- Email: [email protected]
Iron in the blood plasma of the ascidian Pyura chilensis
Crimp. Eiorhem. I)/2nM. Printed in Great B&m
Vol. 94A,
No. 4, pp. 777-781.
0300-9629189 $3.00 + 0.00 lC> 1989 Pergamon Press plc
1989
IRON IN THE BLOOD PLASMA OF THE ASCIDIAN PYURA
~~I~E~~~~
JAIME PIZARRO, CARLOS ANDRADE and IRMA CRIVELLI* Department of Chemistry, Faculty of Sciences, University of Chile, Casilla 653, Santiago, Chile
In order to study the iron-binding in the blood plasma of the ascidian Pyura chilensisMolina 1782, the plasma components were separated by Sephadex G-75 chromatography, gel electrophoresis and fractionated precipitation combined with Miissbauer spectroscopy iron detection. 2. Iron-binding in the plasma was also investigated by fractionation of 59Fe-labeled plasma, using gel chromatography. 3. Proteins and N-acetylamino sugars were found as plasma components. 4. iron was detected in protein component(s) by calorimetric methods and Mcssbauer measurements. 5. The results, which strongly suggest a high-iron molecular weight plasma protein association, are compared with other iron concentrating ascidians. Abstract-l.
INTRODUCTION
It is well known that certain marine animals within the class Ascidiacca of the subphylum Tunic&a concentrate iron and/or vanadium in their blood, besides a number of other metals (manganese, chromium, etc.), related to the surrounding sea water (Goodbody, 1974; Biggs and Swinehart, 1976). Although ascidian biology has been of considerable interest in the last two decades, most of the studies were carried out in the vanadium-containing species and principally in blood cells (Biggs and Swinehart, 1976; Macara et al., 1979; Hawkins et al., 1980a,b). The compounds formed by the metals in the blood cells are presently unknown although there is some evidence that vanadium is present as hydrated vanadium(II1) (Carlson, 1975; Tullius et al., 1980). Less frequent are studies on species containing iron (suborder Aplousobranchia and Stolidobranchia) (Biggs and Swinehart, 1976; Agudelo et al., 1983). Hawkins et al. (1983) published a preliminary report on the formation of an iron-sulfur cluster by the reduction of sulfate with the blood pigment of Pyura sto~on~er~ in the presence of iron similar to the 2-Fe ferredoxins. Systematic studies of the moiety associated with the meta in blood plasma are lacking except for the studies reported by Hawkins et al. (1980b) and Webb and Chrystal (1981). Recently. through an article of Finch and Heubers (1986), we have access to the work of Martin et al. (1984) in which the authors report the finding of an iron-binding protein in the plasma of Pyura stolonifera, characterized as a “transferrin”. Pyura stolonifera seems to be somewhat unusual amongst the iron-containing species (Molgula munhattensis, Pyurn chiiensis, etc.); most of the works reported were made with this species, maybe due to a more intense color presented by its blood suggesting a higher iron-chromophore concentration. and con-._ *To whom all correspondence
_.._ should be addressed.
-
sequently, the possibility of a more complete characterization of the iron compounds in the ascidian’s blood. The present work reports the results of iron binding studies in blood plasma of the ascidian Pyura chilensis Molina 1782 (family Pyuridae, suborder Stolidobranchia) which has a relatively high iron content and is used as food in Chile. The results are comparatively analyzed with those for Pyura stolo~i~era and Her~rnan~~ momus.
MATERIALS AND METHODS Specimens of the ascidian Pyura chilensis were collected from Quintero Bay (Pacific Ocean, 32”47’ lat., 47”32’ long.) and maintained in sea water until removal of the blood samples (approximately 3 hr) by cutting the base and rupturing the blood vessels in the ventral visceral zone. A small pressure on the body makes the blood flow out, which is collected in a flask maintained in an ice bath and occasionally stirred. Orange colored blood cells were removed from the pink-yellow plasma by centrifugation. From this stage on, all the experiments were performed in environmental conditions to biological systems (in an ice bath or in a cold room). The whole plasma and the plasma fractions eluted from a column were characterized by UV-visible spectrophotometry, tested for proteins (Biuret test), N-acetvlamino sugars (Reissig et al., 1955) and iron using i,4,6-tripiridyl-striazvne (TPTZ) (Collins et al.. 1959) and atomic absorntion spec&ometry (AkS) after digestion’ with an HClO,-GNO, mixture. This last method (AAS) was also used to determine the iron content in blood cells. Whole plasma and fractions were concentrated by freeze drying when it was needed. Radioactivity experiments were carried out by labeling the plasma with addition of 0.5 ml of S9FeC1,(1.6 pCi/ml) and allowing it to reach equilibrium for several hours. The plasma was dialyzed against distilled water for 75 min and the activity measured directly, or after filtration by gel chromatography (Sephadex G-75). in a BiogammaBeckman radiation counter. Electrophoresis of the whole plasma and of the gel filtration fractions was carried out in 4% and 7% polyacrylamide gels (Davis technique) (Davis et al., 1964) using Bromophenol Blue as the tracking dye
777
JAIME PIZARRO et al.
