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Metallothionein and cu-chelatin: characterization of metal-binding proteins from tissues of four marine animals
Comp. Biochem. Physiol. Vol. 7011,pp. 93 to 104, 1981
0305-0491/81/090093-12S02.00/0 Copyright © 1981 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
METALLOTHIONEIN AND CU-CHELATIN: CHARACTERIZATION OF METAL-BINDING PROTEINS FROM TISSUES OF FOUR MARINE ANIMALS* J. W. RIDLINGTON,D. C. CHAPMAN,D. E. GOEGERand P. D. WHANGER Department of Agricultural Chemistry, Oregon State University, Corvallis, OR 97331, U.S.A.
(Received 2 February 1981) Abstract--1. Metal binding proteins with mol. wt in the range of 10,000 were isolated from liver and kidney of four different marine species by column chromatography using gel filtration and ionic exchange resins. The proteins were purified with respect to copper, cadmium and zinc. Amino acid analysis was performed on each of the purified proteins. 2. The amino acid compositions of these metal-binding proteins isolated from fish liver and crab hepatopancreases were very similar to that of metallothionein from rat tissues. Other metal-binding proteins were purified from sea lion kidney and liver, whale liver, crab tissues, and fish liver with amino acid composition similar to rat liver copper chelatin. 3. Even though some of these metal-binding proteins were found with mol. wt and amino acid compositions similar to rat metallothionein or Cu-chelatin, there were some metal-binding proteins present which appear to be unique to marine animals.
INTRODUCTION
Metallothionein, a low mol. wt soluble protein rich in cysteine and containing various metals such as Cd, Cu, Zn and Hg was originally isolated and characterized from equine kidney by Margoshes & Vallee (1957). Similar proteins have also been characterized in livers and kidneys of other mammalian species (Pulido et al., 1966, Bremner & Marshall, 1974; Nordberg et al., 1974; Winge & Rajagopalan, 1972). Although the physiological function of metallothionein has not been elucidated, several investigators (Nordberg, 1972; Webb, 1972) proposed its role in the detoxification of heavy metals, whereas others suggested its involvement in Zn metabolism (Chen et al., 1977; Bremner & Davies, 1975). Cu-chelatin, similar in mol. wt to metallothionein, has been identified in a number of mammalian tissues (Winge et al., 1975; Riordan & Gower, 1975; Evans et al., 1975; Shapiro et al., 1961). Cu-chelatin is very different from metallothionein in that it binds predominantly Cu and possesses a much lower cysteine content. Another low mol. metal binding protein has been characterized from the American oyster (Ridlington & Fowler, 1979) which binds predominantly Cd, but has an amino acid composition similar to Cu-chelatin. Metal binding proteins from other non-mammalian sources have been partially characterized from flounder (Brown, 1977), eel (N~Sel-Lambot et al., 1978) and crab (Olafson et al., 1979). Metallothionein like proteins have been found by Olafson & Thompson (1974) in fur and grey seals while Lee et al. (1977) reported proteins similar to metallothionein in mol. wt from the soluble fraction of liver and kidney of California sea lions. * Supported by NIH Grant AM 19285 from NIAMDP. Oregon Agricultural Experiment Station Technical Paper No. 5742. 93
In the present investigation, soluble low mol. wt metal binding proteins were purified from four marine organisms and their characteristics compared to rat Cu-chelatin and metallothionein. Metal binding proteins were isolated by subcellular fractionation and column chromatography from crab hepatopancreases, sea lion liver and kidney, staghorn sculpin liver and whale liver. MATERIALS AND METHODS
Materials and chemicals Fresh crabs (Cancer maoester) were purchased from a commercial source in Newport, OR and kept alive on ice until sacrifice. Sea lion tissues were taken from healthy California sea lions (Zalophus californianus californianus) which were obtained along the Oregon Coast under permit. Sea lion kidneys and livers were removed and kept at O°C for immediate use. Immature Pacific staghorn sculpin (Leptocottus armatus) were collected from tidal channels in Yaquina Bay, OR by beach seine and maintained on a diet of Oregon Moist Pellet for at least 90 days before sacrifice. Whale liver was obtained from sperm whales (Physeter catodon) which beached near Florence, OR in 1979. The whale tissue was stored at -20°C. Biochemicals were purchased from Sigma Chemical Company (St. Louis, MO) and all other chemicals were analytical grade.
Isolation of metal-bindin(i proteins For the isolation of the metal binding proteins, the livers, or in the case of the crabs, the hepatopancreases, were quickly removed from the animals after sacrifice, weighed, and placed onto ice. The tissues were homogenized with 2 vol of 0.05 M Tris buffer, pH 8.4; centrifuged at 10,000 g for 20 rain; and at 100,000g for 90 rain to obtain the final supernatant soluble fraction. This final supernarant fraction was applied to a column (2.0 × 90 cm) of Sephadex (3-75 and eluted with Tris-C1 buffer (pH 8.5, 0.05 M) at- a flow rate of 20 ml/hr. Cadmium, zinc and copper concentrations of column fractions were deter-
J. W. RIDLINGTON et al.
94
mined by direct aspiration of the solution into a Jarrell Ash atomic absorption spectrophotometer (model 3375). Metal binding proteins in the 10,000 tool. wt range from the G-75 Sephadex chromatography were pooled, dialyzed and further chromatographed on DEAE Sephacel using a 2.0 x 8.0 cm column. Fractions were eluted at a flow rate of 20 ml/hr using a linear gradient from 0.003 M Tris to 0.3 M Tris at a constant pH of 8.4. Following analysis of the fractions for metal concentrations, appropriate samples were pooled and freeze dried for additional protein characterization. Disc gel electrophoresis and amino acid analysis. Disc gel electrophoresis was used to assess homogeniety of the metal binding protein preparations using 7% polyacrylamide gels (Jovin et al., 1964). The gels were electrophoresed at 4 mA/gel until the tracking dye had proceeded 90% through the gel. The gels were stained with Coomassie Brilliant blue and destained overnight in a 10% acetic acid solution. Amino acid analysis of the metal binding proteins isolated from DEAE column chromatography were performed using a Beckman model 120 amino acid analyzer with ion exchange resin Dionex DC 6a. Analyses were performed on samples following performic acid oxidation and hydrolysis in HCI (Hirs, 1967).
