Comp, Biochem. Physiol. Vol. 83A, No. 1, pp. 157 177, 1986 0300-9629/86 $3.00+0.00 Printed in Great Britain © 1986 Pergamon Press Ltd 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 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. Reducing conditions must be maintained throughout the procedure of isolating metal- lothioneins from crabs. Dithiothreitol is preferred to 2-mercaptoethanol for long-term protection. 2. Two metallothioneins (I0,100 and 4100 mol. wt, respectively) in the hepatopancreas of the crab Carcinus maenas showed great variability between individual crabs as to their presence and to their contents of Zn, Cu and Cd. 3. The 10,100 mol. wt metallothionein was induced in the laboratory by exposure to Cu and Cd, and variably by Zn-exposure. Laboratory induction did not raise significantly the total metal content of 0.88_ 1.13 g atoms/mol protein of this metallothionein in crabs from the Firth of Clyde, Scotland. 4. The 4100 mol. wt metallothionein was not induced in the laboratory by exposure to Cu, Cd or Zn. This metallothionein in crabs from the Firth of Clyde. Scotland, contained 0.27 + 0.34 g atoms of total Cu, Cd and Zn per mole of protein. INTRODUCTION Varying numbers of different metallothioneins (MTs) including various isoforms have been reported in different crab species. For example, the presence of a single MT (ca 10-12,000mol. wt) separated by Sephadex G-75 chromatography, resolving after ion- exchange and electrophoresis into two isoforms, has been reported in Scylla serrata (Olafson et al., 1979), Cancer magister (Olafson et al., 1979) and Cancer pagurus (Overnell and Trewhella, 1979). More re- cently, Overnell (1982a,b, 1984a,b) has reported resolution of such an MT into three isoforms in Cancer pagurus. Jennings et al. (1979) and Rainbow and Scott (1979), however, reported the presence of two MTs of about 27,000 and 12,000 mol. wt in Carcinus maenas as separated by Sephadex G-75. Engel and Brouwer (1984) similarly report metallothionein-like proteins of about 30,000 and 40,000 tool. wt in addition to one of about 10,000mol. wt in the lobster Homarus americanus. The previous paper (Wong and Rainbow, 1986) has shown that C. maenas does contain two MTs but these are of 10,100 and 4100 mol. wt, respectively, as estimated on Sephadex G-50. It is therefore sur- prising to see how divergent the results are within and between different crab species whereas in vertebrate systems, two MTs have been clearly identified (Kagi and Nordberg, 1979). *Abbreviations: DTT, dithiothreitol; 2-M, 2-Mercapto- ethanol; MT, metallothionein; PMSF, phenylmethyl- sulphonyl fluoride. Many studies on MTs have been performed with molecular-sieve chromatography using Sephadex G-75. The resolving properties of this particular gel type, however, are not totally suitable for separating MTs from other low mol. wt impurities for MTs elute near the lower limit (5000mol. wt) of the linear fractionating range. Moreover, many such elutions have been performed under oxidizing conditions (see Minkel et al., 1980) causing artifactual changes to elution profiles. Correspondingly many published elution profiles of invertebrate metal-binding ligands show considerable variation attributable to both ox- idation artefacts and poor resolution. Minkel et al. (1980) have shown that a reducing agent such as 2-M* can prevent the formation of oxidation artefacts. Once the effects of oxidation have been minimized, it would seem appropriate to switch to a gel type with a more suitable linear fractioning range such as Sephadex G-50 (30,000-1500 mol. wt). By using reducing conditions and Sephadex G-50 we hope to be able to distinguish between apparent and real variability in the presence and metal contents of MTs in the common shore crab Carcinus maenas. MATERIALS AND METHODS Carcinus maenas (L.) were obtained from the University Marine Biological Station, Millport, Scotland, and held immersed in the laboratory aquarium at 6°C for more than a week in an attempt to reduce physiological variability caused by entrained tidal rhythms. Induction experiments: Crabs were exposed individually for 14 days at 6°C under 12:12 light/dark regime to one of four media at 33.3 ppt salinity: (i) artificial sea-water as control [Tropical Marin New containing 2.5 ~zg Zn, 6/~g Cu 157