778
and Tris-glycine (pH 8.3) as buffer; the current was approximately 4mA/gel. The gels were stained with Coomassie Brilliant Blue for proteins, Schiff base stain (PAS) for carbohydrate and TPTZ for iron (this last method was previously verified with electrophoresis and staining of an aqueous iron standard solution).
,,6,6,5
3,7
7,4
111 m(
RESULTS
The iron content in blood cells was in the range of 568-5300 mg iron/kg dry weight and l-2 ppm in plasma; the results indicate concentration factors of 5 x lo5 to 5 x lo3 and 1 x lo3 to 2 x lo3 for cells and plasma respectively related to sea water iron concentration. The pink-yellow colored plasma showed a pH of 6.8 and a density of l.O214g/ml (20°C). Figure 1 shows the plasma UV-visible spectra: a band at 260-300 nm, shoulders in the 30&400 and 360-380 nm regions and a broad band from 400 to 750 nm. Since gel filtration dilutes the plasma, this must be concentrated several times to reveal the presence of iron in plasma fractions. As a consequence, a precipitate was obtained whose analysis tested positive for proteins and iron. The supernatant was chromatographed through Sephadex G-75 with 0.05 M NaCl (Hawkins et al., 1980a) as eluent. The elution pattern of concentrated plasma detected at 278 and 300-330 nm (Fig. 2) shows two major peaks. The ratio of the absorbance at the two wavelengths varies with the fraction number; furthermore, the absorbance at 300-330 nm is a shoulder in the spectra of fractions 2-5; however, it appears more resolved in fractions 8-12. These facts suggest that more than one compound is responsible for the absorptions at 278 and 300-330 nm. The elution pattern obtained from the determination of proteins, N-acetylamino sugars and iron concentrations are shown in Fig. 3; in some experiments a second iron maximum was obtained around fraction 10. The pattern in Figs 2 and 3 shows that the elution profile maxima of the absorptions at 278 and 300-330 nm and the concentrations of proteins, iron and N-acetylamino sugars are coincident, suggesting a possible iron-protein or iron-glycoprotein association. The results of j9Fe-labeled plasma chro-
250
350
450
0.6 0.4
2
4
6 7.4ml
8
10
12
14
16
FRACTION
Fig. 2. Elution pattern of concentrated plasma of Pyura chilensis from Sephadex G-75 (1.6 x 40 cm column) with 0.05 M NaCl as eluent at 278 nm (solid line) and at 318 nm (dashed line). Arrows indicate proper scale. Volumes and not fractions must be compared.
matography in Sephadex G-75, measuring iron activity (Fig. 4), shows the same pattern as Fig. 3 with the first maximum centered at fraction 4 and the second one at fraction 9 (a control experiment showed that an aqueous iron solution, 59FeC13, elutes at fraction 9). The first maximum confirms the presence of iron-binding molecules in the blood plasma. Fractions 4 and 9 were measured after a further dialyzing time of 1.3 hr in a first experiment and for 4 hr in a second one. The activity was reduced about 18% and 50% in relation to the original activity for fractions 4 and 9 respectively in the first case (1.3 hr) and to about 40% and 75% for the same fractions after 4 hr, indicating the labile character of at least part of the iron-binding moiety. Electrophoresis
in polyacrylamide
gel
Experiments (7% polyacrylamide) were made with whole plasma three-fold concentrated (A) and with fractions eluted from a G-75 column (16/40).
550
WAVELENGTH,
650
750
ntn
Fig. I. Ultraviolet and visible spectra of plasma from Pyura chilensis concentrated five-fold. Cell path length: 1 cm (A, B), 10 cm (C). Dilution shown where applicable. Arrows indicate proper scale.
719
Iron in blood plasma of an ascidian
Concentrated whole plasma (A) and fraction 4 (B) (Fig. 5) show two major protein bands (R,= 0.1 and 0.3) and two weakly staining bands (R, = 0.04 and 0.06). Furthermore, when the gel (B) is stained with PAS a weak band shows up at R, = 0.1. Gel electrophoretic experiments (4% polyacrylamide) were made with fractions from a 16/70 G-75 column. Since the plasma was used directly as extracted, fractions 21-30 from three equivalent experiments were mixed and then concentrated by freeze drying. Three electrophoretic experiments were run in parallel; the development process was applied to one of the gels, showing five protein bands (the band with R,= 0.63 was the most intense; Fig. 6); the other two gels were cut in appropriate sections and eluted separately; determination of Fe in the eluents indicates the presence of the metal in the R,= 0.53 protein band (60% of the original pool iron content); the major concentration of proteins in the Rf = 0.63 band (30%) was also confirmed from the eluents. Thus, the electrophoresis experiments reinforced or perhaps glycoprotein-iron the protein-iron interaction.
