Biomedical subjects

R E Weber

Publications and source records attributed to R E Weber.

At least 37 records · Page 2Linked to original sources

PubMed1997

The anodic hemoglobin of Anguilla anguilla. Molecular basis for allosteric effects in a root-effect hemoglobin.

The functional and structural basis for the Root effect has been investigated in the anodic hemoglobin of the European eel, Anguilla anguilla. This hemoglobin exhibits a large Bohr effect, which is accounted for by oxygen-linked binding of seven to eight protons in the presence of GTP at pH 7.5. Oxygen equilibrium curves show nonlinear lower asymptote of Hill plots, indicating the occurrence of heme-heme interactions within the T state. Analysis of the curves according to the co-operon model (Brunori, M., Coletta, M., and Di Cera, E. (1986) Biophys. Chem. 23, 215-222) reveals that T state cooperativity is positive at high pH and in the stripped hemoglobin (where the T --> R allosteric transition is operative) and negative at low pH and in the presence of organic phosphate (where the molecule is locked in the low affinity structure), indicating site heterogeneity. The complete amino acid sequence of eel anodic hemoglobin has been established and compared with that of other fish hemoglobins. The presence of the Root effect correlates with a specific configuration of the alpha1beta2 switch interface, which at low pH would stabilize subunit ligation in the T state without changing the quaternary structure. We propose that the major groups involved in the binding of oxygen-linked protons in eel anodic hemoglobin are located on the beta chain and comprise His-HC3 at the C terminus, His-FG4 at the switch interface, and Lys-EF6 and the N terminus at the phosphate-binding site.

Amino Acid Sequence↗

PubMed1997

Oxygen binding and aggregation of hemoglobin from the common European frog, Rana temporaria.

The hemoglobin from the European frog, Rana temporaria, consists of one major and three minor components. The tetramers aggregate upon deoxygenation notably at pH 7:3. Aggregation due to formation of disulphide bridges, as occurs in related species, was observed only in polyacrylamide gels. The hemolysate showed a pronounced Bohr effect. Oxygen affinity decreased with increasing hemoglobin concentration, indicating that aggregation affects the functional properties of the hemolysate. Oxygen binding equilibria of unfractionated hemolysate are insensitive to chloride and show low sensitivity to ATP. Analysis of oxygen equilibrium curves in terms of the two-state allosteric model (MWC) showed that pH change exerted a greater effect on the association constant of the oxygenated state (KR) than that of the deoxy state (KT). The number of interacting binding sites (q) increased with hemoglobin concentration. Cooperativity of oxygen binding, evaluated as Hill coefficient n, never exceeded the value of 3.0. Earlier studies on hemoglobin and blood from this and related species, report significantly higher n values at high O2 saturation. Molecular adaptive aspects are discussed.

Animals↗

PubMed1996

Molecular shape, dissociation, and oxygen binding of the dodecamer subunit of Lumbricus terrestris hemoglobin.

Small angle x-ray scattering of the 213-kDa dodecamer of Lumbricus terrestris Hb yielded radius of gyration = 3.74 +/- 0.01 nm, maximum diameter = 10.59 +/- 0.01 nm, and volume = 255 +/- 10 nm3, with no difference between the oxy and deoxy states. Sedimentation velocity studies indicate the dodecamer to have a spherical shape and concentration- and Ca2+-dependent equilibria with its constituent subunits, the disulfide-bonded trimer of chains a-c and chain d. Equilibrium sedimentation data were fitted best with a trimer-dodecamer model, ln K4 = 7 (association K in liters3/g3) at 1 degrees C and 4 at 25 degrees C, providing DeltaH = -20 kcal/mol and DeltaS = 4.4 eu/mol. Oxydodecamer dissociation at pH 8.0, in urea, GdmCl, heteropolytungstate K8[SiW11O39] and of metdodecamer at pH 7, was followed by gel filtration. Elution profiles were fitted with exponentially modified gaussians to represent the three peaks. Two exponentials were necessary to fit all the dissociations except in [SiW11O39]-8. Equilibrium oxygen binding measurements at pH 6.5-8. 5, provided P50 = 8.5, 11.5-11.9 and 11.9-13.5 torr, and n50 = 5.2-9. 5, 3.2-4.9, and 1.8-2.7 for blood, Hb, and dodecamer, respectively, at pH 7.5, 25 degrees C. P50 was decreased 3- and 2-fold in approximately 100 mM Ca2+ and Mg2+, respectively, with concomitant but smaller increases in cooperativity.