RESULTS
G-75 Sephadex A typical elution profile of the fish liver supernatant material obtained on Sephadex G-75 is shown in Fig. la. There are two Zn peaks, but Cu was only observed in the lower tool. wt peak. In this peak there was approx 6 times as much Zn as Cu. Cd was not found in any of the fractions. The peak containing both Cu and Zn corresponds to the position where metallothionein from rat liver is usually located, Since proteins with characteristics similar to metaUothionein were being investigated, only Cu, Cd and Zn levels were determined. The fractions in the second peak (50--60) were pooled for further examination. The Sephadex G-75 elution profile obtained with the supernatant material obtained from whale liver is presented in Fig. lb. The first and second peaks contain Zn and Cu but no Cd while the third peak, which is in the 10,000 tool. wt region, possesses only Zn and Cd. The Zn and Cd found in this last peak were present in a molar ratio of 2:1 respectively. The fractions in the last peak were pooled for additional characterization. Figure lc and Fig. ld are representative elution profiles obtained on Sephadex G-75 with supernatant material secured from the hepatopancreases of two individual crabs. Both crabs yielded profiles containing three major peaks, but the metal concentration of the individual peaks varied greatly for each crab. Crab II (Fig. ld) had Cu only in the first peak while Crab I (Fig. lc) possessed Cu in all three peaks with the second peak containing the most. With regard to Cd, Crab I had no Cd in any of the fractions, but Crab II had a substantial concentration of Cd in the peak area corresponding to metailothionein. The distribution of Zn was similar for both crabs in the first and third peaks~ however, in the second peak Crab I contained no Zn where the high levels of Cu were located, while Crab II possessed a significant level of Zn in the same second peak containing the Cd. The third peak which corresponds to a tool. wt less than
1000 had substantially more Zn in this area than tissue from other animals examined. Typical elution profiles obtained from the examination of sea lion liver and kidney supernatants by G-75 Sephadex column chromatography are illustrated in Fig. le and Fig. If respectively. The profiles are similar with regard to the peak distribution of Zn and Cu, but the sea lion liver contains very little Cd while a considerable concentration of Cd is found in both peaks of the kidney. Fractions from the Sephadex G-75 column chromatography corresponding to a mol. wt of 60(~3-10,000 were pooled separately from each animal for additional purification by DEAE-Sephacel column chromatography.
DE AE-Sephacel Further purification of the G-75 Sephadex fractions pooled in the metallothionein area was conducted using DEAE-Sephacel. Figure 2a represents the DEAE Sephacei profile obtained with fish liver. All the metal and protein were bound to the column prior to starting the elution gradient which eluted three distinct peaks, each containing Zn and Cu, but no Cd. These peaks eluted in a position in which rat metallothionein is routinely located. The second and third peaks were pooled and examined by disc gel electrophoresis for purity. The gels are shown in Fig. 3a and illustrate the near homogeniety of each sample and the Rs's of 0.48 and 0.41, respectively, for the second and third peaks. Given in Fig. 2b is the DEAE-Sephacel elution profile obtained with the pooled whale liver fractions after G-75 Sephadex column chromatography. Three predominant peaks were resolved, each containing Cd and Zn in approximately the same proportions, but Cu was not associated with any of the peaks. Figure 3b shows the disc gel obtained with the second peak and reveals a single band with an Rf of 0.38. The profiles obtained with the pooled G-75 Sephadex fractions of Crab I and Crab II on DEAE Sephacel are presented in Fig. 2c and Fig. 2d respectively. Crab I yielded a profile containing only a single Cu containing peak in the usual metallothionein area. No Zn or Cd was detected in any of the Crab I fractions. Similar to the profile of Crab I, Crab II gave only a single peak, but unlike Crab II, it was located in an area where rat metall0thionein is not eluted, and this peak contained predominantly Cd with a small concentration of Zn. The purity of these two pooled peaks was tested by disc gel electrophoresis and provided single bands with Rs's of 0.21 and 0.93, respectively, for Crabs I and II. Only the gel for Crab II is presented in Fig. 3c since the single band for Crab I was too faint to photograph. The two species of metallothionein usually have Rs's of 0.4 and 0.6 on disc gels, thus the metal containing protein of Crab II appears very different from rat metallothionein. The elution pattern obtained with the pooled sea lion liver and kidney after the DEAE Sephacel chromatography step is shown in Fig. 2e and Fig. 2f. Each tissue preparation was fractionated into a number of different peaks. The sea lion liver fractions contained predominantly Cu with a small concentration of Zn and no Cd (Fig. 20. The majority of the Cu was eluted through the DEAE column with the wash buffer prior to the gradient in a manner very
Characterization of metal-binding proteins similar to that observed for the rat Cu-chelatin. The examination of this Cu containing fraction by disc gel electrophoresis revealed the presence of more than one protein and no amino acid analysis was performed. However, the other major Cu containing peak contained only a single protein band with an R s of 0.5 and its amino acid composition was determined. The elution profile of the sea lion kidney on DEAE-Sephacel, similar to the liver material, also exhibited quite a number of peaks, but unlike the liver, a number of these peaks possessed Cd. The majority of all the metals (Cu, Cd, Zn) came through the column in the initial wash and an examination of this first peak by gel electrophoresis showed only a single major protein band with an R s of 0.19 (Fig. 3d). There were two other major metal peaks, the first of these contained mostly Cu and Cd, while the second possessed predominantly Zn and Cu with some Cd. Disc gel electrophoresis demonstrated the near homogeniety of these two proteins with Rs's of 0.51 for both of these proteins (Fig. 3e). Again, as in the liver, the elution position of the first peak and the observed Rf is similar to that observed for rat liver Cu-chelatin.
