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Identity of blue pigments obtained from different tissues of the sculpin, Pseudoblennius percoides Günther
Comp. Biochem. Physiol., 1976, Vol. 55B.pp. 85 to 87. Pergamon Press. Printed in Great Britain
IDENTITY OF BLUE PIGMENTS OBTAINED FROM DIFFERENT TISSUES OF THE SCULPIN, PSE UDOBLENNIUS PERCOIDES GUNTHER KATSUMI YAMAGUCHI, KANEHISA HASHIMOTO AND FUMIO MATSUURA* Laboratory of Marine Biochemistry, Faculty of Agriculture, The University of Tokyo, Tokyo, Japan (Received 24 N o v e m b e r 1975)
Blue chromoproteins were isolated from the skin, muscle, digestive tract and eggs of the sculpin, Pseudoblennius percoides Giinther. 2. They showed the same absorption spectrum and almost the identical molecular weights (38,000-42,000) and isoelectric points (3.4-3.7). 3. The identity of these chromoproteins was confirmed by isoelectric focussing with mixed samples. 4. Their chromophores were identified as being solely biliverdin IX~. 5. It may be concluded that one and the same biliverdin-protein exists in the different tissues of this fish. Abstract--1.
INTRODUCTION
The sculpin, Pseudoblennius percoides Giinther, living in the coastal waters of Japan, displays light blue to deep blue--green pigmentation not only in the skin but in the muscle and the digestive tract, and also in the eggs. This paper deals with isolation and comparison of these pigments from different tissues of the fish.
Sephadex thin layer method (Morris, 1964). The measurement of the isoelectric point was performed with an LKB isoelectric focussing apparatus model 8100. The chromic acid-dichromate degradation method (Riidiger, 1969; Rtidiger & O'Carra, 1969) was used for identification of the chromophores. Biliverdin IX~ was prepared from a commercial bilirubin according to the method of Tixier (1945) and purified by silicic acid column chromatography with a solvent system, methylethylketone/water/acetic acid (100:10: 1, by vol).
MATERIALS AND METHODS
RESULTS
Three sculpins were caught with rod and line off Kanagawa Prefecture, Japan. Two were males weighing 50 and 20g. The 50,g fish showed light blue pigmentation over the skin, muscle and digestive tract, whereas the 20 g one was almost colourless in the skin but deep blue-green in the muscle and digestive tract. The remaining fish was a female weighing 35 g and displaying a light blue skin and digestive tract along with blue-green eggs and almost colourless muscle. The skin, muscle, digestive tract and eggs were removed from the three individuals. Each tissue sample was cut into small pieces, minced in a mortar with cold distilled water, and centrifuged. The pigment that passed into the aqueous layer was then fractionated with ammonium sulphate, precipitation occurring between 40 and 80% saturation. The precipitates were collected by suction, dissolved in a small volume of cold distilled water and dialysed overnight against 0.04 M phosphate buffer, pH 6.8. When subjected to further purification by starch block electrophoresis (Kunkel, 1954) in the same buffer, the pigment, irrespective of the tissue, migrated anodally as a single narrow band. The pigmented zone was removed, extracted with, and finally dialysed against, cold distilled water to obtain a purified blue chromoprotein. All the purification procedures were carried out in a semi-dark cold room at 2-4°C. The u.v.-visible absorption spectrum of each chromoprotein was measured with a Cary 14 scanning spectrophotometer. The molecular weight was determined by the
The blue chromoproteins purified from the different tissues showed an identical absorption spectrum revealing a characteristic maximum at 670 nm and a dull one at 268-70 nm together with an inflexion at 328-9 nm (Fig. 1). The extinction ratios, E 2 7 o / E 6 7 o , ranged between 1.55 and 1.60.
0.31-70 O.I
200
3o0
I 400
I 500
I 600
I 700
Wovelengfh, nrn * Present address: Sch. of Fish. Sci., Kitasato Univ., Sanriku, Iwate, Japan.
Fig. 1. Absorption spectrum of the purified blue chromoprotein of sculpin in 0.1 M phosphate buffer, pH 7.0. 85
KATSUMIYAMAGUCHLKANEHISAHASHIMOTOAND FUMIOMATSUURA
86
Table 1. Molecular weight and isoelectric point of blue chromoproteins purified from various tissues of the sculpin Tissues
Molecular weight
IX~ and it may be concluded that one and the same biliverdin-protein is distributed widely within the body of this species of fish.