Animals↗

PubMed1996

The crystal structures of trout Hb I in the deoxy and carbonmonoxy forms.

We have determined the X-ray crystallographic structure of trout Hb I in both the deoxy and carbonmonoxy forms to resolution limits of 2.3 angstroms and 2.5 angstroms, respectively. The overall fold of the molecule is highly similar to that of human HbA despite the low level of sequence identity between these proteins. Trout Hb I is unusual in displaying almost no pH dependence of oxygen binding affinity, and (at most) very weak interactions with heterotropic effector ligands such as organic phosphates. Comparison of the two quaternary states of the protein indicates how such effects are minimised and how the low-affinity T state of the protein is stabilised in the absence of heterotropic interactions.

Allosteric Regulation↗

PubMed1996

Spectroscopical and functional characterization of the hemoglobin of Nostoc commune (UTEX 584 (Cyanobacterial).

Structural analysis of a monomeric hemoglobin from the cyanobacterium Nostoc commune strain UTEX 584, cyanoglobin (Potts et al. (1992) Science 256, 1690-1692), is presented. Cyanoglobin binds molecular oxygen reversibly, with high oxygen affinity and non-cooperativity. There was no evidence for decreased stability of the pigment at 37 degrees C. Cyanoglobin-specific antibodies showed no cross-reactivity with two reference hemoglobins, leghemoglobin a and sperm whale myoglobin. The absorption spectral properties of cyanoglobin differ significantly from those of the two reference hemoglobins. The spectrum of oxy-cyanoglobin most closely resembles that of an oxy-hemoglobin from the protozoan Tetrahymena pyriformis, a hemoprotein that shares substantial amino-acid sequence identity with cyanoglobin. Met-cyanoglobin possesses spectral characteristics at pH 7.0-9.0 that resemble those of the alkaline met-hemoglobin (a putative hemichrome) of another protozoan, Paramecium caudatum. The spin-state character of met-cyanoglobin is pH-dependent. Met-cyanoglobin does not coordinate the strong-field ligands, cyanide and azide, at pH 7.0. The capacity of cyanoglobin to coordinate cyanide increased with decreasing pH. Far-UV CD spectra of cyanoglobin are indicative of a protein with a significant amount of alpha-helical structure. Data from Soret-region CD spectra suggest that the orientations of the heme moieties in cyanoglobin and leghemoglobin a are similar to one another.

Blotting, Western↗

PubMed1996

Oxygen equilibria of cathodic eel hemoglobin analysed in terms of the MWC model and Adair's successive oxygenation theory.

Allosteric effects of erythrocytic NTP and proton concentrations on cathodic eel Hb were investigated by precise measurement of Hb-O2 equilibria (including extreme saturation values) and analysis in terms of the MWC two-state model and the Adair four-step oxygenation theory. Stripped cathodic Hb shows a reverse Bohr effect and high sensitivities to ATP and GTP that extend to high pH values (> 8.5). A decrease in pH raises KT and lowers the allosteric constant L, compared to opposite effects in 'normal' Bohr effect Hbs. Phosphates even at low concentrations (GTP/Hb = 0.5) annihilate the reverse Bohr effect. GTP exerts a greater effect than ATP due to greater changes in KT and L, and NTP slightly reduces KR. In the absence of NTP, about 1.1 protons are released on deoxygenation at pH 8.15 (where most protons are released), indicating a pK value of the reverse Bohr group of approximately 8.2 (higher in oxy-Hb and lower in deoxy-Hb). The pH and NTP dependence of the Adair association constants and calculated fractional populations of Hb molecules in different oxygenation stages show that NTP effectors stabilise the T structure and postpone the T-R transition, whereas protons in the absence of NTP have the opposite effect. A molecular mechanism for the reverse Bohr effect is suggested.

Adenosine Triphosphate↗

PubMed1995

Mass spectrometric composition, molecular mass and oxygen binding of Macrobdella decora hemoglobin and its tetramer and monomer subunits.

