We performed the deuterium-exchange reaction on human hemoglobin in its carbon monoixy and deoxy forms at various pH values and 36.5 degrees C. Peptides containing only one histidine residue were separated from tryptic and chymotryptic digests of the deuterated hemoglobin, except for two peptides which contained the alpha-87 and alpha-89 and the beta-116 and beta-117 histidine residues, respectively. The pseudo-first-order rate constant for the exchange reaction of each histidin residue was measured by using the mass spectrometric method. We obtained the following results. The pKa values for the alpha-20, alpha-89, and beta-146 histidine residues in deoxyhemoglobin decreased significantly, while that for the beta-143 histidine residue increased significantly on ligation. The pseudo-first-order rate constants were virtually zero for the alpha-45, alpha-58, alpha-87, beta-63, and beta-92 histidine residues which are linked with a heme group, and also for the alpha-122 histidine residue which is buried at the alpha 1 beta 1 contact in the hemoglobin molecule. No change was detected in the pKa values on ligation for the other histidine residues in deoxyhemoglobin.

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Intracellular hemoglobins of the sea blood clam Anadara broughtonii consist of HbI dimer (33%) and HbII tetramer (60%). The molecular weights of globins of HbI and HbII were determined by sodium dodecyl sulfate (SDS)-gel electrophoresis to be 15,500 and 16,500, respectively. The existence of two dissimilar chains, alpha and beta, in globin from HbII tetramer was confirmed electrophoretically and the chains were separated by CM-cellulose chromatography in 8 M urea. In contrast, globin from HbI dimer showed a single band on two types of electrophoresis. The NH2-terminus and the COOH-terminus of HbI were determined to be proline and leucine, respectively. From the results of finger-printing, the alpha and beta chains from HbII were considered to have a rather similar profile, whereas globin from HbI was very different. The results obtained by amino acid analysis of each chain also supported the above findings. It was thus shown that HbII has an alpha2beta2 subunit structure, which is rare among invertebrate hemoglobins. On the other hand, HbI seems to have two identical subunits, designated as "gamma", and to exist as a "gamma2" dimer structure. Both Anadara Hb's appear to have no functional groups relating to the Bohr effect and to be unable to form a binding site for organic phosphates.