The quarternary structure of an unusual high-molecular-weight intracellular haemoglobin from the bivalve mollusc Barbatia reeveana

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Abstract
The arcid clam B. reeveana contains an intracellular Hb with an unusual structure. Compared with other intracellular Hb, it is extremely large, with a MW of 430,000 and an s20,w of 13.6S. A minor component (MW = 220,000; s20,w = 9.7S) is present as a probable dissociation product of the major component. This Hb has an unusual subunit structure. It contains 1 mol of Heme/16,000g of protein, in common with most other Hb. The smallest polypeptide that could be obtained after treatment with sodium dodecyl sulphate or 6 M-guanidine with reducing agent has a MW of 32,000-37,000. Digestion of the Hb with the proteinase subtilisin produces both 57,000- and 30,000-MW aggregates that contain 1 mol of heme/16,000 g of protein and that can be dissociated into 16,500-MW polypeptides by treatment with sodium dodecyl sulphate. The intact polymer shows slight cooperativity (h = 1.7), lacks a Bohr effect between pH 7 and 8, and has a low O2 affinity [P50 = 4.8 kPa (36 mmHg) at 20.degree. C] relative to other Hb. The 30,000 MW aggregate obtained by digestion of the polymer binds O2 reversibly with an affinity greater than that of the polymer, but with some cooperativity (h = 1.7). These results are consistent with the hypothesis that the subunits of this unusually large intracellular Hb are 32,000 MW polypeptides that in turn are composed of 2 covalently linked heme-containing O2 binding domains. This is the 1st report of an intracellular Hb with such a structure.