Cooperative, Low‐Molecular‐Weight Dimeric Myoglobins from the Radular Muscle of the Gastropod Mollusc Nassa mutabilis L

Abstract

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 ...

Alteration of the proximal bond energy in the unliganded form of the homodimeric myoglobin from Nassa mutabilis Kinetic and spectroscopic evidence

“…N, mutabilis Mb was prepared as previously reported [3]. Horse heart Mb was obtained from Sigma Chemical Co, (St, Louis, Me, USA) and further purified as previously reported (see ref, [1]).…”

“…mutabil/s Mb was recorded, at pH 7.0 (in 0,2 M phosphate buffer) and 20°C, on a Varian Cary 219 double beam spectrophotometer, The concentration of N, mutabllis Mb was determined on the basis of ~ =11.7 mM-~,cm -~ for the unliganded derivative [3], The transient absorption spectrum of the deoxygenated derivative of N, mutabtlis Mb was obtained at pH 3.1 (in 0.2 M phosphate buffer) and 20*C, before the occurrence of the hemoprotein denaturation (t,/; .. I s), from the initial optical density changes after rapidly mixing the deoxygenated derivative of N, mutabilis Mb with degas~d buffer to bring the pH to 3.1 [11], Values of the second-order rate constant for CO binding to the deoxygenated derivative of N. mutabifls Mb were oh.…”

“…Although it is usually formed by a single polypeptide chain [1], dimeric Mb's have been reported during the last few years to be present in the radular muscles of marine gastropods from prosobranchia subclass [2][3][4][5]. In particular, the homodimeric Mb from Nassa mutabilis has raised interest because of a fairly marked cooperativity in oxygen binding (n = 1.5) [3],…”

“…The occurrence of cooperative homodimeric Mb's and Hb's in invertebrates [3,6,7] opens the question whether they might all be representatives of an ancestral class of hemoproteins with a similar primitive hemeAbbreviations: Mb, Myoglobin; Hb, Hemoglobin; N. mutabilis Mb, Nassa mutabilh' homodimerie myoglobin; S. inaequivalvls Hb, Scapharca inaequivalvi~ homodimgrie hemoglobiP. heme interaction mechanism(s) in spite of their different location and physiological role.…”

Evolution of myoglobin

“…Read Read [86]). More recent studies have added 15 myoglobins from Nerita peloronta [92], Littorina littorina, Siphonaria gigas [93], Lunatia heros [94], Acmaea testudinalis, Haliotis kamtschatkana, Tegula funebralis, Fusitriton oregonensis, Thais lamellosa [95], Aplysia kurodai [96], A. juliana [97], Cerithidea rhizophorarum [98], Dolabella auriculaia [99], Nassa mutabilis [100] and Bursatella leachii [101]. In contrast to monomeric vertebrate myoglobins, some gastropod myoglobins exist as dimers.…”