Root effect hemoglobins in marine fish

• 1.1. The Root effect was evaluated in hemolysates from 26 species of bony fish and 20 species of cartilaginous fish found on the Brazilian southeastern coast.
• 2.2. Teleost Root shifts, with a single exception, are correlated with the presence of the choroid rete mirabile but not with its counterpart in the swimbladder.
• 3.3. Five ray species displayed weak and moderate Root effects despite the absence of choroid and swimbladder rete.
• 4.4. The presence and intensity of the Root effect is probably primarily related to the high oxygen demand of the retina and with the importance of visual perception in fish.
• 5.5. In marine teleosts the magnitude of the Root effect seems to be associated with the presence and size of both the choroid rete and the pseudobranch.
• 6.6. An antioxidant protection of the fish eyes can be advocated for the pseudobranch.

     

Role of tertiary structures on the Root effect in fish hemoglobins

Many fish hemoglobins exhibit a marked dependence of oxygen affinity and cooperativity on proton concentration, called Root effect. Both tertiary and quaternary effects have been evoked to explain the allosteric regulation brought about by protons in fish hemoglobins. However, no general rules have emerged so far. We carried out a complementary crystallographic and microspectroscopic characterization of ligand binding to crystals of deoxy-hemoglobin from the Antarctic fish Trematomus bernacchii (HbTb) at pH 6.2 and pH 8.4. At low pH ligation has negligible structural effects, correlating with low affinity and absence of cooperativity in oxygen binding. At high pH, ligation causes significant changes at the tertiary structural level, while preserving structural markers of the T state. These changes mainly consist in a marked displacement of the position of the switch region CD corner towards an R-like position. The functional data on T-state crystals validate the relevance of the crystallographic observations, revealing that, differently from mammalian Hbs, in HbTb a significant degree of cooperativity in oxygen binding is due to tertiary conformational changes, in the absence of the T–R quaternary transition. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Steric factors moderate conformational fluidity and contribute to the high proton sensitivity of Root effect hemoglobins

The structural basis of the extreme pH dependence of oxygen binding to Root effect Hbs is a long-standing puzzle in the field of protein chemistry. A previously unappreciated role of steric factors in the Root effect was revealed by a comparison of pH effects on oxygenation and oxidation processes in human Hb relative to Spot (Leiostomus xanthurus) and Carp (Cyprinodon carpio) Hbs. The Root effect confers five-fold increased pH sensitivity to oxygenation of Spot and Carp Hbs relative to Hb A(0) in the absence of anionic effectors, and even larger relative elevations of pH sensitivity of oxygenation in the presence of 0.2M phosphate. Remarkably, the Root effect was not evident in the oxidation of the Root effect Hbs. This finding rules out pH-dependent alterations in the thermodynamic properties of the heme iron, measured in the anaerobic oxidation reaction, as the basis of the Root effect. The alternative explanation supported by these results is that the elevated pH sensitivity of oxygenation of Root effect Hbs is attributable to globin-dependent steric effects that alter oxygen affinity by constraining conformational fluidity, but which have little influence on electron exchange via the heme edge. This elegant mode of allosteric control can regulate oxygen affinity within a given quaternary state, in addition to modifying the T-R equilibrium. Evolution of Hb sequences that result in proton-linked steric barriers to heme oxygenation could provide a general mechanism to account for the appearance of the Root effect in the structurally diverse Hbs of many species.     

Ligand-dependent Bohr effect of Chrionomus hemoglobins.

The O2 and CO Bohr effects of monomeric and dimeric hemoglobins of the insect Chironomus thummi thummi were determined as proton releases upon ligation. For the O2 Bohr effect of the monomeric hemoglobin III a maximum value of 0.20 H+/heme was obtained at pH 7.5. Upon ligation with CO, however, only 0.04 H+/heme were released at the same pH. In agreement with this finding isoelectric focusing experiments revealed different isoelectric points for O2-liganded and CO-liganded states of hemoglobin III. Analogous results were obtained in the cases of the monomeric hemoglobin IV and the dimeric hemoglobins of Chironomus thummi thummi; here O2 Bohr effects of 0.43 and 0.86 H+/heme were observed. For the corresponding CO Bohr effects values of 0.08 and 0.31 H+/heme were obtained respectively. On the basis of the available structural data the reduced CO Bohr effect in hemoglobin III is discussed as arising from a steric hindrance of the CO ligand by the side chain of isoleucine-E11, obstructing the movement of the heme-iron upon reaction with carbon monoxide. It should, however, be noted that ligands, according to their different electron donor and acceptor properties, may generally induce different conformational changes and thus different Bohr effects, in those hemoglobins in which distinct tertiary and/or quaternary constraints have not evolved. The general utilization of CO instead of O2 as allosteric effector is ruled out by the results reported here.     

