Two metallothioneins in the shore crab Carcinus maenas

Comp. Biochem. Physiol. Vol. 83A, No. 1, pp. 149 156, 1986 0300-9629/86 $3.00+0.00 ,© 1986 Pergamon Press Ltd Printed in Great Britain TWO M E T A L L O T H I O N E I N S IN THE SHORE CRAB CARCINUS MAENAS V. W. T. WONG and P. S. RAINBOW School of Biological Sciences, Queen Mary College, University of London, Mile End Road, London E1 4NS, UK. Telephone: 01-980-4811 (Received 8 May 1985) Abstract--1. Two proteins (mol. wt 10,100 and 4100 respectively, as estimated by Sephadex G-50 chromatography) occur in the hepatopancreas of Carcinus maenas, binding variable amounts of Cu, Zn and Cd. 2. Both proteins give UV absorbance traces with a shoulder at 254nm (characteristic of metal-mercaptide bonds), reversibly lost on acidification. 3. It is concluded that both proteins are metallothioneins. INTRODUCTION Low mol. wt metal-binding ligands have been reported from many invertebrates (see Roesijadi, 1981) but their identities have remained elusive. Some have been shown to possess metallothionein (MT)-like properties and their identifications as such have been accepted, while others appear to differ from MTs. Much of the confusion over identities has, however, arisen from masking by other impurities present as a result of inadequate purification. Consequently, the reported absence of MTs in particular invertebrates (Coombs, 1974; Howard and Nickless, 1978; George et al., 1979; Marshall and Talbot, 1979; Ridlington and Fowler, 1979; Rainbow et al., 1980; Roesijadi, 1981; Lyon et al., 1983) should be treated with some scepticism. For example, of the crustaceans studied, crabs (Jennings et al., 1979; Rainbow and Scott, 1979; Olafson et al., 1979a,b; Overnell and Trewhella, 1979; Overnell, 1982a,b, 1984a,b; Engel and Brouwer, 1984), lobsters (Ray and White, 1981; Engel and Brouwer, 1984) and shrimps (Olafson et al., 1979; White and Rainbow, 1986) appear to possess MTs while crayfish (Lyon et al., 1983) and barnacles (Rainbow et al., 1980) have been reported as not doing so. It is possible that in at least some of the above-mentioned cases, insufficient purifications have led to erroneous conclusions. Working on the shore crab Carcinus maenas, Jennings et al. (1979) and Rainbow and Scott (1979) concluded that two MT-like ligands were present, of about 12,000 and 27,000mol. wt, as estimated by molecular-sieve chromatography. More recent studies (Minkel et al., 1980) have, however, shown that in preparative gel chromatography, oxidation artefacts can be brought about by the aggregation of low mol.wt metal-binding ligands unless reducing conditions are maintained. MTs appear to be particularly susceptible to this phenomenon (Minkel et al., 1980), possessing a very high cysteine content and forming cross-linking disulphide bridges. Recent work has also implicated lysine residues in aggregation artefacts (Templeton and Cherian, 1984). It can be postulated, then, that oxidative aggregation may lead 149 to the apparent discovery of artifactually produced different forms of MT including dimers and other polymers, or even to the apparent absence of any MT whatsoever. The aim of this paper is to describe the identity and number of MT-like ligands occurring naturally in Carcinus m a e n a s from the Firth of Clyde, Scotland, adopting certain parameters for identification: (1) Reducing conditions must be maintained during preparative gel chromatography to prevent the formation of aggregation artefacts. (2) Sufficient purification must be performed to remove other impurities before investigation of MT-like