New Zealand's five endemic mudfish (Neochanna spp.) species have distributions that differ both geographically and by habitat type. Differences in habitat preferences between species have led to the proposal of an evolutionary series within the group. A morphological cline can be observed from the galaxiform Chatham Island and Canterbury species inhabiting lakes and streams, respectively, to the anguilliform Northland and brown mudfishes of ephemeral wetlands. Morphological specializations proposed for wetland dwelling include loss of pelvic fins, reduced eyes, enlarged nostrils, development of caudal flanges, and elongation of dorsal and anal fin bases to become almost confluent with the caudal fin. Another expectation of adaptation to wetland dwelling is specializations in respiratory physiology to obtain oxygen from highly hypoxic or acidic waters, and the ability to cope with seasonal exposure to air during the drought season. Expected respiratory specializations to wetland dwelling include high oxygen affinity haemoglobins, high levels of cooperative oxygen binding, the presence of multiple haemoglobins and the ability to aestivate and survive long periods of emersion. The four mainland Neochanna species were examined to determine if differences in haemoglobin expression as well as differences in haemoglobin oxygen binding correlated with differing habitats and treatments. Whole blood oxygen affinity was determined at several pH levels (6.5, 7.0, 7.5 and 8.0) and temperatures (10'C, 15'C and 20'C), as well as different treatments (aestivating, fasting and control) using a Hemox analyzer. The presence of multiple haemoglobins was determined by isoelectric focusing. All four species displayed high oxygen affinities (p50 = 6.5 to 9.5 mm Hg at pH 7.5 15'C), moderate levels of cooperativity (Hill coefficients = 1.75 to 2.00 at pH 7.5 15'C), pH sensitivity (Bohr coefficients = -0.62 to -0.94 between pH 7.5 and 7.0 at 15'C), temperature sensitivity (ΔH = -2.20 to -15.78 k cal mol-1 between 10'C and 15'C) and the presence of multiple haemoglobins. Black, brown and Northland mudfish were able to survive aestivation for six weeks but there were no changes between air-breathing and water-breathing individuals with respect to oxygen binding characteristics. Although there is evidence of habitat specialization in haemoglobin physiology between mudfish species, differences between species did not correlate with the evolutionary series proposed for specialization to dwelling in ephemeral wetlands and latitudinal distributions of mudfish species appear to strongly dictate oxygen binding properties of mudfish whole blood.

The major pathway for O 2 binding to mammalian myoglobins (Mbs) and hemoglobins (Hbs) involves transient outward movements of the distal histidine (HisE7), which allows ligand migration into the distal portion of the heme pocket. This E7 gate pathway appears dominant in vertebrate Hbs and Mbs. However, a number of invertebrate globins, including the dimeric hemoglobin from the blood clam Scapharca inaequivalvis (ScHbI), have an inverted quaternary structure in which an EF:FE dimer interface appears to block the HisE7 gate. Another set of globins, including the mini-hemoglobin from the Nemertean sea worm Cerebratulus lacteus (CerHb), are missing the N-terminal A-helix, which results in an internal tunnel between the E- and H-helices. This apolar channel has been suggested to represent an alternative to the HisE7 gate pathway. To determine the roles of the E7 gate and alternative pathways, we have systematically examined the effects of mutations at the E7 position in ScHbI and CerHb and at 21 other positions along the polar channel in CerHb. As was observed for SwMb and HbA, there is a progressive decrease in the bimolecular rate constants for O 2 binding to ScHbI as the size of the amino acid at position E7 is increased from Ala to Trp. This pattern is unaffected when ScHbI is completely converted to the R- or high affinity quaternary state by the F97Y mutation or when the dimer interface is completely disrupted by the K30D mutation. In contrast, E7 mutations have little affect on the rates of ligand entry and escape in CerHb. Instead, ligands diffuse through the apolar channel between the E- and H-helices as judged by decreases in both overall association and dissociation rate constants and increases in the extent of geminate recombination when the channel is blocked by small to large amino acid mutations. In SwMb, these trends are only observed when the small to large mutations are constructed at or near the E7 gate or directly in the distal pocket where ligands are captured. Thus, it is clear that globins have evolved more than one pathway for rapid O 2 uptake and release.

Cooperative oxygen binding by vertebrate tetrameric hemoglobins (Hbs) has been extensively studied and is relatively well understood. Nonetheless, Hill coefficients greater than four have been reported for adult avian, amphibian, and reptilian red blood cells. Such reports also exist for embryonic red cells from various animals. These results are controversial and not yet convincingly established. Oxygen binding studies on avian Hb D, which is known to undergo deoxygenation-dependent self-association, were carried out to answer this question. The goal was to determine unequivocally whether Hill coefficients greater than four occur. Such high Hill coefficients were observed but only at very high Hb D concentrations. Moreover, the early model of avian deoxy Hb D self-association was found to be incomplete. The model has now been expanded to describe better the observed sedimentation data at high Hb concentrations. The possibility that embryonic deoxy Hbs self-associate was also assessed by sedimentation studies of deoxygenated Hb solutions from a marsupial, the tammar wallaby. The results obtained show unambiguously that these embryonic Hbs self-associate upon deoxygenation. Recent phylogenetic analyses suggest that the avian [alpha superscript D]-globin originated from embryonic [alpha]-globins. This finding suggests that the propensity to self-associate upon deoxygenation is an intrinsic property of tetrameric Hbs with embryonic [alpha]-globins. Furthermore the residues mediating the inter-tetramer interactions in adult avian deoxy Hb D and embryonic deoxy Hbs are likely to be the same. Recombinant globins were expressed in bacteria and protocols for the assembly of avian recombinant tetrameric Hb D developed. Initial measurements by sedimentation were carried out to verify the role of a conserved glutamate residue previously speculated to be involved in inter-tetramer interactions. The present studies provide a framework for future investigations of deoxygenation-dependent Hb self-association. In particular the need to carry out oxygen equilibrium measurements at high Hb concentrations as well as sedimentation studies of the deoxygenated Hb solutions is stressed. / text