Isoelectric point
DISCUSSION Skin
40,000
3.6
Muscle
41,000
3.4
Digestive tract
38~000
3.7
Eggs
42,000
3.6
Each flqure indicates mean of tWO determinations.
As shown in Table 1 the molecular weights and isoelectric points exhibit no marked differences between the chromoproteins of the different tissues. The identity of the chromoproteins was further established by isoelectric focussing of a mixture consisting of equal amounts of the four chromoproteins from different origins; only a single band appeared at isoelectric point 3.6. On account of the small quantities of chromoprotein available for the identification of the chromophores, the chromic acid-dichromate micro-degradation technique, which enables direct degradation of the chromophores while still attached to the proteins, was used. In chromic acid degradation, dark blue spots of methyl vinyl maleimide and haematinic acid appeared on the thin layer chromatogram and in the dichromate degradation orange-red spots of 3-methyl-4-carboxyethylpyrrole-dialdehyde(2,5) appeared in all the four chromoproteins of the different tissues. Such a result was indistinguishable from that of the biliverdin IX, used as a control (Fig. 2). Thus the chromophores were identified exclusively as biliverdin
It has long been known that some species of the family Cottidae exhibit blue to blue green colours in the skin, oral tegument, bone and serum (Willstaedt, 1941; Augustinsson, 1959). Bada (1970) isolated a blue green pigment from the blood plasma of the Arctic sculpin, Myoxocephalus scorpioides, and characterized it as a biliprotein of which absorption spectrum had only one maximum at 658--60nm in the visible region. The isoelectric point and molecular weight were found to be 3.1 and ~46,000, respectively. Bada (1970) also noted the presence of the identical biliprotein in the skin. Subsequently, in the sera of several species of cottids, Low & Bada (1974) have reported that occurrence of green, blue, lavender and red chromoproteins of which the chromophores are biliverdin and/or an unidentified biladiene. The same pigments were manifested in the cartilages of the mouth and gills and in the skin. The present authors noticed by chance that the eggs of cabezon, Scorpaenichthys marmoratus, one of the family Cottidae, are of a red-purple colour supposedly due to the chromoprotein(s) found in the serum (Low & Bada, 1974). Such accumulation of knowledge along with the present results strongly suggests that the presence of biliproteins, mainly biliverdin-protein, in cottids might be a fairly widespread phenomenon. Occurrence of biliverdin protein in fish is not limited to cottids but has been demonstrated in the serum of eel, Anyuilla japonica (Yamaguchi et al., 1966), in the fins of wrasse, Crenilabrus pavo C.V. (Abolin~ & RiJdiger, 1966: AbolinL 1970) and in the muscle of Napoleon fish (also called "hirosa"), Cheilinus undulatus RiJppell (Yamaguchi & Matsuura. 1969). The physiological function of these biliproteins is still a matter of speculation as already remarked by
(a)
(b)
0.5 H r
0
Q
0
0
COOH
0 CO0 H
OH~HO
I e I
H
H
@ ~
@ •
2
4
3
.5
9I ¢ ? 2¢ ?
3
4
5
Fig. 2. Typical thin layer chromatograms of imides (a) and pyrrole
Blue pigments obtained from different tissues of the sculpin Low & Bada (1974). However; judging from the fact that the same biliverdin-protein exists in various tissues of the sculpin, it may be suggested that biliverdin, perhaps derived from haemoglobin and myoglobin a n d linked with a protein for protection from isomerization a n d oxidation, is stored temporarily in various parts of the body a n d subsequently at an o p p o r t u n e m o m e n t transported via the serum to the skin and eggs for the purpose of camouflage or sexual dimorphism, as a protector against u.v. rays or as a photoreceptor for hatching and so on.
Acknowledgements--We wish to express our sincere thanks to Prof. Y. Hashimoto of the The Univ. of Tokyo for supply of the sculpins which he himself fished up, and to Dr. T. Abe, Tokai Reg. Fish. Res. Lab. for the identification of the fish. Cordial thanks are also due to Dr, R. M. Love, Torry Research Station, Aberdeen, Scotland for his kind revision of this manuscript. This work was supported in part by a grant from the Ministry of Education, Japan. REFERENCES
ABOLINg L. (1970) The preparation and identification of the chromoprotein Crenilabrus blue. Pubbl. Staz. Zool. Napoli 3g, 229-248. ABOLINg L. & Ri~DIGER W. (1966) Uber die farbgebende Gruppe yon Crenilabrus-Blau. Experientia 22, 298-299. AUGUSTINSSON K.-B. (1959) Electrophoresis studies on blood plasma esterases. Acta Chem. Scand. 13, 1097-1105.