1.0 * ‘0 ‘; % ”
0 Fraction
number
pattern of 59Fe-labeled plasma from Pyura was labeled with 0.5ml of S9FeCl, (1.6 pCi/ml) and allowed to reach equilibrium, then dialyzed against distilled water and chromatographed on Sephadex G-75 (1.6 x 40 cm column). The arrow shows the fraction where an aqueous control solution of 59FeCl, elutes. Fig. 4. Elution
chilensis. Plasma
Fractionated
precipitation
of proteins
The proteins were fractionated by adding different volumes of 4 M (NH,)2SOP to a fixed volume of plasma. The iron concentration (determined by AAS) in the supernatant of the 50/58.5% fraction was the lowest (20% of the total iron concentration in the plasma) and the corresponding precipitate was used for further characterization of the iron compound in the plasma. The presence of iron was also detected by Mossbauer spectroscopy, but the low concentration of the metal was not enough to determine the Mbssbauer parameters nor to obtain structural and electronic information.
DISCUSSION
The plasma UV spectrum for Pyura chilensis is similar to the plasma spectra of other iron- and such as Pyura vanadium-containing ascidians stolonifera and Ascidia ceratodes (Hawkins et al., 1980b), i.e. a band at 26&280nm, and one at 300-350 nm which appears like a shoulder for
Rf ---_--_
-_--_ -_---_
_-__--------c--_-
--__-
----
--
--_
-----_--_-
_--_--__-_
--
, -__-____
0.04
2
___---_-
0.06
3
---_----
0.1
4
-_______
0.3
lj
_--_--_-
0.5
6
________
0,--J
--,-___ --------l-3 ---_---_-_ -------_--_-------_--_-
7.4ml
FRACTIONS
Fig. 3. Elution pattern of concentrated plasma of Pyura chilensis from Sephadex G-75 (1.6 x 40 cm column) with 0.05 M NaCl as eluent. Fractions were collected and analyzed for (1) iron concentration with TPTZ, (2) protein concentration with Biuter test, (3) N-acetylamino sugar concentration (Reissig et al., 1955). Arrows indicate proper scale.
---_--_
--
42 3
-___ __-_----_-__--_
--_-_
5
-___-___
Fig. 5. Gel electrophoretograms: 7% of concentrated whole plasma (A) (1.6 x 40cm column) eluted fraction chilensis. Protein bands stained with shown as: n very strong; II0 strong; weak.
---_
0.1, 0.04 0.30 0.06
0.8,
polyacrylamide gels and Sephadex G-75 4 (B) from Pyura Coomassie Blue are N weak; __ very
780
JAIME PKARRO et al.
P. chilensis and A. ceratodes; P. chilensis shows a further absorption band at 350-400 nm, but P. stolonifera and A. ceratodes only show a tail at the longer wavelength side of the 300-350 nm band. The most clear spectral differences between the species are observed in the visible region. The vanadiumcontaining species A. ceratodes shows no absorption, whereas P. stolonifera shows a broad and symmetrical band centered at 500 nm and P. chilensis an asymmetrical band centered at 450 nm with some reproducible bumps between 450 and 500 nm and with a tail between 450 and 750 nm. If the observations of Martin et al. (1984) concerning the ironbinding protein are considered, the absorption spectra exhibited by P. chilensis (Fig. 1) show more characteristics to look for the presence of a hemerythrin-like structure (Okamura and Klotz, 1975) in its plasma instead of a transferrin-type structure. When the elution pattern of the plasma for the three species are compared, some similarities and differences may be observed. Pyura chilensis shows two major elution bands which are coincident when 27&280 and 300-330 nm absorptions are detected (volumes and not fractions must be considered). Pyura stolonifera shows two bands (fractions l-3 and 8-13) when detection is made in the 278 nm region but only one (fractions 8-13) when detection is at 300-330 nm. A major difference between the two species appears in the first elution band which gives positive tests for proteins, N-acetylamino sugars and iron for P. chilensis; however, the test is positive only for protein in P. stolomfiru. For the second elution band the results are again different for both species: P. chilensis gives a positive test for proteins and N-acetylamino sugars and in some experiments a positive test for iron is obtained. Pyura stolon@ra tests positive only for N-acetylamino sugars. Taking into account that in the experiments made with plasma only proteins were found in both elution bands, it may be considered that not only do the distributions seem to be different in both species, but the concentrations of N-acetylamino sugars and proteins appear to be as well. In the case of iron the difference may not arise; the results for concentrated plasma must be compared in both species before any conclusion can be drawn, but it is important to point out that the results have not only shown the iron-binding capacity of the plasma protein through the iron radiotracer experiments, but also give evidence of an iron-protein association in iron plasma occurring naturally. When the results of metal association studies (radiotracer experiments) for plasma of P. stolonfera, Herdmania momus (Webb and Chrystal, 1981) and P. chilensis are compared, the most clear similarity is that in all three species the plasma proteins of the highest molecular weight bind iron. For P. chilensis the first band in the elution pattern for iron plasma occurring naturally and iron incubation plasma are coincident. In the electrophoretic experiments the principal interest was not only to have more information about protein components but also to get more and new evidence for a protein-iron association; in consequence, it was decided to work with a 16170 column
-----_--_
---
5
_---__-
0.85
Fig. 6. Gel electrophoretograms: 4oio polyacrylamide gels of G-75 (1.6 x 70 cm column) eluted fraction 21 30 pool from concentrated Prura chi1ensi.s plasma. Protein bands stained with Coomassie Blue are shown as: n very strong; R strong. The results shown correspond to the mixture of three pools
of fractions
21 30.
