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.

A stopped-flow apparatus was constructed for x-ray scattering study at subzero temperature. It can be operated over a wide temperature range down to −20 °C with highly viscous solution (20 cP) successfully. We have applied the stopped-flow x-ray scattering method to many biological reactions. In particular, the association of E. coli ribosomal subunits was detected at −10 °C which was too fast to be detected at room temperature. Dissociation of E. foetida hemoglobin was measured by the stopped-flow x-ray scattering method combined with a time-resolved imaging plate as a detector.

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.

The complete amino acid sequence of a dimeric hemoglobin (HbI) from the marine bivalve mollusc Anadara broughtonii was determined by sequencing of the intact chain and peptide fragments produced by cleavage at two asparaginylglycine bonds and at methionyl, arginyl, and tryptophanyl residues. The clam hemoglobin consists of two identical polypeptide chains. The globin chain has 146 amino acid residues with a proline at the NH2 terminus and a leucine at the COOH terminus. The calculated molecular mass of the native hemoglobin was 32945 daltons. The clam hemoglobin contains only two histidine residues, which correspond to the distal and proximal heme-linked positions. Compared with human beta chain, an additional segment of seven residues is present in the NH2-terminal region and also five less residues in the COOH-terminal region. Although such an amino-terminal elongation has been known to be characteristic of hemoglobins from the most primitive living vertebrates Cyclostomata, a very similar structure was found to occur in the hemoglobin from the primitive invertebrate arcid clam.