87
BADA J. L. (1970) A blue-green pigment isolated from blood plasma of the Arctic sculpin (Myoxocephalus scorpioides). Experientia 26, 251-252. KUNKEL H. G. (1954) Zone electrophoresis. In Methods of Biochemical Analysis Vol. 1 (Edited by GLICK D.) pp. 141-170, Interscience, New York. Low P. S. & BAOA J. L. (1974) Bile pigments in the blood serum of fish from the family Cottidae. Comp. Biochem. Physiol. 47A, 411-418. MORRIS C. J. O. R. (1964) Thin-layer chromatography of proteins on Sephadex G-100 and G-200. J. Chromatogr. 16, 167-175. R~DIGER W. 0969) Chromsgure- und Chromatabbau yon Gallenfarbstoffen. Hoppe-Seyler's Z. Physiol. Chem. 350, 1291-1300. Ri~DIGER W. & O'CARRA P. (1969) Studies on the structures and apoprotein linkages of the phycobilins. Eur. J. Biochem. 7, 509-516. TIXlER R. (1945) Contribution a l'6tude de quelques pigments pyrro!iques naturels des coquilles de Mollusques, de l'oeuf d'Emeu et du squelette du corail bleu (Heliopora caerulea). Ann. Inst. ocdanogr. Monaco 22, 343-397. WILLSTAEDTH. (1941) Zur Kenntnis der griinen Farbstoffe von Seefischen. Enzymologia 9, 260-264. YAMAGUCHI K., KOCHIYAMA Y., HASHIMOTO K. & MATSUURA F. (1966) Studies on a blue-green serum pigment of eel--II. Identification of prosthetic group. Bull. Japan. Soc. Sci. Fish. 32, 873-879. YAMAGUCHI K. & MATSUURA F. (1969) A blue pigment from the muscle of a marine teleost, "hirosa", Cheilinus undulatus Riippell. Bull. Japan. Soc. Sci. Fish. 35, 920-926.
IDENTITY OF BLUE PIGMENTS OBTAINED FROM DIFFERENT TISSUES OF THE SCULPIN, PSE UDOBLENNIUS PERCOIDES GUNTHER KATSUMI YAMAGUCHI, KANEHISA HASHIMOTO AND FUMIO MATSUURA* Laboratory of Marine Biochemistry, Faculty of Agriculture, The University of Tokyo, Tokyo, Japan (Received 24 N o v e m b e r 1975)
Blue chromoproteins were isolated from the skin, muscle, digestive tract and eggs of the sculpin, Pseudoblennius percoides Giinther. 2. They showed the same absorption spectrum and almost the identical molecular weights (38,000-42,000) and isoelectric points (3.4-3.7). 3. The identity of these chromoproteins was confirmed by isoelectric focussing with mixed samples. 4. Their chromophores were identified as being solely biliverdin IX~. 5. It may be concluded that one and the same biliverdin-protein exists in the different tissues of this fish. Abstract--1.
INTRODUCTION
The sculpin, Pseudoblennius percoides Giinther, living in the coastal waters of Japan, displays light blue to deep blue--green pigmentation not only in the skin but in the muscle and the digestive tract, and also in the eggs. This paper deals with isolation and comparison of these pigments from different tissues of the fish.
Sephadex thin layer method (Morris, 1964). The measurement of the isoelectric point was performed with an LKB isoelectric focussing apparatus model 8100. The chromic acid-dichromate degradation method (Riidiger, 1969; Rtidiger & O'Carra, 1969) was used for identification of the chromophores. Biliverdin IX~ was prepared from a commercial bilirubin according to the method of Tixier (1945) and purified by silicic acid column chromatography with a solvent system, methylethylketone/water/acetic acid (100:10: 1, by vol).