with better these were
small volume of fraction in order to obtain resolution in the first major elution band with new conditions. About 46% of the total proteins found to elute in fractions 22228, but the quantification for iron was poor due to low concentration. The electrophoresis of the mixed fractions of this elution band supplied new evidence for ironprotein interaction since iron (determined by AAS) and proteins were found in the eluent of R, = 0.53. In summary, the elution profile results for iron plasma occurring naturally in the “Fe radioactive experiments, the electrophoresis in polyacrylamide gel. as well as the fractioned precipitation combined with Miissbauer spectroscopy detection. strongly suggest the association of Fc to a high molecular weight plasma glycoprotein in P. chilensis. Although this result may seem rather trivial in the light of Martin’s results. it may be pointed out that little or nothing I\ known about the compounds formed by metals in the blood of ascidian species and their biological role(s). Pyura stolon$vx is the only iron-containing species for which not only one but two compounds have been isolated: a metal-free pigment which reacts in the presence of oxygen with iron(H) sulfate to give an iron--sulfur cluster of the 2-Fe ferredoxin-type. and the mono-sited transferrin isolated as an “‘Fe moiety. However, there arc no similar data available for other members of the Pyuridae family concerning the ironbinding moiety and it does not seem to be possible to extrapolate conclusions from one member to another even in the same family. This is reinforced by the results of Sakurai et al. (1987), which indicate that vanadyl ion ligand fields in blood cells of L\cidicr ahodori are different from each other, depending on the places where they were collected. Therefore. it appears to be important to have evidence which supports an iron-protein association in the plasma of P. chilensis. although it has not been possible to gather information about the structure of such a moiety. Acknowledgements-This work was supported by Grant No. Q-948-855 from the Departamento de Investigation y Bibliotecas, Universidad de Chile. The authors are grateful to the Laboratorio de Fisiologia de Montemar tor providing facilities during the work.
Iron in blood
plasma
REFERENCES
Agudelo M., Kustin K., McLeod G. C., Robinson W. and Wang R. (1983) Iron accumulation in Tunicate blood cells. I. Distribution and oxidation state of iron in the blood of Boltenia ooifera, Styela claua and Molgula manhattensis. Biol. Bull. 165, 100-109. Biggs W. R. and Swinehart J. H. (1976) Vanadium in selected biological systems. In Metal Ions in Biology (Edited by H. Sigel), Vol. 6. pp. 141-196. M. Dekker, New York. Carlson R. M. K. (1975) Nuclear magnetic resonance spectrum of living tunicate blood cells and the structure of the nature vanadium chromogen. Proc. natn. Acad. Sci. U.S.A. 12, 2217-2221. Collins P. F., Dielh H. and Smith G. F. (1959) 2,4.6Tripyridyl-s-triazine as a reagent for iron. Analyt. Chem. 31, 1862%1867. Davis B. J. (1964) Disc electrophoresis. 11. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404427. Finch C. A. and Huebers H. A. (1986) Iron metabolism. Clin. Physiol. Biochem. 4, SSIO. Goodbody I. (1974) The physiology of Ascidians. Adc. Mar. Biol. 12, I-149. Hawkins C. J., Parry D. L. and Pierce C. (1980a) Chemistry of the blood of the Ascidian Podoclouella moluccensis. Biol. Bull. 159, 669480. Hawkins C. J., Merifield P. H., Parry D. L., Biggs W. R. and Swinehart J. H. (1980b) Comparative study of the blood
of an ascidian
781
plasma of the Ascidians Pyura stolonijera and Ascidia ceratodes. Biol. Bull. 159, 656-668. Hawkins C. J., Parry D. L., Wood B. J. and Clark P. (1983) Formation of an Iron-Sulfur cluster by the reduction of sulfate with the blood pigment of an Ascidian in the presence of iron. Inorg. Chem. Acta 78, L29-L31. Macara I. G., McLeod G. C. and Kustin K. (1979) Isolation properties and structural studies on a compound iron tunicate blood cells that may be involved in vanadium accumulation. Biochem. J. 181, 457465. Martin A. W., Huebers E., Huebers H., Webb J. and Finch C. A. (1984) A mono-sited transferrin from a representative deuterostome: the Ascidian Pyura stolonifera (Subphylum Urochordata). Blood 984, 1047-1052. Okamura M. Y. and Klotz I. M. (1975) inorganic Biochemistry, pp. 32%343. Elsevier, New York. Reissig J. L., Strominger J. L. and Leloir L. F. (1955) A modified calorimetric method for the estimation of N-acetylamino sugars. J. biol. Chem. 217, 9599966. Sakurai H.. Hirato J. and Michibato H. (1987) EPR characterization of vanadyl (IV) species in blood cells of Ascidians. Biochem. biophys. Res. Commun. 149,411416. Tulhus T. D., Gillum W. W., Carlson R. M. K. and Hodgson K. 0. (1980) Structural study of the vanadium complex in living Ascidian blood cells by X-ray absorption-spectroscopy. J. Am. them. Sot. lOi, 5601567. Webb J. and Chrystal P. (1981) Protein binding of iron in blood plasma of the Ascidian Herdmania momus. Mar. Biol. 63, 107Fl12.
