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 ± 0.2; the apparent molecular weight is 28000 ± 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 15 000 ± 500 by gel filtration and abnormal spectrophotometric properties. This form is incapable of reassociation. S20, w= 2.0 ± 0.05 S for the native oxy derivative by velocity of sedimentation, S20, w= 1.25 ± 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 15 000 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.