The hexagonal bilayer hemoglobin (Hb) of the leech Macrobdella decora has an equilibrium sedimentation mass of 3544(+/- 80) kDa. Maximum entropy analysis of the electrospray ionization mass spectra of the Hb show three groups of peaks: two peaks of equal intensity at approximately 17 kDa, A (16,770.1 Da) and B (16,841.9 Da); three peaks at approximately 24 kDa, C (24,340.1 Da), D (24,398.6 Da) and E (24,420.0 Da) with relative intensities of 1:6:3, respectively; and three peaks of equal intensities at approximately 33 kDa, F (32,586.0 Da), G (32,714.5 Da) and H (32,849.9 Da). Although reduction with dithiothreitol does not affect the masses of peaks A through E, the approximately 33 kDa peaks give rise to four new peaks at approximately 16 kDa, P (16,052.2 Da), Q (16,537.3 Da), R (16,666.7 Da) and S (16,792.9 Da), indicating that F, G and H represent disulfide-bonded dimers of globin chains, P + Q, P + R and P + S, respectively. The relative intensities of the three groups of peaks are (A + B) to (C + D + E) to (F + G + H) = 0.39:0.26:0.32, and the globin to linker ratio 0.71:0.29 is in good agreement with the ratio 0.72:0.28 obtained by HPLC. The largest functional subunit obtained by dissociation at pH 7 in 4 M urea, is a subunit lacking linker chains with apparent mass 63(+/- 3) kDa. The equilibrium sedimentation profile of this subunit is fitted best as a monomer-dimer-tetramer equilibrium, with association constants K1,2 = 365 l g-1 and K1,4 = 8.1 x 10(5) l3 g-3. A model of the Hb consisting of a hexagonal bilayer of 36 tetramer and 42 linker subunits provides a total mass and globin to linker ratio closest to the experimental values. Equilibrium O2 binding measurements of the native Hb and its tetramer and monomer subunits were carried out over the pH range 6.6 to 8.0 at 10 and 25 degrees C, and in the absence and presence of Na+, Mg2+ and Ca2+. The Hb exhibits a moderately high O2 affinity, P50 = 4.4 torr at pH 7.5 and 25 degrees C, a high cooperativity (n50 approximately 3) and a substantial Bohr effect, phi = delta log P50/delta pH = -0.38. The tetramer subunit has a higher affinity, lower cooperativity and smaller Bohr effect, 1.9 torr, 1.3 to 1.5 and -0.30, respectively. The monomer subunit has a much higher affinity (P50 = 0.29 torr) and no cooperativity or Bohr effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗

PubMed1995

The cathodic hemoglobin of Anguilla anguilla. Amino acid sequence and oxygen equilibria of a reverse Bohr effect hemoglobin with high oxygen affinity and high phosphate sensitivity.

As in other fish, the cathodic hemoglobin of the eel Anguilla anguilla is considered to play an important role in oxygen transport under hypoxic and acidotic conditions. In the absence of phosphates this hemoglobin shows a reverse Bohr effect and high oxygen affinity, which is strongly modulated over a side pH range by GTP (whose concentration in the red blood cells varies with ambient oxygen availability). GTP obliterates the reverse Bohr effects in the cathodic hemoglobin. The molecular basis for the reverse Bohr effect in fish hemoglobins has remained obscure due to the lack of structural data. We have determined the complete amino acid sequence of the alpha and beta chains of the cathodic hemoglobins of A. anguilla and relate it to the oxygen equilibrium characteristics. Several substitutions in crucial positions are observed compared with other hemoglobins, such as the replacement of the C-terminal His of the beta chain of Phe (that suppresses the alkaline Bohr effect) and of residues at the switch region between alpha and beta subunits (that may alter the allosteric equilibrium, thus causing the high intrinsic oxygen affinity and low cooperativity). The residues binding organic phosphate in the beta cleft of fish hemoglobins are conserved, which explains the strong effect of GTP on oxygen affinity and suggests that these residues contribute to the reverse Bohr effect in the absence of alkaline Bohr groups. Moreover, His beta 143 that is considered to be responsible for the reverse Bohr effect in human and tadpole Hbs is replaced by Lys.