95
metal levels of the whole tissue basically reflected those found in the supernatant fractions. DISCUSSION
To date the majority of research dealing with metal binding proteins has been conducted employing mammalian tissue obtained from rats, humans, horses and rabbits (Bremner & Davies, 1975; Lee et al., 1977; Nordberg, 1972; Olafson et al., 1979; Webb, 1972). In this study we investigated the metal binding proteins found in a number of marine organisms to determine how these proteins compare to those previously characterized from rats and other animals. Our studies revealed that the marine species possess metal-binding proteins with characteristics very similar to either metallothionein or Cu-chelatin, as well as proteins which bind metals that are very different from either of these proteins. The marine fish, staghorn sculpin, yielded two metal binding peaks from DEAE Sephacel which eluted in locations where rat metallothionein I and II are usually found. Peak II contained approx 10 times as much Zn as peak I and both peaks had some Cu but no Cd. The amino acid analysis of these two homogeneous peaks showed that peak II contained 26~o cysteine acid and an overall Amino acid analysis In Table 1 the amino acid compositions are amino acid composition nearly identical with rat reported for those protein fractions which were essen- metallothionein, while peak I possessed only 8~o cystially homogeneous using disc gel electrophoresis as teic acid and an amino acid composition quite unlike the criterion of judgement. Two protein fractions were metallothionein. On the basis of elution position from analyzed from the fish diver and gave very different Sephadex-G-75 and DEAE-Sephacel and the amino amino acid compositions. The first protein contained acid composition of peak II, it appears that the stag8 ~ cysteine whereas the second protein had 265/o horn sculpin does possess a predominately Zn bindcysteine which is similar to the cysteine content found ing protein similar to rat metallothionein, but only a in metailothioneins. The protein analyzed from the single metallothionein species, unlike the two found in whale liver possessed 12~o cysteine which is in the the rat. The whale liver yielded two metal binding peaks range for levels frequently reported for Cu-chelatin. from DEAE and an overall elution pattern that Analysis of the Crab I protein revealed a 3~o cysteine content and a 7 ~ tyrosine concentration while the appeared very similar to that obtained with the fish, Crab II protein had an amino acid composition re- except that Cd was present in the whale peaks rather sembling metallothionein with a 335/o cysteine and than Cu as in the fish. Zn however was still the preessentially no aromatic amino acids. The three pro- dominant metal present in all peaks. The metal bindtein fractions analyzed from sea lion kidney yielded ing proteins eluted from Sephadex G-75 and DEAEone fraction containing 15~o cysteine, similar to Cu- Sephacel in a manner similar to rat metallothionein, chelatin while the other two protein fractions con- but the amino acid analyses of one of these homotalned approx 3 ~ cysteine and an amino acid compo- geneous metal-binding proteins yielded only 12~o cyssition unlike either Cu-chelatin or metallothionein. teic acid and thus may not be considered metallothioSea lion liver protein had a low cysteine content of nein. However, further examination of the amino acid 3 ~ and an overall amino acid composition similar to composition does reveal a strong resemblance to Cuchelatin, which has been isolated from the rat (Evans one of the sea lion kidney fractions. et al., 1975; Ridlington & Fowler, 1979; Winge & Rajagopolan, 1972), as well as a close resemblance to Metal analyses of tissues the Cd-binding protein identified in the oyster (Pulido The concentrations of Cu, Zn and Cd in the whole et al., 1966). Perhaps further purification of one of the tissue and the 10sg supernatant fractions were deter- minor metal-binding proteins from whale liver would mined; this data is presented in Table 2. For all reveal a metallothionein like protein. tissues analyzed, more than 50% of the metal was The G-75 Sephadex elution profiles obtained from always present in the supernatant material. Nearly all the hepatopancreases of the two crabs differ dramatithe Cd was located in the supernatant material. The cally in that Crab I contains high Cu and no Cd in Cu concentrations in the supernatant fractions exhi- the metaUothionein area while Crab II has high Cd bited a 15 fold range with a low of 1.9 ppm in the and no Cu in the same fraction range. Since these whale liver to a high of 30 ppm in the sea lion liver, crabs were obtained commercially, they apparently while the Zn concentrations had an 8 fold range with came from very different aquatic environments which the fish liver containing the highest with 56 ppm. would account for the striking differences in metal There was no Cd detected in the sea lion liver super- elution patterns. This observed difference in metal natant material, but the kidney retained 6.3 ppm. The content of the two crabs also represents a potential
96
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(f) Fig. 2. DEAE-Sephacel column chromatography profile of the low real. wt fractions from the G-75 Sephadex chromatography of fish fiver (a), whale ]ivcr (b), Crab ! hepatopancrcas¢ (c) Crab ]I hepatopar=crease (d), sea ]ion liver (e) and sea ]ion kidney (~. (A A): A2s0; ( H ) : Zn; (11 II): Cu; 0 O: Cd.
