Traute Kleinschmidt

Phylogenetic Analysis of Reptilian Hemoglobins: Trees, Rates, and Divergences

Abstract. Phylogenetic relationships among reptiles were examined using previously published and newly determined hemoglobin sequences. Trees reconstructed from these sequences using maximum-parsimony, neighbor-joining, and maximum-likelihood algorithms were compared with a phylogenetic tree of Amniota, which was assembled on the basis of published morphological data. All analyses differentiated α chains into αA and αD types, which are present in all reptiles except crocodiles, where only αA chains are expressed. The occurrence of the αD chain in squamates (lizards and snakes only in this study) appears to be a general characteristic of these species. Lizards and snakes also express two types of β chains (βI and βII), while only one type of β chain is present in birds and crocodiles. Reconstructed hemoglobin trees for both α and β sequences did not yield the monophyletic Archosauria (i.e., crocodilians + birds) and Lepidosauria (i.e., Sphenodon+ squamates) groups defined by the morphology tree. This discrepancy, as well as some other poorly resolved nodes, might be due to substantial heterogeneity in evolutionary rates among single hemoglobin lineages. Estimation of branch lengths based on uncorrected amino acid substitutions and on distances corrected for multiple substitutions (PAM distances) revealed that relative rates for squamate αA and αD chains and crocodilian β chains are at least twice as high as those of the rest of the chains considered. In contrast to these rate inequalities between reptilian orders, little variation was found within squamates, which allowed determination of absolute evolutionary rates for this subset of hemoglobins. Rate estimates for hemoglobins of lizards and snakes yielded 1.7 (αA) and 3.3 (β) million years/PAM when calibrated with published divergence time vs. PAM distance correlates for several speciation events within snakes and for the squamate ↔ sphenodontid split. This suggests that hemoglobin chains of squamate reptiles evolved ∼3.5 (αA) or ∼1.7 times (β) faster than their mammalian equivalents. These data also were used to obtain a first estimate of some intrasquamate divergence times.

Oxygen binding properties, capillary densities and heart weights in high altitude camelids

SummaryThe oxygen binding properties of the blood of the camelid species vicuna, llama, alpaca and dromedary camel were measured and evaluated with respect to interspecific differences. The highest blood oxygen affinity, not only among camelids but of all mammals investigated so far, was found in the vicuna (P50=17.6 Torr compared to 20.3–21.6 Torr in the other species). Low hematocrits (23–34%) and small red blood cells (21–30 μm3) are common features of all camelids, but the lowest values are found in theLama species. Capillary densities were determined in heart and soleus muscle of vicuna and llama. Again, the vicuna shows exceptional values (3720 cap/mm2 on average in the heart) for a mammal of this body size. Finally, heart weight as percent of body weight is higher in the vicuna (0.7–0.9%) than in the other camelids studied (0.5–0.7%). The possibility that these parameters, measured in New World tylopodes at sea level, are not likely to change considerably with transfer to high altitude, is discussed.In the vicuna, a unique combination of the following features seems to be responsible for an out-standing physical capability at high altitude: saturation of blood with oxygen in the lung is favored by a high blood oxygen affinity, oxygen supply being facilitated by low diffusion distances in the muscle tissue. Loading, as well as unloading, of oxygen is improved by a relatively high oxygen transfer conductance of the red blood cells, which is due to their small size and which compensates the negative effect of a low hematocrit on the oxygen conductance of blood. Blood oxygen transport is presumably favored by two factors: a relatively large heart mass and, as a result of low hematocrit, a low blood viscosity. Both are advantageous for achieving a high maximal cardiac output.