properties; a widely used 2-step purification procedure consisting of molecular-sieve chromatography followed by ion-exchange chromatography will therefore be employed. (3) In the absence of other protein impurities, the identification of MT is to be based on a characteristic UV absorbance at 254nm, reversibly altered by acidification. MATERIALS AND METHODS Reducing conditions were maintained throughout extraction with either 2-mercaptoethanol (2-M) or dithiothreitol (DTT) (see following paper, Wong and Rainbow, 1986). Large male crabs, Carcinus maenas (L.), were obtained from the University Marine Biological Station, Millport, Scotland, after collection sublittorally, in creels or by trawling. Crabs were killed by freezing overnight, then thawed and the hepatopancreas dissected out and placed into a beaker kept on ice. An approximately equal volume of homogenizing buffer was then added, Tris-HCl (0.02 M Tris, 0.01 M NaC1, HC1 added to adjust the pH to 8.6) with 0.1mM phenylmethylsulphonyl fluoride (PMSF) to prevent protease activity and either 14mM 2-M or 1 mM DTT to maintain reducing conditions equivalent to the 5 mM concentration of thiol groups in liver tissue (Jocelyn, 1972; Minkel et al., 1980). The mixture was homogenized before centrifuging at 25,000g for 3 hr. The supernatant was applied onto either a Sephadex G-75 or a Sephadex G-50 column and eluted with Tris-HCl buffer (pH 8.6) with either 2 mM 2-M or 0.5 mM DTT to maintain a reducing environment. 150 V.W.T. Woyc, and P. S. RAINBOW Selected fractions were used for ion-exchange chromatography on DEAE-32 cellulose, being eluted off with a 0.02 M Tris HCI buffer gradient of increasing ionic strength (c. 2-35 mS, 0.0l-0.4 M NaCl). Metal analysis (atomic absorption spectrophotometry, Varian AA 375 spectrophotometer) of selected eluted fractions was performed after each purification step to check for the eluting position of the metal-binding ligands. Ultraviolet absorbance scanning was performed after ionexchange chromatography. Polyacrylamide gel electrophoresis was also performed on samples after ion exchange chromatography according to Ornstein and Davis (1962) and silver-stainedaccording to the technique of Merril et al. (1981) modified by extensive washing in double-distilled water between treatment stages. of the longer period of protection (about a month) provided by the former (Cleland, 1964; Mao, 1967). In the low mol. wt range, resolution is greatly improved with material in this range now eluting off the column in the linear separation range. Three metal peaks are clearly resolved. The first two peaks binding Cu, Zn and Cd have been termed MT-like peaks (MT-I and MT-2) whereas the third peak appears to bind only Zn. MT-1, MT-2 and the Zn peak have tool. wts of 10,100, 4100 and < 1500 respectively as estimated on Sephadex G-50. Figure 3 is a preliminary ion-exchange profile from a short column (0.8 x 20 cm) of fractions pooled from the low mol. wt range on Sephadex G-75 (e.g. 60(~1000ml, Fig. 1). Three metal peaks are clearly seen. The Zn peak (l) eluted first to be followed by the 2 MT-like peaks (now labelled II and III). Figure 4 is an ion-exchange profile of similar pooled fractions from Sephadex G-75 (equivalent to 600-1000 ml, Fig. 1) performed on a longer column (1.6 × 40cm) in an attempt to achieve better separation indicated by the 254 nm absorbance profiles. Comparison of the absorbance profiles of Figs 3 and 4 suggests that resolution has been improved. The 254 absorbance trace will reflect the presence of MT as a result of the characteristic absorbance