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Table 2. Metal concentration in whole tissue and superngtant fraction /zg metal/g tissue Supernatant Fraction Cu Zn Cd
Sea lion kidney Sea lion liver Whale liver Crab I hepatopancreas Crab II hepatopancreas Fish liver
3.4 30.2 19 26.2 7.8 8.1
16.9 36.3 29.2 7.9 12.7 56.1
6.3 0.0 11.2 0.9 11.7 0.1
Cu 5.4 40.5 3.0 40.8 9.3 ND
Whole Tissue Cd Zn 32.1 62.9 39.7 14.2 16.4 75.9
9.2 0 12.4 1.5 13.2 0.1
utilization of crabs for environmental monitoring of a protein. The only sure way is to purify the protein metals since the metal content of the crab hepatopan- and perform an amino acid analysis on it. Even creas probably merely reflects the metal content of the though none of these proteins are identical to metalcrab's feeding milieu. The DEAE-Sephacel elution lothionein, the amino acid composition of the first profiles obtained from the two crabs were starkly dif- peak from the sea lion kidney tissue from DEAEferent with Crab I having a single Cu containing peak Sephacel shows a close similarity to Cu-chelatin. Not early in the gradient while Crab II yielded a single Cd only is the amino composition nearly identical to Cucontaining peak very near the end of the eluting chelatin, but its R s on disc gels and behavior on G-75 gradient. The elution pattern from Crab I showed no Sephadex and DEAE-Sephacel is the same as that Zn, while Crab II contained some Zn in the same area reported by Irons and Smith for rat Cu-chelatin where the Cd eluted. The observed elution profile (Winge et al., 1975). Another interesting aspect of the from neither crabs resembled those obtained for rat sea lion kidney proteins is that the two metal binding metallothionein, but the amino acid analysis of the proteins which eluted from the DEAE Sephacel single Cd containing peak of Crab II produced a cys- column in positions similar to rat metallothioneins A teic acid content of 33% and an overall amino acid and B respectively have identical amino acid compocomposition virtually identical to rat liver metallo- sitions in which both consist of approx 3% cysteic thionein. This protein of 33% cysteic acid had an R r acid. It thus appears that the sea lion kidney contains of 0.93 whereas rat metallothionein would have an R I metal binding proteins, which like rat metallothioof 0.4 or 0.6 (Webb, 1972). The elution characteristics nein, exists in two isomeric forms. and amino acid composition are still consistent with On the basis of this study, metal binding proteins identifying this Cd-binding crab protein as metallo- exist in marine animals which are very similar in thionein. The Cu-binding protein from Crab I gave an weight and amino acid composition to the rat proamino acid composition unlike Cu-chelatin or metal- teins Cu-chelatin and metallothionein. In addition, lothionein. It thus appears that in a high Cd environ- there are other metal-binding proteins with mol. wt of ment the crab accumulates Cd and this Cd is found approx 10,000 which are perhaps unique to marine associated with a protein that is very similar in amino animals. Finally, as illustrated by the crab study in acid content to rat metallothionein. Evidence for this paper, even though a protein elutes in a position metallothionein in tissues of marine animals has been very different from where rat metallothionein is presented by others (Nordberg et al., 1974; Olafson & usually found (Fig. 2c and d), the protein may still Thompson, 1974). have an amino composition resembling metallothioThe sea lion G-75 Sephadex elution profiles for the nein (Table I). liver and kidney were very different with respect to the metal composition of the metal peak in the REFERENCES 10,000 mol. wt range. The kidney contained both Cu and Cd (Table 2) while the liver had essentially only BREMNER I. & MARSHALL R. B. (1974) Hepatic Cu- and Cu and both tissues possessed only a small concenZn-binding proteins in ruminants. 2. Relationship between Cu and Zn concentrations and the occurrence tration of Zn. Lee et al. (1977) have also observed the of a metallothionein-like fraction. Br. J. Nutr. 32, selective accumulation of Cd in the sea lion kidney as 293-300. opposed to the liver. DEAE-Sephacel chromaBREMnER I. & DAWESN. T. (1975) The induction of metaltography resolved several metal containing peaks lothionein in rat liver by zinc injection and restriction of from the pooled G-75 Sephadex fractions. The amino food intake. Biochem. J. 149, 733-738. acid analysis of those fractions which were homo- BROWND. A. (1977) Increase of Cd and Cd:Zn ratio in the geneous showed the liver fraction possessing 3% cyshigh molecular weight protein pool from apparently norteic acid and those of the kidney constituting 2,4 and mal liver of tumor-bearing flounders (Parophrys vetulus). 15% respectively (Table 1). On account of the low Mar. Biol. 44, 203-209. cysteine content, these proteins cannot be classed as CHEN R. W., VASEYE. J. & WHANGERP. D. (1977) Accumulation and depletion of zinc in rat liver and kidney metallothionein in spite of similarities to metallothiometaliothionein. J. Nutr. 107, 805-813. nein such as metal binding characteristics and position of elution from both G-75 Sephadex and DEAE EVANS G. W., WOLENETZ M. L. & GRACE C. I. (1975) Copper-binding proteins in neonatal and adult rat liver Sephacel. This indicates that even though a metal soluble fraction. Nutr. Rep. Int. 12, 261-269. bound to a protein elutes at the position of metallo- Hms C. H. W. (1967) Determination of cysteine as cysteic thionein on gel filtration, the elution position cannot acid. In Methods in Enzymol, Vol. II, p. 59. Academic be used as the only evidence for identification of such Press, New York.