Functional multiplicity and structural correlations in the hemoglobin system of larvae of Chironomus thummi thummi (insecta, diptera): Hb components CTT I, CTT IIβ, CTT III, CTT IV, CTT VI, CTT VIIB, CTT IX and CTT X

Larvae of the dipteran insect Chironomus thummi thummi that burrow in fresh-water muds, contain at least 12 hemoglobin (Hb) components of which the functional properties have not been systematically documented, although their amino acid sequences have been elucidated, showing mutually distinct primary structures. We isolated eight components (the monomeric Hbs CTT I, CTT III and CTT IV and the dimeric Hbs CTT II beta, CTT VI, CTT VIIB, CTT IX and CTT X) and measured in each O2 affinity and cooperativity and their pH dependence, and the effects of temperature, NaCl and ATP. The O2 affinities, Bohr- and temperature effects of the isohemoglobins are discussed in relation to mode of life and the microenvironmental conditions to which the larvae are subjected in nature, and with regard to the molecular mechanisms underlying the Hb-oxygenation reactions.

Adaptation of hemoglobin function to subterranean life in the mole, talpa europaea

In order to understand the mechanism responsible for the high oxygen affinity of mole blood, we investigated in the mole. Talpa europaea, red cell parameters that determine hemoglobin function. We have found that the oxygen half saturation pressure (P50) of mole blood is 2.85 kPa (21.4 Torr) at pCO2 4.7 kPa, pH 7.4 and 37 degree C. The concentration of 2,3-diphosphoglycerate (2,3-DPG) averaged 5.3 mmol/l in red cells. In addition, we have determined P50 in hemoglobin solutions at various concentrations of 2,3-DPG at an assumed intraerythrocytic pH of 7.2 and 37 degree C. These data were used to calculate the association constants of 2,3-DPG to mole hemoglobin. P50 was 1,89 kPa (14.2 Torr) in hemoglobin solutions without 2,3-DPG. The response to 2,3-DPG was relatively low. Noteworthy, CO2 did not affect the oxygen affinity at constant pH in the presence of 2,3-DPG. Our results suggest that the high blood oxygen affinity of the mole can be attributed to a weak interaction of its hemoglobin with 2,3-DPG.

A Molecular View of Primate Supraordinal Relationships from the Analysis of Both Nucleotide and Amino Acid Sequences

The fossil record suggests that the orders of eutherian mammals arose in a burst of adaptive radiation at the dawn of the Cenozoic (Savage and Russell, 1983). Possibly because of the apparent bushlike pattern of this radiation, establishing the course of phylogenetic branching that led to the orders and suborders of eutherian mammals has proven difficult. Although some regard this radiation as an almost simultaneous emergence of major clades (Simpson, 1978), most feel that such phylogenies are not actually bushlike (Gingerich, 1985; Novacek, 1990), although several splitting events may be close enough in time and in character to represent a considerable challenge for molecular and morphological analyses.

Embryonic pig hemoglobins Gower I (ζ2ϵ2), Gower II (α2ϵ2), Heide I (ζ22) and Heide II (α22): oxygen-binding functions related to structure and embryonic oxygen supply

The common pig lacks a fetal hemoglobin but has four embryonic hemoglobins: Gower I (zeta 2 epsilon 2), Gower II (alpha 2 epsilon 2), Heide I (zeta 2 theta 2) and Heide II (alpha 2 theta 2) as well as adult Hb A (alpha 2 beta 2) and the amino acid sequence for each of the five constituent polypeptide chains has been established. The oxygenation characteristics of the five components, measured in relation to pH, temperature and the erythrocytic ligand 2,3-diphosphoglycerate (DPG), together with the changes in their relative concentrations during early embryonic life, are given. The findings indicate a progressive decrease in maternal-fetal oxygen affinity difference and thus in oxygen transfer efficacy at a given diffusion gradient that correlates with the development of the gas exchange structures. The functional properties of the individual hemoglobins are additionally discussed in relation to molecular structure.

