochromine A

Fig. 1. In adenochromines B and C, the 5-thioihistidine residues are in posifons 5,6 and 2,6, respectively. In secoadenochromines B and C, the 5-thiolhistidine residue is in position 6 and 2, respectively. 353

354

ANNA PALUMBOet al. MATERIALS AND METHODS

(a) Bioloftical material Specimens of Octopus vulgaris were collected in the Bay of Naples. Branchial hearts, white bodies, gills, kidneys, hepatopancreas, ovaries after egg deposition, skin, eyes, branchial glands, amoebocytes and hemolymph were used fresh or frozen at -20°C. (b) Analytical procedures Ultra-violet spectra were recorded with a Beckman model 25 spectrophotometer. Amino acid analyses were carried out with a Beckman model 120 B amino acid analyzer. Thin-layer chromatography (TLC) was carried out on cellulose coated glass plates (Merck, Darmstadt, West Germany) using as solvent n-propanol-1 M HC1 (3:2, v/v). Low voltage electrophoresis was performed on Whatman no. 3 MM paper in a pH 6.5 buffer, pyridine-acetic acidwater (50:2:950, v/v). Chromatograms and electrophoretograms were examined under a u.v. lamp (254 nm) and then sprayed with either ninhydrin solution, ferric chloride reagent (1% ferric chloride in 95% ethanol followed by ammonia vapour), or Pauly reagent. (c) General procedure for extraction of desferriadenochrome (DFA) from tissues Whenever possible, 20 g of tissues were homogenized in 200 ml of 0.5 M HCIO 4 containing 2 ml of 80% thioglycolic acid, and extracted for 1 hr under vigorous stirring. After centrifugation at 17,300g for 15 min, the supernatant was adjusted to pH 1.5 with solid KHCOa. The precipitated KCIO 4 was removed by centrifugation and the supernatant was concentrated to ca. 10 ml under reduced pressure and clarified by a further centrifugation to give a clear yellow solution. When the extract was cloudy due to lipids (or lipoproteins), it was extracted with chloroform before the final centrifugation; the lipids floated on the boundary between the aqueous and the chloroform layers. The aqueous solution was chromatographed on a column (3.5 x 85cm) of Sephadex G-10 fPharmacia, Uppsala, Sweden) using 0.02 M HC1 as the eluent. Fractions of ca. 10ml were collected and monitored spectrophotometrically at 305 nm. Fractions containing DFA (in general, fractions 22-26) were combined and evaporated to dryness at 40~C. The residue, taken up in 0.02 M HCI (7-8 ml), was centrifuged to remove insoluble materials and chromatographed on a column (2.5 x 93 cm) of Sephadex LH-20, developed with 0.02 M HC1 at a flow rate of 60 ml/hr. Fractions of ca. 10ml were collected. DFA generally emerged in fractions 20-22, which were combined and evaporated to dryness. The preparations of DFA by this procedure contained impurities, mostly proteins, the amounts of which varied from one organ to another. The yields of DFA obtained from the various tissues examined were determined spectrophotometrically and are reported in Table 1. (d) Purification of DF A-WB from white bodies When examined by paper electrophoresis, the DFA fraction (84 nag) obtained from white bodies (20g) as above, was found to contain, in addition to traces of the usual DFA, a major, more acidic, component designated as DFA-WB. The mixture was then fractionated by ion exchange chromatography on a column (0.7 x 54cm) of Dowex 50 W-X8 (200-400 mesh) developed at a flow rate of 26 ml/hr with 1 M pyridine acetate buffer, pH 4.5, containing 0.2% mercaptoethanol to minimize oxidation of the substance. Fractions of 4 ml were collected and monitored spectrophotometrieally at 305 rim. Fractions 5-7, 8-16 and 19-36 were combined separately and concentrated to small volumes under reduced pressure. Addition of acetone to these solutions gave DFA as an