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IRONS R. D. & SMITH J. C. (1977). Isolation of a nonthionein copper-binding protein from liver of copperinjected rats. Chem.-Biol. Interact. 18, 83-89. JOVlN T., CHRAMBACHA. & N^UOHTON M. A. (1964) An apparatus from preparative temperature-regulated polyacylamide gel electrophoresis. Analyt. Biochem. 9, 351-369. LEE S. S., MATE B. R. VON DER TRENCH K. T., RIMERM^S R. A. & BUHLERD. R. (1977) Metallothionein and subcellular localization of mercury and cadmium in the Californian sea lion. Comp. Biochem. Physiol. 57, 45-53. MARGOSHESM. & VALLEEB. L. (1957) A Cd protein from equine kidney cortex. J. Am. Chem. Soc. 79, 4813-4814. N6EL-LAMBOTF., GERDAYC. & DISTECttEA. (1978) Distribution of Cd, Zn and Cu in liver and gills of the eel anouilla anouilla with special reference to metallothionein. Comp. Biochem. Physiol. 61, 177-189. NORDBFJtOG. F. (1972) Cadmium metabolism and toxicity. Environ. Physiol. Biochem. 2, 7-36. NORDeERG M., TROJENOWSKAB. & NORDBERG G. (1974) Studies on metal-binding proteins of low molecular weight from renal tissue of rabbits exposed to Cd or Hg. Environ. Physiol. Biochem. 4, 149-158. OLAFSON R. W. & THOMPSO~ J. A. (1974) Isolation of heavy metal binding proteins from marine vertebrates. Mar. Biol. 28, 83-86.
OLd'SON R. W., SIM R. G. & BOTO K. G. (1979) Isolation and chemical characterization of the heavy metal-binding protein metallothionein from marine invertebrates. Comp. B~ochem. Physiol. 62, 407-416. PULIDO P., K],G! J. H. R. & VALLEEB. L. (1966) Isolation and some properties of human metallothionein. Biochemistry 5, 1768-1777. RIDLINGTONJ. W. & FOWLER, B. A. (1979). Isolation and partial characterization of a cadmium-binding protein from the American oyster (Crassostrea viroinica). Chem.Biol. Interact. 25, 127-138. RIORD^NJ. R. & GOWER I. (1975) Purification of low molecular weight copper proteins from copper loaded liver. Biochem. biophys. Res. Commun. 66, 678-686. SHAPIRO J., MORELL A. G. & SCHEINBERG I. H. (1961) A copper protein of human liver. J. clin. Invest. 40, 1081. WEBe M. (1972) Protection by zinc against cadmium toxicity. Biochem. Pharmacol. 21, 2767-2771. WINGED. & RAJAGOPALANK. V. (1972) Purification and some properties of Cd-binding protein from rat liver. Archs Biochem. Biophys. 153, 755-762. WINGE D. R., PREMAKUMARR., WILEY R. D. & RAJAGOPAL^N K. V. (1975) Copper-chelatin purification and properties of a copper-binding protein from rat liver. Archs Biochem. Biophys. 170, 253-266.
0305-0491/81/090093-12S02.00/0 Copyright © 1981 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
METALLOTHIONEIN AND CU-CHELATIN: CHARACTERIZATION OF METAL-BINDING PROTEINS FROM TISSUES OF FOUR MARINE ANIMALS* J. W. RIDLINGTON,D. C. CHAPMAN,D. E. GOEGERand P. D. WHANGER Department of Agricultural Chemistry, Oregon State University, Corvallis, OR 97331, U.S.A.
(Received 2 February 1981) Abstract--1. Metal binding proteins with mol. wt in the range of 10,000 were isolated from liver and kidney of four different marine species by column chromatography using gel filtration and ionic exchange resins. The proteins were purified with respect to copper, cadmium and zinc. Amino acid analysis was performed on each of the purified proteins. 2. The amino acid compositions of these metal-binding proteins isolated from fish liver and crab hepatopancreases were very similar to that of metallothionein from rat tissues. Other metal-binding proteins were purified from sea lion kidney and liver, whale liver, crab tissues, and fish liver with amino acid composition similar to rat liver copper chelatin. 3. Even though some of these metal-binding proteins were found with mol. wt and amino acid compositions similar to rat metallothionein or Cu-chelatin, there were some metal-binding proteins present which appear to be unique to marine animals.
INTRODUCTION
Metallothionein, a low mol. wt soluble protein rich in cysteine and containing various metals such as Cd, Cu, Zn and Hg was originally isolated and characterized from equine kidney by Margoshes & Vallee (1957). Similar proteins have also been characterized in livers and kidneys of other mammalian species (Pulido et al., 1966, Bremner & Marshall, 1974; Nordberg et al., 1974; Winge & Rajagopalan, 1972). Although the physiological function of metallothionein has not been elucidated, several investigators (Nordberg, 1972; Webb, 1972) proposed its role in the detoxification of heavy metals, whereas others suggested its involvement in Zn metabolism (Chen et al., 1977; Bremner & Davies, 1975). Cu-chelatin, similar in mol. wt to metallothionein, has been identified in a number of mammalian tissues (Winge et al., 1975; Riordan & Gower, 1975; Evans et al., 1975; Shapiro et al., 1961). Cu-chelatin is very different from metallothionein in that it binds predominantly Cu and possesses a much lower cysteine content. Another low mol. metal binding protein has been characterized from the American oyster (Ridlington & Fowler, 1979) which binds predominantly Cd, but has an amino acid composition similar to Cu-chelatin. Metal binding proteins from other non-mammalian sources have been partially characterized from flounder (Brown, 1977), eel (N~Sel-Lambot et al., 1978) and crab (Olafson et al., 1979). Metallothionein like proteins have been found by Olafson & Thompson (1974) in fur and grey seals while Lee et al. (1977) reported proteins similar to metallothionein in mol. wt from the soluble fraction of liver and kidney of California sea lions. * Supported by NIH Grant AM 19285 from NIAMDP. Oregon Agricultural Experiment Station Technical Paper No. 5742. 93
In the present investigation, soluble low mol. wt metal binding proteins were purified from four marine organisms and their characteristics compared to rat Cu-chelatin and metallothionein. Metal binding proteins were isolated by subcellular fractionation and column chromatography from crab hepatopancreases, sea lion liver and kidney, staghorn sculpin liver and whale liver. MATERIALS AND METHODS
Materials and chemicals Fresh crabs (Cancer maoester) were purchased from a commercial source in Newport, OR and kept alive on ice until sacrifice. Sea lion tissues were taken from healthy California sea lions (Zalophus californianus californianus) which were obtained along the Oregon Coast under permit. Sea lion kidneys and livers were removed and kept at O°C for immediate use. Immature Pacific staghorn sculpin (Leptocottus armatus) were collected from tidal channels in Yaquina Bay, OR by beach seine and maintained on a diet of Oregon Moist Pellet for at least 90 days before sacrifice. Whale liver was obtained from sperm whales (Physeter catodon) which beached near Florence, OR in 1979. The whale tissue was stored at -20°C. Biochemicals were purchased from Sigma Chemical Company (St. Louis, MO) and all other chemicals were analytical grade.