Primary structures of Arenicola marina isomyoglobins: Molecular basis for functional heterogeneity

The primary structures of isomyoglobins MbI and MbII from the body wall musculature of the polychaete Arenicola marina were investigated, aiming to trace the molecular basis for their functional differentiation. Unexpectedly, five chains, MbIa, MbIb, MbIIa, MbIIb and MbIIc, each consisting of 145 amino-acid residues and occurring in a ratio of = 33:17:25:12.5:12.5 were found. All substitutions can be explained by one-point mutations. With the exception of the 41(C6)Asn-->Asp(MbI/MbII) exchange that appears to be the basis for the electrophoretic separation of MbI and MbII, the substitutions do not involve drastic changes in the character of the side-chains. Pairwise comparison of MbIa and MbIIa with other invertebrate globin chains indicate the following sequence of decreasing identities: Aplysia (mollusc) Mb, Chironomus (insect) CTT III hemoglobin, whale Mb and Ascaris (nematode) Mb. The marked difference in O2 affinities between MbI and MbII appears attributable to 62Pro which distorts the E helix around E6 and occurs in all MbII chains, but in only 33% of the MbI chains (MbIb).

The Primary Structure of Three Hemoglobin Chains from the Indigo Snake (Drymarchon corais erebennus, Serpentes): First Evidence for αD Chains and Two β Chain Types in Snakes

Abstract The hemoglobin of the indigo snake (Drymarchon corais erebennus, Colubrinae) consists of two components, HbA and HbD, in the ratio of 1:1. They differ in both their α and β chains. The amino acid sequences of both α chains (αA and αD) and one β chain (βI) were determined. The presence of an αDchain in a snake hemoglobin is described for the first time. A comparison of all snake β chain sequences revealed the existence of two paralogous β chain types in snakes as well, which are designated as βI and βII type. For the discussion of the physiological properties of Drymarchon hemoglobin, the sequences were compared with those of the human α and β chains and those of the closely related water snake Liophis miliaris where functional data are available. Among the heme contacts, the substitution αD58(E7)His->Gln is unusual but most likely without any effect. The residues responsible for the main part of the Bohr effect are the same as in mammalian hemoglobins. In each of the three globin chains only two residues at positions involved in the α1/_2 interface contacts, most important for the stability and the properties of the hemoglobin molecule, are substituted with regard to human hemoglobin. On the contrary, nine, eleven, and six α1/β1 contact residues are replaced in the αA, αD, βI chains, respectively.

Studies on the evolutionary relationships between hemoglobins inChironomus pallidivittatus andC. Tentans

SummaryThe monomeric hemoglobins ofChironomus tentans andC. pallidivittatus have been isolated and separated into their respective components by gel chromatography on Sephadex G-75 and ion-exchange chromatography on DEAE-Sephacel. The amino acid compositions of the purified components are given. The sequence of the 30 N-terminal amino acid residues of one of the monomeric components (Hb I fromC. pallidivittatus) was determined and found to be identical in almost all of its parts with the monomeric hemoglobins ofC. thummi (CTT III and CTT IV).Antibodies against the monomeric hemoglobins Hb I and Hb IIc and the dimeric fraction were highly specific and no cross reaction between dimeric and monomeric hemoglobins could be demonstrated. The antibodies against the monomers crossreact with the monomeric hemoglobins CTT III and CTT IV ofC. thummi. Taken together with genetic data, the immunological results indicate that divergence of monomeric from dimeric forms was an early event in the evolution of the various hemoglobins inChironomus.

Allosteric Transition in Hemoglobin (αA2β12) from the Rhynchocephalian Reptile Relict Sphenodon Punctatus

The major hemoglobin component Hb A of the tuatara, Sphenodon punctatus. a relict of the rhynchocephalian reptiles that lived 200 million years ago, was investigated in the light of the apparent contradiction inherent in an effect of organic phosphate cofactors on the oxygen affinity of hemoglobins exhibiting hyperbolic oxygen equilibrium curves. The heterotropic allosteric effect of ATP, the major erythrocytic cofactor in the tuatara, is shown to be correlated with distinct homotropic interactions (Hills cooperativity coefficient at half-saturation, n50, attaining 1.3–1.5 above pH 7.9, and with free energies of heme-heme interaction (4.7 kJ.mole−1 at pH 7.2) which resemble those in other vertebrate hemoglobins that exhibit higher n50 values. Curiously, chloride ions increase hemoglobin-oxygen affinity below pH ± 7.2. The possible mechanisms underlying the effects of ATP and chloride are discussed.