MATERIALS AND METHODS
RESULTS
Three sculpins were caught with rod and line off Kanagawa Prefecture, Japan. Two were males weighing 50 and 20g. The 50,g fish showed light blue pigmentation over the skin, muscle and digestive tract, whereas the 20 g one was almost colourless in the skin but deep blue-green in the muscle and digestive tract. The remaining fish was a female weighing 35 g and displaying a light blue skin and digestive tract along with blue-green eggs and almost colourless muscle. The skin, muscle, digestive tract and eggs were removed from the three individuals. Each tissue sample was cut into small pieces, minced in a mortar with cold distilled water, and centrifuged. The pigment that passed into the aqueous layer was then fractionated with ammonium sulphate, precipitation occurring between 40 and 80% saturation. The precipitates were collected by suction, dissolved in a small volume of cold distilled water and dialysed overnight against 0.04 M phosphate buffer, pH 6.8. When subjected to further purification by starch block electrophoresis (Kunkel, 1954) in the same buffer, the pigment, irrespective of the tissue, migrated anodally as a single narrow band. The pigmented zone was removed, extracted with, and finally dialysed against, cold distilled water to obtain a purified blue chromoprotein. All the purification procedures were carried out in a semi-dark cold room at 2-4°C. The u.v.-visible absorption spectrum of each chromoprotein was measured with a Cary 14 scanning spectrophotometer. The molecular weight was determined by the
The blue chromoproteins purified from the different tissues showed an identical absorption spectrum revealing a characteristic maximum at 670 nm and a dull one at 268-70 nm together with an inflexion at 328-9 nm (Fig. 1). The extinction ratios, E 2 7 o / E 6 7 o , ranged between 1.55 and 1.60.
0.31-70 O.I
200
3o0
I 400
I 500
I 600
I 700
Wovelengfh, nrn * Present address: Sch. of Fish. Sci., Kitasato Univ., Sanriku, Iwate, Japan.
Fig. 1. Absorption spectrum of the purified blue chromoprotein of sculpin in 0.1 M phosphate buffer, pH 7.0. 85
KATSUMIYAMAGUCHLKANEHISAHASHIMOTOAND FUMIOMATSUURA
86
Table 1. Molecular weight and isoelectric point of blue chromoproteins purified from various tissues of the sculpin Tissues
Molecular weight
IX~ and it may be concluded that one and the same biliverdin-protein is distributed widely within the body of this species of fish.
Isoelectric point
DISCUSSION Skin
40,000
3.6
Muscle
41,000
3.4
Digestive tract
38~000
3.7
Eggs
42,000
3.6
Each flqure indicates mean of tWO determinations.
As shown in Table 1 the molecular weights and isoelectric points exhibit no marked differences between the chromoproteins of the different tissues. The identity of the chromoproteins was further established by isoelectric focussing of a mixture consisting of equal amounts of the four chromoproteins from different origins; only a single band appeared at isoelectric point 3.6. On account of the small quantities of chromoprotein available for the identification of the chromophores, the chromic acid-dichromate micro-degradation technique, which enables direct degradation of the chromophores while still attached to the proteins, was used. In chromic acid degradation, dark blue spots of methyl vinyl maleimide and haematinic acid appeared on the thin layer chromatogram and in the dichromate degradation orange-red spots of 3-methyl-4-carboxyethylpyrrole-dialdehyde(2,5) appeared in all the four chromoproteins of the different tissues. Such a result was indistinguishable from that of the biliverdin IX, used as a control (Fig. 2). Thus the chromophores were identified exclusively as biliverdin
It has long been known that some species of the family Cottidae exhibit blue to blue green colours in the skin, oral tegument, bone and serum (Willstaedt, 1941; Augustinsson, 1959). Bada (1970) isolated a blue green pigment from the blood plasma of the Arctic sculpin, Myoxocephalus scorpioides, and characterized it as a biliprotein of which absorption spectrum had only one maximum at 658--60nm in the visible region. The isoelectric point and molecular weight were found to be 3.1 and ~46,000, respectively. Bada (1970) also noted the presence of the identical biliprotein in the skin. Subsequently, in the sera of several species of cottids, Low & Bada (1974) have reported that occurrence of green, blue, lavender and red chromoproteins of which the chromophores are biliverdin and/or an unidentified biladiene. The same pigments were manifested in the cartilages of the mouth and gills and in the skin. The present authors noticed by chance that the eggs of cabezon, Scorpaenichthys marmoratus, one of the family Cottidae, are of a red-purple colour supposedly due to the chromoprotein(s) found in the serum (Low & Bada, 1974). Such accumulation of knowledge along with the present results strongly suggests that the presence of biliproteins, mainly biliverdin-protein, in cottids might be a fairly widespread phenomenon. Occurrence of biliverdin protein in fish is not limited to cottids but has been demonstrated in the serum of eel, Anyuilla japonica (Yamaguchi et al., 1966), in the fins of wrasse, Crenilabrus pavo C.V. (Abolin~ & RiJdiger, 1966: AbolinL 1970) and in the muscle of Napoleon fish (also called "hirosa"), Cheilinus undulatus RiJppell (Yamaguchi & Matsuura. 1969). The physiological function of these biliproteins is still a matter of speculation as already remarked by
(a)
(b)
0.5 H r
0
Q
0
0
COOH
0 CO0 H
OH~HO
I e I
H
H
@ ~
@ •
2
4
3
.5
9I ¢ ? 2¢ ?