amorphous precipitate which was collected by centrifugation, washed twice with acetone, and eventually dried in a desiccator. Thus, fractions 5-7 gave 11 nag of oxidized DFA-WB, 8-16 20 mg of pure DFA-WB and 17-36 6 mg of DFA-WB contaminated with peptides, as evidenced by spectrophotometric and electrophoretic analyses, as well as by amino acid analysis. Subsequent elution of the column with 2 M pyridine acetate buffer, pH 5, yielded a further fraction with ~,m~ at 305 nm, containing ca. 4 mg of DFA, identical in all respects (electrophoresis, acid degradations) to DFA typical of branchial hearts. (e) Conversion of DFA-WB into DFA by partial hydrolysis A solution of DFA-WB (8 nag) in 4 ml of 0.03 M HC1 was heated at 105°C for 24 hr in an evacuated, sealed tube. The hydrolysate was evaported at 40°C, taken up in 0.02 M HCI, and chromatographed on a column (1.5 x 78 cm) of Sephadex G-10, using 0.02 M HCI as the eluent. Fractions of 4 ml were collected and monitored spectrophotometrically. Fractions 14-16 and 21-25 containing DFA and aspartic acid, respectively, were combined separately and evaporated to dryness under reduced pressure. The yield of DFA determined spectrophotometrically at 305 nm was 5 mg. (f) Acid degradations For the characterization of the isolated DFA or DFA-WB two degradative procedures were used under the following conditions: 1. HCI hydrolysis. A sample of DFA or DFA-WB (2-5 rag) was heated at 110°C for 24 hr with 1 ml of 6 M HCI containing 10#1 of 80% thioglycolic acid in an evacuated sealed tube. 2. HI reductive hydrolysis. A mixture of DFA or DFA-WB (2-5 rag) and red phosphorus (4-8 nag) in 1 ml of freshly distilled 57% HI was heated at 110°C for 48 hr in an evacuated, sealed tube. In both cases, the reaction mixture was evaporated to dryness under reduced pressure and the residue, taken up in 0.1 M (0.5 ml) was analyzed by TLC and/or amino acid analysis. RESULTS

Preliminary experiments revealed that adenochrome may occur in various organs in both forms--ferr/- and desferriadenochrome (FA and D F A ) - - i n a ratio depending on the local iron concentration. Typically, the branchial hearts, containing iron between 0.1 and 0.3% of dry weight (Nardi & Steinberg, 1974) appear brownish purple as they are heavily loaded with ferriadenochrome, whereas the white bodies, which normally have little or no iron (Muzii, unpublished results), contain predominantly desferriadenochrome. Because of the usual coexistence of the iron-bound and iron-free forms, determination of adenochrome content in different tissues was conveniently carried out using an isolation procedure involving the complete conversion of the material into a single form. This was achieved by direct extraction of tissues with 0.5 M H C I O 4 containing thioglycolic acid which reduces Fe(III) to Fe(II), thus converting FA into DFA. Subsequenfly, the extract is chromatographed on Sephadex G-10 and the fraction containing DFA, separated from salts and other low molecular weight compounds, is further purified by chromatography on Sephadex LH-20. Although D F A preparations thus obtained invariably contained impurities, mainly pepfides, this caused few problems for

Isolation of a possible biosynthetic precursor of adenochrome Table 1. Adenochrome distribution in Octopus tmlgaris Adenochrome content*

Source

(mUg wet wt~"

Branchial hearts White bodies Ovaries (after egg deposition) Gills Hepatopancreas