of the mercaptide metal bond, and also of other proteins if present in sufficient quantity, although these would be better detected at 280 nm (characteristic absorbance by constituent aromatic amino acid residues). Absorbance scales are arbitrary and may not be used for comparison of absolute levels between elutions. RESULTS Figure 1 is a typical Sephadex G-75 (linear separation range 70,00(~5000 mol. wt) elution profile derived from hepatopancreas tissue pooled from male crabs, reducing conditions being maintained throughout by the presence of fresh 2-M. No significant levels of Cd occur in the void volume, confirming that no oxidation has occurred (Minkel et al., 1980). In the low mol. wt range, at least two or even three metal peaks can be distinguished but resolution here is poor. It was therefore decided to use Sephadex G-50 (linear separation range 30,00(~1500mol. wt) to achieve better resolution. Figure 2 is a typical Sephadex G-50 elution profile of a single crab hepatopancreas obtained under reducing conditions, using DTT instead of 2-M because Cu,Zn 2.5 KEY Cu 7n 2-0 E --.-- Cd --+-A b s _2_5_4nm_ 1-'3- / 0 / -6 .10' 10k,,. j / / / / /F" x \ \\ \ \ I f I I \ \k \ \ \ , x / "--" // """ \/.\. \\ , x •05 0-5' \ L • 0 O" .~'*"J 20O .f" +_4- + ~,~ ~oo 66o e6o ~o'oo Elution volumem[ Fig. 1. Typical Sephadex G-75 elution profile from crab hepatopancreas. Sample was homogenized in Tris HCI buffer with 14mM 2-M and eluted in Tris HC1 buffer with 2raM 2-M. Absorbance at 254nm in arbitrary units, bed vol. - 860 ml, flow rate = 77 ml hr ~. Carcinus Two metallothioneins in 151 Cu~Zn 2.5. I I I KEY I Cu Zn --.-Cd --+-Abs. _2.5_4_nm_ I | I i I I 2.0. I I I I I 'T E I | I i I 1.5. I I I I I I I I I I i Cd ~)-10 1.0' i I I \ d \ u MT I \\ I I[ "(I I .05 0.5 ,, MT 2 + / I ~, \\ /,. "+ -- ÷ --~ -'~.lll-- i x O" 0 ~ 2OO I'eIF "÷ / Zn peak / ,, / / .N ~" .<~b~"1 -+ +\ ..,,, \ "~,~ ~ t I x + \ 600 400 800 Elation volume rnt 10'00 Fig. 2. Sephadex G-50 elution profile for a single male crab hepatopancreas. Sample was homogenized in Tris-HC1 buffer with 1 mM DTT added and eluted in Tris-HC1 buffer with 0.5 mM DTT added. Absorbance at 254 nm in arbitrary units, bed vol. = 860 ml, flow rate = 27 ml h r - t. Cu,Zn li.] KEY fl // 1.2 / I / 10- 1.0. Cu Zn --.-- 1 Cd --~ -- i X I / Cd I / Abs. _2_5_~n_m_ 3prlmol el~q,~q po,nt i I -/.0 +7 E I 2 o / / / Og 08" / // o / -30 d / ,~ :E 06 0.6" A / i L// /~ / / / 0L 04" -20 :'; / / Ill u J I t 02 0.2" a. 0 / .10 _.~_-- iI °" 6 11 ,b + 2'o 3'0 ~\ +~+~+ go ÷ 5b Elution rot. 60 .0 rnl Fig. 3. Elution profile of selected (see text) Sephadex G-75 fractions on small ion-exchange column (gradient = 5 × bed vol.). Absorbance at 254nm in arbitrary units, bed vol. = 10ml, flow rate = 40ml hr ~. 152 V.W.T. WONG and P. S. RAINBOW Cu,Zn KEY Cu ,-40 I -.- rl Zn II If It Cd --+-Abs. 254nm ..... 'l ~ X opt~ol ebting ~ t .80 / [~ _l I\ . i[~ d "~ ~ ..... / III , £x I ~ E a / / -" I .6C I "~ Cd / / t I t ' "7 04 ', / / ,%--:~ ',L .~,~--~-- , / b~ ~ AI b°',l ', / ,'' ', .4 l I / \\,, > (,9 ; o .o2 .2 i\ I. It, , 0 .~ ~ " / --".Z--~--I-Cp *\ I / II +\ ". . . . . "-* * ~ . ,...,,'--~ " ~ .... l ~'>"+~-r~'*',~ +;;~'~'~--* ~ " * - : - * - * ' ' ~ + ~ t - + 0 100 200 300 .,o X, /'-_ \+ . ~.. \ ~+ _-~"~,__~ .~. ...... . 