Vol. 94A,
No. 4, pp. 777-781.
0300-9629189 $3.00 + 0.00 lC> 1989 Pergamon Press plc
1989
IRON IN THE BLOOD PLASMA OF THE ASCIDIAN PYURA
~~I~E~~~~
JAIME PIZARRO, CARLOS ANDRADE and IRMA CRIVELLI* Department of Chemistry, Faculty of Sciences, University of Chile, Casilla 653, Santiago, Chile
In order to study the iron-binding in the blood plasma of the ascidian Pyura chilensisMolina 1782, the plasma components were separated by Sephadex G-75 chromatography, gel electrophoresis and fractionated precipitation combined with Miissbauer spectroscopy iron detection. 2. Iron-binding in the plasma was also investigated by fractionation of 59Fe-labeled plasma, using gel chromatography. 3. Proteins and N-acetylamino sugars were found as plasma components. 4. iron was detected in protein component(s) by calorimetric methods and Mcssbauer measurements. 5. The results, which strongly suggest a high-iron molecular weight plasma protein association, are compared with other iron concentrating ascidians. Abstract-l.
INTRODUCTION
It is well known that certain marine animals within the class Ascidiacca of the subphylum Tunic&a concentrate iron and/or vanadium in their blood, besides a number of other metals (manganese, chromium, etc.), related to the surrounding sea water (Goodbody, 1974; Biggs and Swinehart, 1976). Although ascidian biology has been of considerable interest in the last two decades, most of the studies were carried out in the vanadium-containing species and principally in blood cells (Biggs and Swinehart, 1976; Macara et al., 1979; Hawkins et al., 1980a,b). The compounds formed by the metals in the blood cells are presently unknown although there is some evidence that vanadium is present as hydrated vanadium(II1) (Carlson, 1975; Tullius et al., 1980). Less frequent are studies on species containing iron (suborder Aplousobranchia and Stolidobranchia) (Biggs and Swinehart, 1976; Agudelo et al., 1983). Hawkins et al. (1983) published a preliminary report on the formation of an iron-sulfur cluster by the reduction of sulfate with the blood pigment of Pyura sto~on~er~ in the presence of iron similar to the 2-Fe ferredoxins. Systematic studies of the moiety associated with the meta in blood plasma are lacking except for the studies reported by Hawkins et al. (1980b) and Webb and Chrystal (1981). Recently. through an article of Finch and Heubers (1986), we have access to the work of Martin et al. (1984) in which the authors report the finding of an iron-binding protein in the plasma of Pyura stolonifera, characterized as a “transferrin”. Pyura stolonifera seems to be somewhat unusual amongst the iron-containing species (Molgula munhattensis, Pyurn chiiensis, etc.); most of the works reported were made with this species, maybe due to a more intense color presented by its blood suggesting a higher iron-chromophore concentration. and con-._ *To whom all correspondence
_.._ should be addressed.
-
sequently, the possibility of a more complete characterization of the iron compounds in the ascidian’s blood. The present work reports the results of iron binding studies in blood plasma of the ascidian Pyura chilensis Molina 1782 (family Pyuridae, suborder Stolidobranchia) which has a relatively high iron content and is used as food in Chile. The results are comparatively analyzed with those for Pyura stolo~i~era and Her~rnan~~ momus.
MATERIALS AND METHODS Specimens of the ascidian Pyura chilensis were collected from Quintero Bay (Pacific Ocean, 32”47’ lat., 47”32’ long.) and maintained in sea water until removal of the blood samples (approximately 3 hr) by cutting the base and rupturing the blood vessels in the ventral visceral zone. A small pressure on the body makes the blood flow out, which is collected in a flask maintained in an ice bath and occasionally stirred. Orange colored blood cells were removed from the pink-yellow plasma by centrifugation. From this stage on, all the experiments were performed in environmental conditions to biological systems (in an ice bath or in a cold room). The whole plasma and the plasma fractions eluted from a column were characterized by UV-visible spectrophotometry, tested for proteins (Biuret test), N-acetvlamino sugars (Reissig et al., 1955) and iron using i,4,6-tripiridyl-striazvne (TPTZ) (Collins et al.. 1959) and atomic absorntion spec&ometry (AkS) after digestion’ with an HClO,-GNO, mixture. This last method (AAS) was also used to determine the iron content in blood cells. Whole plasma and fractions were concentrated by freeze drying when it was needed. Radioactivity experiments were carried out by labeling the plasma with addition of 0.5 ml of S9FeC1,(1.6 pCi/ml) and allowing it to reach equilibrium for several hours. The plasma was dialyzed against distilled water for 75 min and the activity measured directly, or after filtration by gel chromatography (Sephadex G-75). in a BiogammaBeckman radiation counter. Electrophoresis of the whole plasma and of the gel filtration fractions was carried out in 4% and 7% polyacrylamide gels (Davis technique) (Davis et al., 1964) using Bromophenol Blue as the tracking dye
777
JAIME PIZARRO et al.