12

1.7t 0.52 0.14 Traces 0.24

Amoeboeytes

Hemolymph Branchial glands§ Kidneys§ Skin Eyes * Determined as DFA. t A solution of 1.0mg of DFA in lml of 0.1M HCI has an absorbance (at 305 nm) 1.8. In the form of DFA-Asp2 (DFA-WB). § The discrepancy between these results and the previously reported occurrence of adenochrome in these organs (Nardi & Steinberg, 1974) may be attributable to individual variations. their characterization which was based upon comparison of the physical as well as chemical properties with those of the authentic DFA extracted from branchial hearts and extensively purified by ion exchange chromatography (Prota et al., 1976). The occurrence and yields of DFA from the organs examined are reported in Table 1. When examined on paper electrophoresis at pH 6.5, the DFAs from amoebocytes, ovary after egg deposition, gills and hepatopancreas gave single spots positive both to FeCI3 and to ninhydrin, with mobilities identical to the DFA from branchial hearts. They also exhibited the typical u.v. spectrophotometric behaviour of authentic DFA characterized bY an absorption maximum around 306 nm (in 0.1 M HCI) shifting to ca. 320 nm at pH 10. Conclusive evidence for their identity was derived from the identification of the products obtained by acid degradation. Analogous to DFA from branchial heart (Ito et al., 1976; Prota et al., 1976) all the DFAs from other organs gave, on acid hydrolysis with 6 M HCI, glycine, adenochromine and secoadenochromine. Moreover, on reductive hydrolysis with 57% HI, they afforded, as expected (Ito et al., 1976) glycine, dopa and 5-thiolhistidine. As shown in Fig. 2, electrophoresis of preparations from white bodies revealed, in addition to some DFA, a major band, positive to FeC13, moving toward the anode. This DFA analogue, designated as DFA-WB (desferriadenochrome from white bodies), was then further purified by ion exchange chromatography on Dowex 50. It emerged from the column at pH 4.5,

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355

ahead of the small quantity of DFA which was eluted at pH 5.0. On hydrolysis in 6 M HCI DFA-WB yielded adenoehromine, secoadenochromine, 5-thiolhistidine, glycine and aspartic acid, the ratio between glycine and aspartic acid being 1:1 as determined by amino acid analysis. Reductive hydrolysis in 579/0 HI yielded dopa, 5-thiolhistidine, glycine and aspartic acid in the ratio 1:2:2:2. These results indicated that DFA-WB differed from DFA by two aspartic acid residues, which is consistent with its more acidic behaviour in electrophoresis and ion exchange chromatography. Since peptides containing aspartic acid are known to be cleaved preferentially at peptide bonds of aspartic acid by heating in 0.03 M HC1 (Schultz, 1967), DFA-WB was hydrolyzed under these conditions. Fractionation of the reaction mixture on Sephadex G-10 column gave the expected aspartic acid and the shortened peptide identified as DFA by paper electrophoresis. Moreover, no aspartic acid was detected among the products of 6 M HC1 hydrolysis of DFA obtained from DFA-WB. DISCUS~ON The results of this study provide evidence that the same adenochrome found in large amounts in the branchial hearts occurs also in gills, ovaries after egg deposition, hepatopancreas and amoebocytes, the latter being the circulating cells of hemolymph. With regard to distribution a unique position is held by white bodies which contain the analogue, DFA-WB, consisting of a DFA unit linked to two aspartic acid residues through peptide bonds. The easy conversion of DFA-WB to DFA by partial hydrolysis suggests that the former represents a possible biosynthetic precursor of the adenochrome distributed throughout the organism. This view would indicate the white bodies as an important site in the biosynthesis of adenochrome, and this would imply also the existence of a mechanism of transport accounting for its wide distribution thoughout the body. Since neither DFA-WB nor DFA are apparently present in the hemolymph, the circulation of adenochrome could take place through the amoebocytes which are indeed reported as originating from the white bodies (Cowden, 1972). Such a mechanism would require that, at some stage of the cellular differentiation from immobile preamoebocytes in the white bodies to mature circulating amoebocytes, a biochemical change takes place in these cells, by which either the ability to convert DFA-WB to DFA, or to synthesize DFA in place of DFA-WB is acquired. Acknowledgements--The authors wish to thank Mr Vincenzo Saggiomo for his skilful technical assistance. This work was in part supported by Consigiio Nazionale delle Ricerche. REFERENCES

Fig. 2. Paper electrophoresis at pH 6.5 of DFA preparations from: 1. white bodies; 2. branchial hearts; 3. amoebocytes; 4. ovaries; 5. gills; 6. hepatopancreas. Stain: ferric chloride solution.

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