Biophysical Studies of the Erythrocruorin of Abarenicola Assimilis

Abstract

The physico-chemical properties of the erythrocruorin from the marine polychaete, Abarenicola assimilis have been studied. In gel filtration and sedimentation velocity, three components, A,B, and C, representing approximately 1%, 8% and 91% respectively of the total material at pH 7.0 are evident. (1) Fraction C This component has a sedimentation coefficient (s°20,w) of 57.5s, and molecular weight as determined by sedimentation equilibrium of 3.29 x 106 at pH 7.0. Under the electron microscope it appears to be composed of 12 subunits arranged in 2 superimposed hexagonal rings. The amino acid composition, absorption spectrum, isoelectric point and N-terminal groups are reported. The haem and iron contents of the molecule are 2.77% and 0.23% respectively, corresponding to an average minimum molecular weight of 23,500 daltons. Gel filtration at pH 9.0 shows that the molecule dissociates into subunits with molecular weights of 240,000, 100,000 and 23,000 to 41,000 daltons. In polyacrylamide gel electrophoresis, components with molecular weights of 270,000, 120,000 to 135,000 60,000 to 100,000, 36,000 to 41,000 and 28,800 daltons are evident. In buffers containing 0.01 M EDTA, dissociation of Abarenicola erythrocruorin beings at pH 7.8, while in the presence of 0.01 M Ca2+, no evidence of dissociation is apparent until until the pH reaches 8.6 In the presence of 0.1% sodium dodecyl sulphate (SDS), the erythrocruorin is dissociated into 9 subunits (1 to 9) with molecular weights ranging from 12,800 (1) to 99,000 (9) daltons. On the other hand, in the presence of SDS and 2-mercaptoethanol, polypeptide chains with molecular weights of 13,700 (I) 15,800 (II), 24,700 (III), 27,800 (IV) and 33,600 (V)daltons are present. Re-electrophoresis of the subunits isolated by detergent electrophoresis in the absence of 2-mercaptoethanol and gel filtration in SDS suggests that the dissociation pattern is complex. Abarenicola enythrocrouorin is shown to bind oxygen co-operatively. Fox example, at pH 7.0 and 25°C, n1/2 is 4.0, and P50 is 6.0 mm Hg. Over the pH range 7.0 to 7.8, however, it lacks an alkaline Bohr effect. Co-operativity is also retained in the 10s subunit. In addition, the 10s subunit has been shown to possess similar absorption spectrum, amino acid composition, isoelectric point, and N-terminal groups as the 57.5S components. (2) Fractions A and B These are higher molecular weight polymers of fraction C at pH 7.0. They are similar to the 57.5S component in possessing similar absorption spectra and electrophoretic patterns in sodium dodecyl sulphate polyacrylamide gel electrophoresis. Under the electron microscope, fraction B shows that stacks containing 2 to 8 molecules are apparent from their side views. These polymers are not in association-dissociation equilibrium with the 57.5S monomer. Polymerisation is apparently not ionic in origin, and is also unlikely to be a result of meterythrocruorin formation.