The Root effect

Considering the presently available data it is clear that the Root effect represents an exaggerated alkaline Bohr effect which occurs in the absence of a normal acid Bohr effect and is associated with a loss of oxygen binding capacity at low pH. Undoubtedly at the molecular level the presence of a Ser residue at position F9(94) beta in these haemoglobin is of primary importance. No Root effect haemoglobin has yet been identified which lacks this substitution. On the other hand however many haemoglobins are known which possess this Ser residue and at the same time lack a Root effect. Other factors arising from interactions at other sites in the haemoglobin molecule are obviously sufficient to negate the otherwise stabilizing effect of this critical Ser residue. The loss of cooperativity of Root effect systems as the pH is lowered is readily explained as due to stabilization of the low affinity T state to such a degree that the switch to the high affinity R state is suppressed even in the fully liganded molecule. The observation of Hill coefficients of less than unity requires that within the T state chain heterogeneity exists such that the alpha and beta chain haems demonstrate significantly different affinities for ligand. The physiological role of Root effect haemoglobins is demonstrably not inevitably linked to the swim bladder but more probably arose from the need to oxygenate the poorly vascularized retina of many fishes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Allosteric effect of water in fish and human hemoglobins

Prompted by the reported lack of solvation effects on the oxygen affinity of fish (trout I) hemoglobin that questioned allosteric water binding in human hemoglobin A (Bellelli, A., Brancaccio, A., and Brunori, M. (1993) J. Biol. Chem. 268, 4742-4744), we have investigated solvation effects in fish and human hemoglobins by means of the osmotic stress method and allosteric analysis. In contrast to the earlier report, we demonstrate that water potential does affect oxygen affinity of trout hemoglobin I in the presence of inert solutes like betaine. Moreover, we show that upon oxygenation electrophoretically anodic hemoglobin from trout and eel bind a similar number of water molecules as does human hemoglobin A, whereas the cathodic hemoglobins of trout and eel bind smaller, but mutually similar, numbers of water molecules. Addition of cofactors strongly increases the number of water molecules bound to eel hemoglobin A (as in human hemoglobin) but only weakly affects water binding to eel hemoglobin C.     

The evolution of Root effect hemoglobins in the absence of intracellular pH protection of the red blood cell: insights from primitive fishes

The Root effect, a reduction in blood oxygen (O₂) carrying capacity at low pH, is used by many fish species to maximize O₂ delivery to the eye and swimbladder. It is believed to have evolved in the basal actinopterygian lineage of fishes, species that lack the intracellular pH (pH_i) protection mechanism of more derived species’ red blood cells (i.e., adrenergically activated Na⁺/H⁺ exchangers; βNHE). These basal actinopterygians may consequently experience a reduction in blood O₂ carrying capacity, and thus O₂ uptake at the gills, during hypoxia- and exercise-induced generalized blood acidoses. We analyzed the hemoglobins (Hbs) of seven species within this group [American paddlefish (Polyodon spathula), white sturgeon (Acipenser transmontanus), spotted gar (Lepisosteus oculatus), alligator gar (Atractosteus spatula), bowfin (Amia calva), mooneye (Hiodon tergisus), and pirarucu (Arapaima gigas)] for their Root effect characteristics so as to test the hypothesis of the Root effect onset pH value being lower than those pH values expected during a generalized acidosis in vivo. Analysis of the haemolysates revealed that, although each of the seven species displayed Root effects (ranging from 7.3 to 40.5% desaturation of Hb with O₂, i.e., Hb O₂ desaturation), the Root effect onset pH values of all species are considerably lower (ranging from pH 5.94 to 7.04) than the maximum blood acidoses that would be expected following hypoxia or exercise (pH_i 7.15–7.3). Thus, although these primitive fishes possess Hbs with large Root effects and lack any significant red blood cell βNHE activity, it is unlikely that the possession of a Root effect would impair O₂ uptake at the gills following a generalized acidosis of the blood. As well, it was shown that both maximal Root effect and Root effect onset pH values increased significantly in bowfin over those of the more basal species, toward values of similar magnitude to those of most of the more derived teleosts studied to date. This is paralleled by the initial appearance of the choroid rete in bowfin, as well as a significant decrease in Hb buffer value and an increase in Bohr/Haldane effects, together suggesting bowfin as the most basal species capable of utilizing its Root effect to maximize O₂ delivery to the eye.     