778
and Tris-glycine (pH 8.3) as buffer; the current was approximately 4mA/gel. The gels were stained with Coomassie Brilliant Blue for proteins, Schiff base stain (PAS) for carbohydrate and TPTZ for iron (this last method was previously verified with electrophoresis and staining of an aqueous iron standard solution).
,,6,6,5
3,7
7,4
111 m(
RESULTS
The iron content in blood cells was in the range of 568-5300 mg iron/kg dry weight and l-2 ppm in plasma; the results indicate concentration factors of 5 x lo5 to 5 x lo3 and 1 x lo3 to 2 x lo3 for cells and plasma respectively related to sea water iron concentration. The pink-yellow colored plasma showed a pH of 6.8 and a density of l.O214g/ml (20°C). Figure 1 shows the plasma UV-visible spectra: a band at 260-300 nm, shoulders in the 30&400 and 360-380 nm regions and a broad band from 400 to 750 nm. Since gel filtration dilutes the plasma, this must be concentrated several times to reveal the presence of iron in plasma fractions. As a consequence, a precipitate was obtained whose analysis tested positive for proteins and iron. The supernatant was chromatographed through Sephadex G-75 with 0.05 M NaCl (Hawkins et al., 1980a) as eluent. The elution pattern of concentrated plasma detected at 278 and 300-330 nm (Fig. 2) shows two major peaks. The ratio of the absorbance at the two wavelengths varies with the fraction number; furthermore, the absorbance at 300-330 nm is a shoulder in the spectra of fractions 2-5; however, it appears more resolved in fractions 8-12. These facts suggest that more than one compound is responsible for the absorptions at 278 and 300-330 nm. The elution pattern obtained from the determination of proteins, N-acetylamino sugars and iron concentrations are shown in Fig. 3; in some experiments a second iron maximum was obtained around fraction 10. The pattern in Figs 2 and 3 shows that the elution profile maxima of the absorptions at 278 and 300-330 nm and the concentrations of proteins, iron and N-acetylamino sugars are coincident, suggesting a possible iron-protein or iron-glycoprotein association. The results of j9Fe-labeled plasma chro-
250
350
450
0.6 0.4
2
4
6 7.4ml
8
10
12
14
16
FRACTION
Fig. 2. Elution pattern of concentrated plasma of Pyura chilensis from Sephadex G-75 (1.6 x 40 cm column) with 0.05 M NaCl as eluent at 278 nm (solid line) and at 318 nm (dashed line). Arrows indicate proper scale. Volumes and not fractions must be compared.
matography in Sephadex G-75, measuring iron activity (Fig. 4), shows the same pattern as Fig. 3 with the first maximum centered at fraction 4 and the second one at fraction 9 (a control experiment showed that an aqueous iron solution, 59FeC13, elutes at fraction 9). The first maximum confirms the presence of iron-binding molecules in the blood plasma. Fractions 4 and 9 were measured after a further dialyzing time of 1.3 hr in a first experiment and for 4 hr in a second one. The activity was reduced about 18% and 50% in relation to the original activity for fractions 4 and 9 respectively in the first case (1.3 hr) and to about 40% and 75% for the same fractions after 4 hr, indicating the labile character of at least part of the iron-binding moiety. Electrophoresis
in polyacrylamide
gel
Experiments (7% polyacrylamide) were made with whole plasma three-fold concentrated (A) and with fractions eluted from a G-75 column (16/40).
550
WAVELENGTH,
650
750
ntn
Fig. I. Ultraviolet and visible spectra of plasma from Pyura chilensis concentrated five-fold. Cell path length: 1 cm (A, B), 10 cm (C). Dilution shown where applicable. Arrows indicate proper scale.
719
Iron in blood plasma of an ascidian
Concentrated whole plasma (A) and fraction 4 (B) (Fig. 5) show two major protein bands (R,= 0.1 and 0.3) and two weakly staining bands (R, = 0.04 and 0.06). Furthermore, when the gel (B) is stained with PAS a weak band shows up at R, = 0.1. Gel electrophoretic experiments (4% polyacrylamide) were made with fractions from a 16/70 G-75 column. Since the plasma was used directly as extracted, fractions 21-30 from three equivalent experiments were mixed and then concentrated by freeze drying. Three electrophoretic experiments were run in parallel; the development process was applied to one of the gels, showing five protein bands (the band with R,= 0.63 was the most intense; Fig. 6); the other two gels were cut in appropriate sections and eluted separately; determination of Fe in the eluents indicates the presence of the metal in the R,= 0.53 protein band (60% of the original pool iron content); the major concentration of proteins in the Rf = 0.63 band (30%) was also confirmed from the eluents. Thus, the electrophoresis experiments reinforced or perhaps glycoprotein-iron the protein-iron interaction.
1.0 * ‘0 ‘; % ”
0 Fraction
number
pattern of 59Fe-labeled plasma from Pyura was labeled with 0.5ml of S9FeCl, (1.6 pCi/ml) and allowed to reach equilibrium, then dialyzed against distilled water and chromatographed on Sephadex G-75 (1.6 x 40 cm column). The arrow shows the fraction where an aqueous control solution of 59FeCl, elutes. Fig. 4. Elution
chilensis. Plasma
Fractionated
precipitation
of proteins
The proteins were fractionated by adding different volumes of 4 M (NH,)2SOP to a fixed volume of plasma. The iron concentration (determined by AAS) in the supernatant of the 50/58.5% fraction was the lowest (20% of the total iron concentration in the plasma) and the corresponding precipitate was used for further characterization of the iron compound in the plasma. The presence of iron was also detected by Mossbauer spectroscopy, but the low concentration of the metal was not enough to determine the Mbssbauer parameters nor to obtain structural and electronic information.