3
4
5
Fig. 2. Typical thin layer chromatograms of imides (a) and pyrrole
Blue pigments obtained from different tissues of the sculpin Low & Bada (1974). However; judging from the fact that the same biliverdin-protein exists in various tissues of the sculpin, it may be suggested that biliverdin, perhaps derived from haemoglobin and myoglobin a n d linked with a protein for protection from isomerization a n d oxidation, is stored temporarily in various parts of the body a n d subsequently at an o p p o r t u n e m o m e n t transported via the serum to the skin and eggs for the purpose of camouflage or sexual dimorphism, as a protector against u.v. rays or as a photoreceptor for hatching and so on.
Acknowledgements--We wish to express our sincere thanks to Prof. Y. Hashimoto of the The Univ. of Tokyo for supply of the sculpins which he himself fished up, and to Dr. T. Abe, Tokai Reg. Fish. Res. Lab. for the identification of the fish. Cordial thanks are also due to Dr, R. M. Love, Torry Research Station, Aberdeen, Scotland for his kind revision of this manuscript. This work was supported in part by a grant from the Ministry of Education, Japan. REFERENCES
ABOLINg L. (1970) The preparation and identification of the chromoprotein Crenilabrus blue. Pubbl. Staz. Zool. Napoli 3g, 229-248. ABOLINg L. & Ri~DIGER W. (1966) Uber die farbgebende Gruppe yon Crenilabrus-Blau. Experientia 22, 298-299. AUGUSTINSSON K.-B. (1959) Electrophoresis studies on blood plasma esterases. Acta Chem. Scand. 13, 1097-1105.
87
BADA J. L. (1970) A blue-green pigment isolated from blood plasma of the Arctic sculpin (Myoxocephalus scorpioides). Experientia 26, 251-252. KUNKEL H. G. (1954) Zone electrophoresis. In Methods of Biochemical Analysis Vol. 1 (Edited by GLICK D.) pp. 141-170, Interscience, New York. Low P. S. & BAOA J. L. (1974) Bile pigments in the blood serum of fish from the family Cottidae. Comp. Biochem. Physiol. 47A, 411-418. MORRIS C. J. O. R. (1964) Thin-layer chromatography of proteins on Sephadex G-100 and G-200. J. Chromatogr. 16, 167-175. R~DIGER W. 0969) Chromsgure- und Chromatabbau yon Gallenfarbstoffen. Hoppe-Seyler's Z. Physiol. Chem. 350, 1291-1300. Ri~DIGER W. & O'CARRA P. (1969) Studies on the structures and apoprotein linkages of the phycobilins. Eur. J. Biochem. 7, 509-516. TIXlER R. (1945) Contribution a l'6tude de quelques pigments pyrro!iques naturels des coquilles de Mollusques, de l'oeuf d'Emeu et du squelette du corail bleu (Heliopora caerulea). Ann. Inst. ocdanogr. Monaco 22, 343-397. WILLSTAEDTH. (1941) Zur Kenntnis der griinen Farbstoffe von Seefischen. Enzymologia 9, 260-264. YAMAGUCHI K., KOCHIYAMA Y., HASHIMOTO K. & MATSUURA F. (1966) Studies on a blue-green serum pigment of eel--II. Identification of prosthetic group. Bull. Japan. Soc. Sci. Fish. 32, 873-879. YAMAGUCHI K. & MATSUURA F. (1969) A blue pigment from the muscle of a marine teleost, "hirosa", Cheilinus undulatus Riippell. Bull. Japan. Soc. Sci. Fish. 35, 920-926.