500 600 Elutionrot. mI "x~ • 400 -0 Fig. 4. Elution profile of selected (see text) Sephadex G-75 fractions on large ion-exchange column (gradient=6×bed vol.). A b s o r b a n c e at 2 5 4 n m in arbitrary units, bed v o l . = 6 0 m l , flow rate = 70 ml h r - ~. The absorbance profile in Fig. 4 can be approximately classified into a large broad peak eluting between the start of the gradient to 17 mS conductivity, a low broad peak between 17 and 24mS conductivity, and a sharp terminal peak at about Cu,Z I I f .8C / \ -, i! / i I I I I t I t~ / ~ , g,II ~I 25 mS. Three metal peaks are again in evidence--the Zn peak (I) followed by the 2 MT-like peaks (II and III both binding Cu, Zn and Cd). In an attempt to further separate peak I (Zn peak) from peak II (MT-1) a longer gradient was used (Fig. KEY Cu Zn -- , - Cd --+-Abs. _2...5_~nm_ ,j~j~ 1.0 X ~timaL ebting point e .30 .60 ¸ ~O t ! E u I / / I ./~0 I ,2 / .7\. / I 0 I I .20 ,, / /" / /. / . -'/ \ I I i ~ \ I ?, " X \~ // - .-"~ E .20~ -'" z "~ Ill X , b " ~ ' ~ ~Z,m-- .~\-"'~ A,,-,\ / .. ~ ~\ -- "% "10 .... ::_:_j~" :. - 1O0 2()0 300 /~00 500 600 Ebtion vol. mI Fig. 5. Elution profile of selected (see text) Sephadex G-75 fractions on large ion-exchange column (gradient = 10 × bed vol.). A b s o r b a n c e at 2 5 4 n m in arbitrary units, bed vol. = 60ml, flow rate = 7 0 m l h r t. 700 Two metallothioneins in Carcinus 153 MT-like fractions from ion-exchange (peaks II and III) were scanned on a UV absorbance spectrophotometer and both found to possess a "shoulder" at about 254 nm with minimal absorbance at 280 nm indicating a lack of aromatic amino acids. In each case, this shoulder disappeared on acidification and reappeared with neutralization (Fig. 7) indicating the presence of a mercaptide-metal bound very characteristic of MT (K/igi and N6rdberg, 1979). Peaks II and III after ion-exchange were also subjected to gel electrophoresis to check for homogeneity and stained with a modified silver-staining technique after Merril e t al. (1981) (Fig. 8). A band recurring in all gels close to the bottom is the bromophenol blue marker, which had thickened during staining. Peak II (Figs 4-6) is homogenous (single band R.F. = 0.90) whereas Peak III (Figs 4-6) is a heterogeneous mixture (three bands R.F.s = 0.05; 0.66; 0.80). In the peak III sample, the uppermost band (R.F. = 0.05) is very sharp and is probably of high mol. wt for Tasheva and Dessev (1983) showed that high mol. wt compounds generally have less 5) but this did not improve the resolution between the two peaks. The peaks are also more diffused and metal concentrations were diluted by the larger eluting volume. Hence, Cd was not detectable. Thus a gradient of about 6 × the bed volume appeared to be most suitable for resolution. Therefore pooled fractions from Sephadex G-50 (equivalent to MT-I and MT-2, Fig. 2) were separately applied to and eluted off the ion-exchange column. Figure 6 confirms the presence of two distinct MT-like peaks in each case after ion-exchange chromatography, showing cross-contamination in both cases. These two peaks correspond to the previous two MT-like peaks on Sephadex G-50. Numerous elutions confirmed that peak MT-1 and peak MT-2 on Sephadex G-50 (Fig. 2) correspond to peaks II and III respectively after ion-exchange. The ionexchange protein profiles for samples from Sephadex G-50 are well resolved into only two distinct peaks as opposed to the multiple peaks on the ion-exchange protein profiles for pooled samples from Sephadex G-75, indicating an improved initial purification with Sephadex G-50. Cu.Z~bC ~ O. II KEY Cu .8c Zn "7 -g -/~0 . • _ Cd - - * - Abs fl_5_4_nm_ / / x optPmolelutingpoint / / ~, 6O .30 / / c Am / / / / / / 40 / / / -20 i~ / / I II ,X/x I/ / 20- \'x I f I . ; ",\ -10 X i/ I O" _2~2JZJ 200 0 ]00 l.O0 m[ 500 [u,Zn,Cd ~.0" b. 20~, 1 /u / E "T > f / 20 / u ! 