DISCUSSION
The plasma UV spectrum for Pyura chilensis is similar to the plasma spectra of other iron- and such as Pyura vanadium-containing ascidians stolonifera and Ascidia ceratodes (Hawkins et al., 1980b), i.e. a band at 26&280nm, and one at 300-350 nm which appears like a shoulder for
Rf ---_--_
-_--_ -_---_
_-__--------c--_-
--__-
----
--
--_
-----_--_-
_--_--__-_
--
, -__-____
0.04
2
___---_-
0.06
3
---_----
0.1
4
-_______
0.3
lj
_--_--_-
0.5
6
________
0,--J
--,-___ --------l-3 ---_---_-_ -------_--_-------_--_-
7.4ml
FRACTIONS
Fig. 3. Elution pattern of concentrated plasma of Pyura chilensis from Sephadex G-75 (1.6 x 40 cm column) with 0.05 M NaCl as eluent. Fractions were collected and analyzed for (1) iron concentration with TPTZ, (2) protein concentration with Biuter test, (3) N-acetylamino sugar concentration (Reissig et al., 1955). Arrows indicate proper scale.
---_--_
--
42 3
-___ __-_----_-__--_
--_-_
5
-___-___
Fig. 5. Gel electrophoretograms: 7% of concentrated whole plasma (A) (1.6 x 40cm column) eluted fraction chilensis. Protein bands stained with shown as: n very strong; II0 strong; weak.
---_
0.1, 0.04 0.30 0.06
0.8,
polyacrylamide gels and Sephadex G-75 4 (B) from Pyura Coomassie Blue are N weak; __ very
780
JAIME PKARRO et al.
P. chilensis and A. ceratodes; P. chilensis shows a further absorption band at 350-400 nm, but P. stolonifera and A. ceratodes only show a tail at the longer wavelength side of the 300-350 nm band. The most clear spectral differences between the species are observed in the visible region. The vanadiumcontaining species A. ceratodes shows no absorption, whereas P. stolonifera shows a broad and symmetrical band centered at 500 nm and P. chilensis an asymmetrical band centered at 450 nm with some reproducible bumps between 450 and 500 nm and with a tail between 450 and 750 nm. If the observations of Martin et al. (1984) concerning the ironbinding protein are considered, the absorption spectra exhibited by P. chilensis (Fig. 1) show more characteristics to look for the presence of a hemerythrin-like structure (Okamura and Klotz, 1975) in its plasma instead of a transferrin-type structure. When the elution pattern of the plasma for the three species are compared, some similarities and differences may be observed. Pyura chilensis shows two major elution bands which are coincident when 27&280 and 300-330 nm absorptions are detected (volumes and not fractions must be considered). Pyura stolonifera shows two bands (fractions l-3 and 8-13) when detection is made in the 278 nm region but only one (fractions 8-13) when detection is at 300-330 nm. A major difference between the two species appears in the first elution band which gives positive tests for proteins, N-acetylamino sugars and iron for P. chilensis; however, the test is positive only for protein in P. stolomfiru. For the second elution band the results are again different for both species: P. chilensis gives a positive test for proteins and N-acetylamino sugars and in some experiments a positive test for iron is obtained. Pyura stolon@ra tests positive only for N-acetylamino sugars. Taking into account that in the experiments made with plasma only proteins were found in both elution bands, it may be considered that not only do the distributions seem to be different in both species, but the concentrations of N-acetylamino sugars and proteins appear to be as well. In the case of iron the difference may not arise; the results for concentrated plasma must be compared in both species before any conclusion can be drawn, but it is important to point out that the results have not only shown the iron-binding capacity of the plasma protein through the iron radiotracer experiments, but also give evidence of an iron-protein association in iron plasma occurring naturally. When the results of metal association studies (radiotracer experiments) for plasma of P. stolonfera, Herdmania momus (Webb and Chrystal, 1981) and P. chilensis are compared, the most clear similarity is that in all three species the plasma proteins of the highest molecular weight bind iron. For P. chilensis the first band in the elution pattern for iron plasma occurring naturally and iron incubation plasma are coincident. In the electrophoretic experiments the principal interest was not only to have more information about protein components but also to get more and new evidence for a protein-iron association; in consequence, it was decided to work with a 16170 column
-----_--_
---
5
_---__-
0.85
Fig. 6. Gel electrophoretograms: 4oio polyacrylamide gels of G-75 (1.6 x 70 cm column) eluted fraction 21 30 pool from concentrated Prura chi1ensi.s plasma. Protein bands stained with Coomassie Blue are shown as: n very strong; R strong. The results shown correspond to the mixture of three pools
of fractions
21 30.