Isolation of metal-bindin(i proteins For the isolation of the metal binding proteins, the livers, or in the case of the crabs, the hepatopancreases, were quickly removed from the animals after sacrifice, weighed, and placed onto ice. The tissues were homogenized with 2 vol of 0.05 M Tris buffer, pH 8.4; centrifuged at 10,000 g for 20 rain; and at 100,000g for 90 rain to obtain the final supernatant soluble fraction. This final supernarant fraction was applied to a column (2.0 × 90 cm) of Sephadex (3-75 and eluted with Tris-C1 buffer (pH 8.5, 0.05 M) at- a flow rate of 20 ml/hr. Cadmium, zinc and copper concentrations of column fractions were deter-
J. W. RIDLINGTON et al.
94
mined by direct aspiration of the solution into a Jarrell Ash atomic absorption spectrophotometer (model 3375). Metal binding proteins in the 10,000 tool. wt range from the G-75 Sephadex chromatography were pooled, dialyzed and further chromatographed on DEAE Sephacel using a 2.0 x 8.0 cm column. Fractions were eluted at a flow rate of 20 ml/hr using a linear gradient from 0.003 M Tris to 0.3 M Tris at a constant pH of 8.4. Following analysis of the fractions for metal concentrations, appropriate samples were pooled and freeze dried for additional protein characterization. Disc gel electrophoresis and amino acid analysis. Disc gel electrophoresis was used to assess homogeniety of the metal binding protein preparations using 7% polyacrylamide gels (Jovin et al., 1964). The gels were electrophoresed at 4 mA/gel until the tracking dye had proceeded 90% through the gel. The gels were stained with Coomassie Brilliant blue and destained overnight in a 10% acetic acid solution. Amino acid analysis of the metal binding proteins isolated from DEAE column chromatography were performed using a Beckman model 120 amino acid analyzer with ion exchange resin Dionex DC 6a. Analyses were performed on samples following performic acid oxidation and hydrolysis in HCI (Hirs, 1967).
RESULTS
G-75 Sephadex A typical elution profile of the fish liver supernatant material obtained on Sephadex G-75 is shown in Fig. la. There are two Zn peaks, but Cu was only observed in the lower tool. wt peak. In this peak there was approx 6 times as much Zn as Cu. Cd was not found in any of the fractions. The peak containing both Cu and Zn corresponds to the position where metallothionein from rat liver is usually located, Since proteins with characteristics similar to metaUothionein were being investigated, only Cu, Cd and Zn levels were determined. The fractions in the second peak (50--60) were pooled for further examination. The Sephadex G-75 elution profile obtained with the supernatant material obtained from whale liver is presented in Fig. lb. The first and second peaks contain Zn and Cu but no Cd while the third peak, which is in the 10,000 tool. wt region, possesses only Zn and Cd. The Zn and Cd found in this last peak were present in a molar ratio of 2:1 respectively. The fractions in the last peak were pooled for additional characterization. Figure lc and Fig. ld are representative elution profiles obtained on Sephadex G-75 with supernatant material secured from the hepatopancreases of two individual crabs. Both crabs yielded profiles containing three major peaks, but the metal concentration of the individual peaks varied greatly for each crab. Crab II (Fig. ld) had Cu only in the first peak while Crab I (Fig. lc) possessed Cu in all three peaks with the second peak containing the most. With regard to Cd, Crab I had no Cd in any of the fractions, but Crab II had a substantial concentration of Cd in the peak area corresponding to metailothionein. The distribution of Zn was similar for both crabs in the first and third peaks~ however, in the second peak Crab I contained no Zn where the high levels of Cu were located, while Crab II possessed a significant level of Zn in the same second peak containing the Cd. The third peak which corresponds to a tool. wt less than
1000 had substantially more Zn in this area than tissue from other animals examined. Typical elution profiles obtained from the examination of sea lion liver and kidney supernatants by G-75 Sephadex column chromatography are illustrated in Fig. le and Fig. If respectively. The profiles are similar with regard to the peak distribution of Zn and Cu, but the sea lion liver contains very little Cd while a considerable concentration of Cd is found in both peaks of the kidney. Fractions from the Sephadex G-75 column chromatography corresponding to a mol. wt of 60(~3-10,000 were pooled separately from each animal for additional purification by DEAE-Sephacel column chromatography.