植物的血红蛋白

近几年来,植物血红蛋白的研究进展十分迅速,豆科植物中与共生固氮无关的血红蛋白基因和包括禾本科植物在内的许多非豆科植物血红蛋白基因的发现使人们对植物血红蛋白有了新的认识,进而把植物血红蛋白分为共生血红蛋白和非共生血红蛋白两种类型。对这两种血红蛋白的性质、功能、基因结构及表达等方面的研究不仅对共生固氮中植物与微生物的相互关系和固氮工程研究;而且对植物细胞的呼吸代谢和耐涝机理等研究有重要价值。     

De novo mutations producing unstable hemoglobins or hemoglobins M

Summary Hemoglobins M and unstable hemoglobins cause clinical syndromes that are transmitted in autosomal dominant fashion. Pedigrees of 50 probands with de novo mutations producing unstable Hb disease or Hb M disease were compiled. Cases were ascertained (1) by screening the relevant literature published from 1950 through 1980 and (2) through personal communication. Additional pedigree data on several published cases were collected, and a depository containing all available information rekated to de novo Hb mutants was established. The 50 probands were born in 14 countries between 1922 and 1976. Paternity was tested in 36% of the cases, and no instance of false paternity was noted.The data were used to test for an association of advanced parental age with the appearance of de novo mutants. Paternal ages at the probands' births ranged from 20 to 50 years, with a mean of 32.7 years. Maternal ages ranged from 18 to 43 years, with a mean of 28.5 years. For each year and country (or, where necessary, for the nearest possible year and/or a demographically similar country), the cumulative frequency distributions of the ages of parents who had a child in that country and year were computed; the ages of each proband's father and mother were then expressed as percentiles on these distributions. The distribution of paternal age percentiles was shifted toward the upper end of the range, with 11 of the 50 paternal ages falling between the 90th and 100th percentiles. The distribution of maternal age percentiles was more complex, with one peak (10 of 50 ages) falling between the 30th and 40th percentiles and a second peak (10 of 50 ages), between the 90th and 100th percentiles. These distributions, though suggestive of an association of advanced parental age and the appearance of de novo mutations that cause unstable Hb disease or methemoglobinemic cyanosis, were not significantly different from those uniform distributions expected in the absence of a parental age effect.     

The measurement of the Bohr effect of fish hemoglobins by gel electrofocusing

• 1. Hemolysates from 16 species of Amazon fish and one amphibian were analyzed by gel electrofocusing. The change in isoelectric point upon deoxygenation provided a reliable estimate of the Bohr effect.
• 2. Certain species of fish had single hemoglobin components whose pI increased significantly upon deoxygenation, as in man. Other fish had hemoglobins whose isoelectric points were unaffected by deoxygenation. Six species of fish had at least two hemoglobin components, one of which had a reduced isoelectric point upon deoxygenation indicating a reversed Bohr effect, whereas the other(s) had an increased isoelectric point on deoxygenation, as occurs with the normal alkaline Bohr effect.
• 3. A close correlation was found between the change in isoelectric point with deoxygenation and the Bohr effect determined by oxygen equilibrium measurements.

     

Allosteric effect of protons and adenosine triphosphate on hemoglobins from aquatic amphibia

Summary The analysis of hemoglobins from two anurans, one semi-aquatic (Leptodactylus labyrinthicus) and the other aquatic (Pipa carvalhoi), showed several components isolated by CM-cellulose chromatography. The two major components (Hb II and Hb III) ofL. labyrinthicus and the major components (Hb III and Hb IV) ofP. carvalhoi possess functional properties as follows: i.P. carvalnoi Hb III and Hb IV andL. labyrinthicus Hb II had very small Bohr effects (–0.05) and a substantial heterotropic effect with polyphosphates. ii.L. labyrinthicus Hb III produced a normal Bohr effect of –0.17, with no influence of polyphosphates.The authors wish to dedicate this paperin memoriam of Prof. Eraldo Antonini who contributed much to the knowledge of hemoglobins     

Alkaline Bohr effect of bird hemoglobins: the case of the flamingo

The hemoglobin (Hb) substitution His-->Gln at position alpha89, very common in avian Hbs, is considered to be responsible for the weak Bohr effect of avian Hbs. Phoenicopterus ruber ruber is one of the few avian Hbs that possesses His at alpha89, but it has not been functionally characterized yet. In the present study the Hb system of the greater flamingo (P. ruber roseus), a bird that lives in Mediterranean areas, has been investigated to obtain further insight into the role played by the alpha89 residue in determining the strong reduction of the Bohr effect. Functional analysis of the two purified Hb components (HbA and HbD) of P. ruber roseus showed that both are characterized by high oxygen affinity in the absence of organic phosphates, a strong modulating effect of inositol hexaphosphate, and a reduced Bohr effect. Indeed, in spite of the close phylogenetic relationship between the two flamingo species, structural analysis based on tandem mass spectrometry of the alpha(A) chain of P. ruber roseus Hb showed that a Gln residue is present at position alpha89.