with better these were
small volume of fraction in order to obtain resolution in the first major elution band with new conditions. About 46% of the total proteins found to elute in fractions 22228, but the quantification for iron was poor due to low concentration. The electrophoresis of the mixed fractions of this elution band supplied new evidence for ironprotein interaction since iron (determined by AAS) and proteins were found in the eluent of R, = 0.53. In summary, the elution profile results for iron plasma occurring naturally in the “Fe radioactive experiments, the electrophoresis in polyacrylamide gel. as well as the fractioned precipitation combined with Miissbauer spectroscopy detection. strongly suggest the association of Fc to a high molecular weight plasma glycoprotein in P. chilensis. Although this result may seem rather trivial in the light of Martin’s results. it may be pointed out that little or nothing I\ known about the compounds formed by metals in the blood of ascidian species and their biological role(s). Pyura stolon$vx is the only iron-containing species for which not only one but two compounds have been isolated: a metal-free pigment which reacts in the presence of oxygen with iron(H) sulfate to give an iron--sulfur cluster of the 2-Fe ferredoxin-type. and the mono-sited transferrin isolated as an “‘Fe moiety. However, there arc no similar data available for other members of the Pyuridae family concerning the ironbinding moiety and it does not seem to be possible to extrapolate conclusions from one member to another even in the same family. This is reinforced by the results of Sakurai et al. (1987), which indicate that vanadyl ion ligand fields in blood cells of L\cidicr ahodori are different from each other, depending on the places where they were collected. Therefore. it appears to be important to have evidence which supports an iron-protein association in the plasma of P. chilensis. although it has not been possible to gather information about the structure of such a moiety. Acknowledgements-This work was supported by Grant No. Q-948-855 from the Departamento de Investigation y Bibliotecas, Universidad de Chile. The authors are grateful to the Laboratorio de Fisiologia de Montemar tor providing facilities during the work.
Iron in blood
plasma
REFERENCES
Agudelo M., Kustin K., McLeod G. C., Robinson W. and Wang R. (1983) Iron accumulation in Tunicate blood cells. I. Distribution and oxidation state of iron in the blood of Boltenia ooifera, Styela claua and Molgula manhattensis. Biol. Bull. 165, 100-109. Biggs W. R. and Swinehart J. H. (1976) Vanadium in selected biological systems. In Metal Ions in Biology (Edited by H. Sigel), Vol. 6. pp. 141-196. M. Dekker, New York. Carlson R. M. K. (1975) Nuclear magnetic resonance spectrum of living tunicate blood cells and the structure of the nature vanadium chromogen. Proc. natn. Acad. Sci. U.S.A. 12, 2217-2221. Collins P. F., Dielh H. and Smith G. F. (1959) 2,4.6Tripyridyl-s-triazine as a reagent for iron. Analyt. Chem. 31, 1862%1867. Davis B. J. (1964) Disc electrophoresis. 11. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404427. Finch C. A. and Huebers H. A. (1986) Iron metabolism. Clin. Physiol. Biochem. 4, SSIO. Goodbody I. (1974) The physiology of Ascidians. Adc. Mar. Biol. 12, I-149. Hawkins C. J., Parry D. L. and Pierce C. (1980a) Chemistry of the blood of the Ascidian Podoclouella moluccensis. Biol. Bull. 159, 669480. Hawkins C. J., Merifield P. H., Parry D. L., Biggs W. R. and Swinehart J. H. (1980b) Comparative study of the blood
of an ascidian
781
plasma of the Ascidians Pyura stolonijera and Ascidia ceratodes. Biol. Bull. 159, 656-668. Hawkins C. J., Parry D. L., Wood B. J. and Clark P. (1983) Formation of an Iron-Sulfur cluster by the reduction of sulfate with the blood pigment of an Ascidian in the presence of iron. Inorg. Chem. Acta 78, L29-L31. Macara I. G., McLeod G. C. and Kustin K. (1979) Isolation properties and structural studies on a compound iron tunicate blood cells that may be involved in vanadium accumulation. Biochem. J. 181, 457465. Martin A. W., Huebers E., Huebers H., Webb J. and Finch C. A. (1984) A mono-sited transferrin from a representative deuterostome: the Ascidian Pyura stolonifera (Subphylum Urochordata). Blood 984, 1047-1052. Okamura M. Y. and Klotz I. M. (1975) inorganic Biochemistry, pp. 32%343. Elsevier, New York. Reissig J. L., Strominger J. L. and Leloir L. F. (1955) A modified calorimetric method for the estimation of N-acetylamino sugars. J. biol. Chem. 217, 9599966. Sakurai H.. Hirato J. and Michibato H. (1987) EPR characterization of vanadyl (IV) species in blood cells of Ascidians. Biochem. biophys. Res. Commun. 149,411416. Tulhus T. D., Gillum W. W., Carlson R. M. K. and Hodgson K. 0. (1980) Structural study of the vanadium complex in living Ascidian blood cells by X-ray absorption-spectroscopy. J. Am. them. Sot. lOi, 5601567. Webb J. and Chrystal P. (1981) Protein binding of iron in blood plasma of the Ascidian Herdmania momus. Mar. Biol. 63, 107Fl12.