DE AE-Sephacel Further purification of the G-75 Sephadex fractions pooled in the metallothionein area was conducted using DEAE-Sephacel. Figure 2a represents the DEAE Sephacei profile obtained with fish liver. All the metal and protein were bound to the column prior to starting the elution gradient which eluted three distinct peaks, each containing Zn and Cu, but no Cd. These peaks eluted in a position in which rat metallothionein is routinely located. The second and third peaks were pooled and examined by disc gel electrophoresis for purity. The gels are shown in Fig. 3a and illustrate the near homogeniety of each sample and the Rs's of 0.48 and 0.41, respectively, for the second and third peaks. Given in Fig. 2b is the DEAE-Sephacel elution profile obtained with the pooled whale liver fractions after G-75 Sephadex column chromatography. Three predominant peaks were resolved, each containing Cd and Zn in approximately the same proportions, but Cu was not associated with any of the peaks. Figure 3b shows the disc gel obtained with the second peak and reveals a single band with an Rf of 0.38. The profiles obtained with the pooled G-75 Sephadex fractions of Crab I and Crab II on DEAE Sephacel are presented in Fig. 2c and Fig. 2d respectively. Crab I yielded a profile containing only a single Cu containing peak in the usual metallothionein area. No Zn or Cd was detected in any of the Crab I fractions. Similar to the profile of Crab I, Crab II gave only a single peak, but unlike Crab II, it was located in an area where rat metall0thionein is not eluted, and this peak contained predominantly Cd with a small concentration of Zn. The purity of these two pooled peaks was tested by disc gel electrophoresis and provided single bands with Rs's of 0.21 and 0.93, respectively, for Crabs I and II. Only the gel for Crab II is presented in Fig. 3c since the single band for Crab I was too faint to photograph. The two species of metallothionein usually have Rs's of 0.4 and 0.6 on disc gels, thus the metal containing protein of Crab II appears very different from rat metallothionein. The elution pattern obtained with the pooled sea lion liver and kidney after the DEAE Sephacel chromatography step is shown in Fig. 2e and Fig. 2f. Each tissue preparation was fractionated into a number of different peaks. The sea lion liver fractions contained predominantly Cu with a small concentration of Zn and no Cd (Fig. 20. The majority of the Cu was eluted through the DEAE column with the wash buffer prior to the gradient in a manner very
Characterization of metal-binding proteins similar to that observed for the rat Cu-chelatin. The examination of this Cu containing fraction by disc gel electrophoresis revealed the presence of more than one protein and no amino acid analysis was performed. However, the other major Cu containing peak contained only a single protein band with an R s of 0.5 and its amino acid composition was determined. The elution profile of the sea lion kidney on DEAE-Sephacel, similar to the liver material, also exhibited quite a number of peaks, but unlike the liver, a number of these peaks possessed Cd. The majority of all the metals (Cu, Cd, Zn) came through the column in the initial wash and an examination of this first peak by gel electrophoresis showed only a single major protein band with an R s of 0.19 (Fig. 3d). There were two other major metal peaks, the first of these contained mostly Cu and Cd, while the second possessed predominantly Zn and Cu with some Cd. Disc gel electrophoresis demonstrated the near homogeniety of these two proteins with Rs's of 0.51 for both of these proteins (Fig. 3e). Again, as in the liver, the elution position of the first peak and the observed Rf is similar to that observed for rat liver Cu-chelatin.
95
metal levels of the whole tissue basically reflected those found in the supernatant fractions. DISCUSSION
To date the majority of research dealing with metal binding proteins has been conducted employing mammalian tissue obtained from rats, humans, horses and rabbits (Bremner & Davies, 1975; Lee et al., 1977; Nordberg, 1972; Olafson et al., 1979; Webb, 1972). In this study we investigated the metal binding proteins found in a number of marine organisms to determine how these proteins compare to those previously characterized from rats and other animals. Our studies revealed that the marine species possess metal-binding proteins with characteristics very similar to either metallothionein or Cu-chelatin, as well as proteins which bind metals that are very different from either of these proteins. The marine fish, staghorn sculpin, yielded two metal binding peaks from DEAE Sephacel which eluted in locations where rat metallothionein I and II are usually found. Peak II contained approx 10 times as much Zn as peak I and both peaks had some Cu but no Cd. The amino acid analysis of these two homogeneous peaks showed that peak II contained 26~o cysteine acid and an overall Amino acid analysis In Table 1 the amino acid compositions are amino acid composition nearly identical with rat reported for those protein fractions which were essen- metallothionein, while peak I possessed only 8~o cystially homogeneous using disc gel electrophoresis as teic acid and an amino acid composition quite unlike the criterion of judgement. Two protein fractions were metallothionein. On the basis of elution position from analyzed from the fish diver and gave very different Sephadex-G-75 and DEAE-Sephacel and the amino amino acid compositions. The first protein contained acid composition of peak II, it appears that the stag8 ~ cysteine whereas the second protein had 265/o horn sculpin does possess a predominately Zn bindcysteine which is similar to the cysteine content found ing protein similar to rat metallothionein, but only a in metailothioneins. The protein analyzed from the single metallothionein species, unlike the two found in whale liver possessed 12~o cysteine which is in the the rat. The whale liver yielded two metal binding peaks range for levels frequently reported for Cu-chelatin. from DEAE and an overall elution pattern that Analysis of the Crab I protein revealed a 3~o cysteine content and a 7 ~ tyrosine concentration while the appeared very similar to that obtained with the fish, Crab II protein had an amino acid composition re- except that Cd was present in the whale peaks rather sembling metallothionein with a 335/o cysteine and than Cu as in the fish. Zn however was still the preessentially no