0 0 / / / /. I u.x" / 100 ~/ / \ / \ / III \ /--~, 200 i 300 Ebticn yd.. ml Fig. 6. Elution profiles of selected Sephadex G-50 fractions corresponding to (a) MT-I and (b) MT-2 (Fig. 2) (see text) on large ion-exchange column (gradient = 6 x bed vol.). Absorbance at 254 nm in arbitrary units, bed vol. = 60 ml, flow rate = 70 ml hr ~. 154 V. W. T. WONGand P. S. RAINBOW peak II high mw oxidation artefact ? peak Ill F-----q 0.05 peak II 6,0 direction of migration 1 0.90 MT2 ~=~ 0.66 MT1 ~ 0.B0 brom-pt~¢~o[ blue I I I i I 260 2ao I I I e I 280 r ~ I I ~~ I 300 Fig. 8. Gel electrophoresis (silver-stained) of ion-exchange fractions equivalent to peaks II and III (see text). Quoted figures are R.F. values. I not being perfectly homogeneous (Tasheva and Dessev, 1983). Therefore the single band in the peak II sample corresponds to MTI and the second band in the peak III sample to MT 2 with the third band very likely to be high mol. wt oxidation artefacts possibly from MTs. Table 1 compares the reported R.F.s of MTs on gel electrophoresis from various sources. An attempt is made here to classify the reported R.F.s into four postulated MT types MT1, MT2 and two forms of oxidized MT and there appears to be consistency based on this tabulation. The characteristics of the Zn peak will be detailed in a forthcoming publication. peok111 g lo #> 2/.,0 260 2BOnm300 DISCUSSION Fig. 7. UV absorbance spectra of ion-exchange fractions equivalent to peaks II and IlI (see text), subjected to acidification and neutralization. Extinction in arbitrary units. mobility than smaller mol. wt compounds. It may therefore represent a high mol. wt oxidation artefact. This band could be stained with either silver or Coomassie blue. The second and third bands are broader and probably represent the presence of considerably more material. The third band (R.F. = 0.90) could correspond to the single band on Sample 1 assuming a slight delay caused by the gel Thus, two MTs labelled MT-1 (10,100 mol. wt) and MT-2 (4100mol.wt) and a Zn-binding peak ( < 1500 mol. wt) are clearly implicated as low mol. wt metal-binding ligands in the hepatopancreas of C a r cinus m a e n a s . The 27,000 MT-like ligand as found by Jennings et al. (1979) and Rainbow and Scott (1979) is most likely an oxidation MT artefact. It is absent in all the elution profiles presented here, reducing conditions being maintained. The 12,000 mol. wt MT-like ligand (Jennings et al., 1979: Rainbow and Scott, 1979) is most probably MT-1, although in these earlier published studies it did not appear to possess a characteristic mercaptide Table I. Comparison of R.F.s of MTs on 7.5% polyacrylamide gel electrophoresis from various sources and classified into MT types Oxidized MT Oxidized MT 0.15 0.2 0.4 0.4 0.5 0.4 0.2 0.4 0.2 0.15 0.4 Postulated MT types MT2 MTI 0.6 0.6 0.85 0.9 0.6 0.6 0.9 MT source References Rat Rat Crab Oystcr PLaice, horse Fish, whale, crab, sealion Fish Crab Winge and Rajagopalan (1972) Winge et al. (1975) Olafson e/ al. (1979b) Ridlington and Fowler (1979) Overnell and Grant (1981) Ridlington et al. (1981) Takeda and Shimizu (1982) Present findings Two metallothioneins in Carcinus 155 (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211, 1437-1438. Minkel D. T., Poulson K., Wielgus S., Shaw III C. F. and Petering D. H. (19803 On the sensitivity of metallothioneins to oxidation during isolation. Biochem. J. 191, 475 485. Olafson R. W., Kearns A. and Sire R. G. (1979a) Heavy metal induction of metallothionein synthesis in the hepatopancreas of the crab Scylla serrata. Camp. Biochem. Physiol. 