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Two chromatographically distinct dimeric myoglobins can be isolated from the radular muscles of the sea-snail Nassa mutabilis L. The purification procedure is based on ammonium sulfate fractionation of the centrifuged homogenate followed by CM-cellulose column chromatography and gel filtration through Sephadex G-100. The total recovery, on a heme basis, is about 50%. The two myoglobins show one band by gel electrophoresis and practically identical physical properties. The isoelectric point is at pH 10.2 k 0.2; the apparent molecular weight is 28000 f 1500 by gel filtration and is not dependent on the ligand state of the hemes. Ageing of the myoglobin solutions produces a form showing an apparent molecular weight of about 15000 f 500 by gel filtration and abnormal spectrophotometric properties. This form is incapable of reassociation. $ 2 0 ,~ = 2.0 f 0.05 S for the native oxy derivative by velocity of sedimentation, s20,w = 1.25 f 0.005 S for the molecule denatured in sodium dodecylsulfate. Acrylamide gel electrophoresis in sodium dodecylsulfate, in reducing conditions, indicates a molecular weight of about 13000. About 15000 g protein/mol heme are found by biuret analysis of the isolated globins. Determinations of the absorption coefficients have been carried out by iron analysis and by spectrophotometric measurements on the CN-metmyoglobin and on the pyridine-chromogen derivatives. Oxygen equilibrium curves show that the myoglobin binds cooperatively oxygen with values of the interaction coefficient, n, = 1.5 with no Bohr effect.Myoglobins of higher organisms are monomeric molecules devoid of cooperativity and of Bohr effect [l]. In some cases, however, the myoglobins of the radular muscles of gastropod molluscs have been found to be dimeric molecules showing cooperative oxygen-binding curves [2,3]. Dimeric myoglobins are interesting from a structural point of view because they should show, with respect to the vertebrate myoglobins, very specific amino acid substitutions at positions corresponding to the exterior of the molecule, in order to permit the protein-protein interactions necessary to stabilize the dimer. They are interesting also because of the cooperative form of their oxygen equilibrium curves. This poses a question of whether a dimer can indeed be cooperative or, as already shown in some cases [4,5], the cooperativity is more simply caused by a ligand-linked association dissociation equilibrium in which monomer and dimer have different ligand binding properties [6]. Recently the amino acid sequence of Busycon canaliculatum myoglobin has been published; but no obvious sites for subunits interactions have been observed and the amino acid sequence is more similar to human p chains than to the myoglobin of Aplysia limacina, another gastropod mollusc [7]. We have looked at myoglobins in the radular muscles of gastropods living in the Mediterranean Sea to determine whether the myoglobins of other gastropods are dimeric and cooperative or whether those reported to be so [2,3] are indeed exceptions. We ...

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