aromatic amino acids. The three pro- dominant metal present in all peaks. The metal bindtein fractions analyzed from sea lion kidney yielded ing proteins eluted from Sephadex G-75 and DEAEone fraction containing 15~o cysteine, similar to Cu- Sephacel in a manner similar to rat metallothionein, chelatin while the other two protein fractions con- but the amino acid analyses of one of these homotalned approx 3 ~ cysteine and an amino acid compo- geneous metal-binding proteins yielded only 12~o cyssition unlike either Cu-chelatin or metallothionein. teic acid and thus may not be considered metallothioSea lion liver protein had a low cysteine content of nein. However, further examination of the amino acid 3 ~ and an overall amino acid composition similar to composition does reveal a strong resemblance to Cuchelatin, which has been isolated from the rat (Evans one of the sea lion kidney fractions. et al., 1975; Ridlington & Fowler, 1979; Winge & Rajagopolan, 1972), as well as a close resemblance to Metal analyses of tissues the Cd-binding protein identified in the oyster (Pulido The concentrations of Cu, Zn and Cd in the whole et al., 1966). Perhaps further purification of one of the tissue and the 10sg supernatant fractions were deter- minor metal-binding proteins from whale liver would mined; this data is presented in Table 2. For all reveal a metallothionein like protein. tissues analyzed, more than 50% of the metal was The G-75 Sephadex elution profiles obtained from always present in the supernatant material. Nearly all the hepatopancreases of the two crabs differ dramatithe Cd was located in the supernatant material. The cally in that Crab I contains high Cu and no Cd in Cu concentrations in the supernatant fractions exhi- the metaUothionein area while Crab II has high Cd bited a 15 fold range with a low of 1.9 ppm in the and no Cu in the same fraction range. Since these whale liver to a high of 30 ppm in the sea lion liver, crabs were obtained commercially, they apparently while the Zn concentrations had an 8 fold range with came from very different aquatic environments which the fish liver containing the highest with 56 ppm. would account for the striking differences in metal There was no Cd detected in the sea lion liver super- elution patterns. This observed difference in metal natant material, but the kidney retained 6.3 ppm. The content of the two crabs also represents a potential
96
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Table 2. Metal concentration in whole tissue and superngtant fraction /zg metal/g tissue Supernatant Fraction Cu Zn Cd
Sea lion kidney Sea lion liver Whale liver Crab I hepatopancreas Crab II hepatopancreas Fish liver
3.4 30.2 19 26.2 7.8 8.1
16.9 36.3 29.2 7.9 12.7 56.1
6.3 0.0 11.2 0.9 11.7 0.1
Cu 5.4 40.5 3.0 40.8 9.3 ND
Whole Tissue Cd Zn 32.1 62.9 39.7 14.2 16.4 75.9
9.2 0 12.4 1.5 13.2 0.1
utilization of crabs for environmental monitoring of a protein. The only sure way is to purify the protein metals since the metal content of the crab hepatopan- and perform an amino acid analysis on it. Even creas probably merely reflects the metal content of the though none of these proteins are identical to metalcrab's feeding milieu. The DEAE-Sephacel elution lothionein, the amino acid composition of the first profiles obtained from the two crabs were starkly dif- peak from the sea lion kidney tissue from DEAEferent with Crab I having a single Cu containing peak Sephacel shows a close similarity to Cu-chelatin. Not early in the gradient while Crab II yielded a single Cd only is the amino composition nearly identical to Cucontaining peak very near the end of the eluting chelatin, but its R s on disc gels and behavior on G-75 gradient. The elution pattern from Crab I showed no Sephadex and DEAE-Sephacel is the same as that Zn, while Crab II contained some Zn in the same area reported by Irons and Smith for rat Cu-chelatin where the Cd eluted. The observed elution profile (Winge et al., 1975). Another interesting aspect of the from neither crabs resembled those obtained for rat sea lion kidney proteins is that the two metal binding metallothionein, but the amino acid analysis of the proteins which eluted from the DEAE Sephacel single Cd containing peak of Crab II produced a cys- column in positions similar to rat metallothioneins A teic acid content of 33% and an overall amino acid and B respectively have identical amino acid compocomposition virtually identical to rat liver metallo- sitions in which both consist of approx 3% cysteic thionein. This protein of 33% cysteic acid had an R r acid. It thus appears that the sea lion kidney contains of 0.93 whereas rat metallothionein would have an R I metal binding proteins, which like rat metallothioof 0.4 or 0.6 (Webb, 1972). The elution characteristics nein, exists in two isomeric forms. and amino acid composition are still consistent with On the basis of this study, metal binding proteins identifying this Cd-binding crab protein as metallo- exist in marine animals which are very similar in thionein. The Cu-binding protein from Crab I gave an weight and amino acid composition to the rat proamino acid composition unlike Cu-chelatin or metal- teins Cu-chelatin and metallothionein. In addition, lothionein. It thus appears that in a high Cd environ- there are other metal-binding proteins with mol. wt of ment the crab accumulates Cd and this Cd is found approx 10,000 which are perhaps unique to marine associated with a protein that is very similar in amino animals. Finally, as illustrated by the crab study in acid content to rat metallothionein. Evidence for this paper, even though a protein elutes in a position metallothionein in tissues of marine animals has been very different from where rat metallothionein is presented by others (Nordberg et al., 1974; Olafson & usually found (Fig. 2c and d), the protein may still Thompson, 1974). have an amino composition resembling metallothioThe sea lion G-75 Sephadex elution profiles for the nein (Table I). liver and kidney were very different with respect to the metal composition of the metal peak in the REFERENCES 10,000 mol. wt range. The kidney contained both Cu and Cd (Table 2) while the liver had essentially only BREMNER I. & MARSHALL R. B. (1974) Hepatic Cu- and Cu and both tissues possessed only a small concenZn-binding proteins in ruminants. 2. Relationship between Cu and Zn concentrations and the occurrence tration of Zn. Lee et al. 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