62B, 417424. Olafson R. W,, Sire R, G. and Boto K~ G. (1979b) Isolation and chemical characterisation of the heavy metal-binding protein metallothionein from marine invertebrates. Camp. Biochem. Physiol. 62B, 407~416. Ornstein L. and Davis B. J. (1962) Disc Electrophoresis (preprinted by Distillation Products Industries, Eastman Kodak Co., Rochester, New York). Overnell J. (1982a) A method for the isolation of metallothionein from the hepatopancreas of the crab Cancer pagurus that minimizes the effect of tissue proteases. Camp. Biochem. Physiol. 73B, 547-553. Overnell J. (1982b) Copper metabolism in crabs and metallothionein: in viz'o effect of copper It on soluble hepatopancreas metal-binding components in the crab Cancer pagurus containing varying amounts of cadmium. Camp. Biochem. Physiol. 73B, 555 564. Overnell J. (1984a) The partition of copper and cadmium between different charge-forms of metallothionein in the Acknowledgements--We wish to thank Mr A. G. Scott and digestive tubules of the crab, Cancer pagurus. Camp. Dr H. B. Chua for their help with biochemical techniques. Biochem. Physiol. 77C, 237-243. Purchase of apparatus for ion-exchange chromatography Overnell J. (1984b) Use of 2-mercaptoethanol during chro~as assisted by a grant to P.S.R. from the Central Research matography of crab (Cancer pagurus) metallothionein on DEAE cellulose. Camp. Biochem. Physiol. 77C, 245-248. Fund, University of London. Overnell J. and Grant P. T. (1981) Reaction of Schiff's reagent with the partially-oxidised forms of metalREFERENCES lothionein and other polypeptides rich in cysteine resiCleland W. W. (1964) Dithiothreitol, a new protective dues. Biochem. Sac. Trans. (593rd Meet), 216-217. reagent for SH groups. Biochemistry 3, 480~482. Overnell J. and Trewhella E. (1979) Evidence for the natural occurrence of (cadmium, copper)-metallothionein in the Coombs T. L. (1974) The nature of zinc and copper crab Cancer pagurus. Camp. Biochem. Physiol. 64C, 69-76. complexes in the oyster Ostrea edulis. Mar. Biol. 28, 1-10. Engel D. W. and Brouwer M. (1984) Trace metal-binding Rainbow P. S. and Scott A. G. (1979) Two heavy metalproteins in marine molluscs and crustaceans. Mar. envir. binding proteins in the midgut gland of the crab Carcinus maenas. Mar, Biol. 55, 143-150. Res. 13, 177-194. George S. G., Carpene E., Coombs T. L., Overnell J. and Rainbow P. S., Scott A. G., Wiggins E. A. and Jackson Youngson A. (1979) Characterization of cadmiumR. W. (19803 Effect of chelating agents on the accumubinding protein from mussels, Mytilus edulis (L.), exposed lation of cadmium by the barnacle Semibalanus balto cadmium. Bioehim. biophys. Acta 580, 225-233. anoides, and complexation of soluble Cd, Zn and Cu. Howard A. G. and Nickless G. (19783 Heavy metal comMar. Ecol. Prog. Set. 29, 143-152. plexation in polluted molluscs. III Periwinkles (Littorina Ray S. and White M. (1981) Metallothionein-like protein in lobsters (Homarus americanus). Chemosphere 10, fittorea ), cockles ( Cardium edule ) and scallops ( Chlamys 1205-1213. opercularis). Chem. Biol. Interact. 23, 227-231. Jennings J. R., Rainbow P. S. and Scott A. G. (1979) Studies Ridlington J. W., Chapman D. C., Gregor D. E. and Whanger P. D. (1981) Metallothionein and Cu-chelatin: on the uptake of cadmium by the crab Carcinus maenas Characterization of metal-binding proteins from tissues in the laboratory. It, Preliminary investigation of of four marine animals. Camp. Biochem. Physiol. 70B, cadmium-binding proteins. Mar. Biol. 50, 141-149. 