Physicochemical Properties of the Hemoglobins from the Common Bloodworm Glycera dibranchiata

Glycera dibranchiata hemoglobin was previously noted to comprise five components separable by electrophoresis, two of which were due to monomeric forms and the remaining three due to polymeric forms. The three polymeric carboxyhemoglobin components had isoelectric points (pI) of pH 5.6, 6.1, and 6.5 while those of the monomeric proteins were pH 7.1 and 7.4. Oxidation of the pigment to methemoglobin caused an increase in each isoelectric point of 0.4 to 0.7. The spectral properties of the oxy-, deoxy-, carboxy-, met-, cyanomet-, and azide forms of the polymeric hemoglobin closely resembled those of vertebrate hemoglobins while analogous forms of the monomeric hemoglobin showed some anomalous properties, most notably in the high spin derivatives. The binding of hydroxide by methemoglobin at 4° indicated the formation of a normal hydroxide complex for the polymer (pK = 9.08, n = 0.98, where n represents the stoichiometry of the reaction) but a mixture of hydroxide and possibly imidazole methemoglobin complexes for the monomer. The pK for the transition in the monomer was 8.99 (n = 1.94) in the presence of methylamine buffer, while in glycine, pK = 10.08, n = 1.10. Studies of the binding of the monomeric methemoglobin with azide, cyanide, and fluoride at 22° gave the following dissociation constants. Kdissmethbcn = 1.2 x 10-3 m, n = 1.05, pH = 6.85 Kdissmethbn3 = 2.2 x 10-3 m, n = 0.97, pH = 6.45 Kdissmethbn3 = 2.7 x 10-3 m, n = 0.96, pH = 7.30 Kdissmethbf = 9.4 x 10-3 m, n = 1.01, pH = 6.85 where MetHbCn is cyanomethemoglobin, MetHbN3 is the azide form of methemoglobin, and MetHbF is fluoromethemoglobin. Amino acid analysis of preparative disc gel purified monomer indicated the presence of 144 amino acids yielding a globin molecular weight of ∼18,200 and a hemoglobin molecular weight of ∼18,800. The amino acid compositions of the nonpurified monomer and polymer were different from each other as well as from the purified monomer. The number of amino acids found in the nonpurified hemoglobin analyses was suggestive of the heterogeneity of these proteins.