93-104. Jocelyn P. C. (1972) Biochemistry o f the SH Group. AcaRidlington J, W. and Fowler B. A. (1979) Isolation and demic Press, London (1972). partial characterization of cadmium-binding protein from K/igi J. H. R. and N6rdberg M. (1979) Metallothionein: the American oyster (Crassostrea virginica). Chem. Biol. Proceedings of the First International Meeting on MetalInteract. 25, 127-138. lothionein and other Low Molecular Weight MetalRoesijadi G. (1981) The significance of low molecular binding Proteins. Birkhauser, Basel. weight, metallothionein-like proteins in marine inLyon R., Taylor M. and Simkiss K. (1983) Metal-binding vertebrates: current status. Mar. envir. Res. 4, 167 179. proteins in the hepatopancreas of the crayfish (Austropatamobius pallipes). Camp. Biochem. Physiol. 74C, Takeda H. and Shimizu C. (1982) Purification of metallothionein from the liver of skipjack and its properties. 51-54. Bull. Jap. Sac. Sci. Fish. 48, 717-723. Mao J. C-H. (1967) Protein synthesis in a cell-free extract Tasheva B. and Dessev G. (1983) Artifacts in sodium from Staphylococcus aureas. J. Bacterial. 94, 80-86. dodecyl sulfate-polyacrylamide gel electrophoresis due to Marshall A. T. and Talbot V. (1979) Accumulation of 2-mercaptoethanol. Analyt. Biochem. 129, 98-102. cadmium and lead in the gills of Mytilus edulis. X-ray microanalysis and chemical analysis. Chem. Biol. Interact. Templeton D. M. and Cherian M. G. (1984) Chemical modifications of metallothionein. Biochem. J. 221, 27, 111 123. 569-575. Merril C. R., Goldman D., Sedman S. A. and Ebert M. H. shoulder n o r have a characteristic M T a m i n o acid composition, p r o b a b l y as a result o f i n a d e q u a t e purification. As can be seen from Fig. 1, M T s isolated on Sephadex G-75 eluted with m a n y other impurities as evidenced by the 254 n m a b s o r b a n c e profile a n d later by the complex n u m b e r o f 254 n m a b s o r b a n c e peaks on ion-exchange profiles. Lyon et al. (1983) a n d R a i n b o w et al. (19803 did not find M T in crayfish and barnacles but their techniques of purification were p r o b a b l y i n a d e q u a t e a n d m a s k i n g of M T characteristics by impurities would have occurred. In s u m m a r y , two MT-like ligands a n d one Znbinding peak have been s h o w n to occur in the h e p a t o p a n c r e a s of C. maenas. The MT-like ligands have mol. wts of 10,100 and 4100 respectively, as estimated by Sephadex G-50, and bind variable a m o u n t s of Cu, Z n a n d Cd. O n ultra-violet absorbance, these ligands each possess the characteristic mercaptide " s h o u l d e r " at 254 n m (peculiar to MTs) which disappears on acidification a n d reappears with neutralization. The two m e t a l - b i n d i n g ligands a p p e a r to be two different forms of metallothioneins, M T I a n d MT2, and m a y be a n a l o g o u s to the two forms of M T s f o u n d in vertebrates. 156 V . W . T . WONG and P. S. RAINBOW White S. L. and Rainbow P. S. (1986) A preliminary study of Cu, Cd and Zn-binding components in the hepatopancreas of Palaemon elegans (Crustacea: Decapoda). Cornp. Biochem. Physiol. (in press). Winge D. R., Premakumar R. and Rajagopalan K. V. (1975) Metalqnduced formation of metallothionein in rat liver. Arch. Biochem. Biophys. 170, 242-252. Winge D. R. and Rajagopalan K. V. (1972) Purification and some properties of Cd-binding protein from rat liver. Arch. Biochem. Biophys. 153~ 755-762. Wong V. W. T. and Rainbow P. S. (1986) Apparent and real variability in the presence and metal contents of metallothioneins in the crab Carcinus maenas including the effects of isolation procedure and metal induction. Comp. Biochem. Physiol. 83A, 157 177.