Amino Acid Sequence↗

PubMed1995

The hemoglobin system of the hagfish Myxine glutinosa: aggregation state and functional properties.

Hemoglobin (Hb) from the hagfish Myxine glutinosa is composed of six major monomeric subunits. Some of these subunits aggregate to dimers at low pH, and to tetramers when deoxygenated and at high protein concentration. The aggregation is inhibited by the presence of KCl. Oxygen equilibrium studies show the presence of a small Bohr effect which is strongly reduced by KCl, indicating that it originates from pH-dependent aggregation. ATP and DPG cause a similar decrease in the Bohr effect. O2 affinity is dependent on protein concentration, temperature and presence of CO2. Cooperativity is practically absent. O2 binding properties of the separated aggregating and non-aggregating Hbs purified at low pH cannot account for the functional properties of the composite hemolysate, suggesting the presence of other subunits interactions. The results are discussed in relation to literature data for other cyclostome Hbs and for M. glutinosa Hb, where the presence of three major monomeric Hbs and a possible CO2-dependent aggregation had been reported.

Animals↗

PubMed1994

Hemoglobins from Plasmodium-infected rat erythrocytes: functional and molecular characteristics.

Aiming to evaluate the mechanisms responsible for altered O2-transporting properties in blood of Plasmodium-infected animals, stripped (cofactor-free) hemoglobin (Hb) solutions were prepared from infected erythrocytes (IE) and noninfected erythrocytes (NIE) of rats inoculated with Plasmodium berghei bergei for functional and structural characterization. At normal intraerythrocytic pH (+/- 7.2), Hb from IE showed a higher affinity, a larger Bohr effect, and lower sensitivities to 2,3-diphosphoglycerate (DPG) and to temperature than did NIE Hb. Moreover, as judged from electrophoresis, isoelectric focusing, and gel filtration experiments, Hb from IE show changes in charge and molecular assembly. The results indicate that the higher O2 affinity and greater Bohr factors observed in IE compared with those for NIE are attributable to chemical modification of the Hb that increases its intrinsic O2 affinity and decreases its sensitivity to DPG as well as to changes in the intracellular physicochemical milieu, including reduced DPG levels.

Animals↗

PubMed1993

Purification and characterization of recombinant polymeric hemoglobin P1 of Glycera dibranchiata.

The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli. The expressed globin was reconstituted with heme and purified. The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem. Biophys. Acta, 1041, 117-123, 1990). Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin. The molar ellipticity at 222 nm provided an estimate of 77% for the alpha-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II. Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4).

Animals↗

PubMed1993

Arterial blood gas tensions during breath-hold diving in the Korean ama.

Korean female unassisted divers (cachido ama) breath-hold dive > 100 times to depths of 3-7 m during a work day. We sought to determine the extent of arterial hypoxemia during normal working dives and reasonable time limits for breath-hold diving by measuring radial artery blood gas tensions and pH in five cachido ama who dove to a fixed depth of 4-5 m and then continued to breath hold for various times after their return to the surface. Eighty-two blood samples were withdrawn from indwelling radial artery catheters during 37 ocean dives. We measured compression hyperoxia [arterial PO2 = 141 +/- 24 (SD) Torr] and hypercapnia (arterial PCO2 = 46.6 +/- 2.4 Torr) at depth. Mean arterial PO2 near the end of breath-hold dives lasting 32-95 s (62 +/- 14 s) was decreased (62.6 +/- 13.5 Torr). Mean arterial PCO2 reached 49.9 +/- 5.4 Torr. Complete return of these values to their baseline did not occur until 15-20 s after breathing was resumed. In dives of usual working duration (< 30 s), blood gas tensions remained within normal ranges. Detailed analysis of hemoglobin components and intrinsic oxygenation properties revealed no evidence for adaptive changes that could increase the tolerance of the ama to hypoxic or hypothermic conditions associated with repetitive diving.

2,3-Diphosphoglycerate↗

PubMed1993

Mutant hemoglobins (alpha 119-Ala and beta 55-Ser): functions related to high-altitude respiration in geese.

The unusually high blood-O2 affinity in the bar-headed and Andean geese is a necessary adaptation for migration across high mountain ranges. The amino acid residues alpha-119 and beta-55, which form an alpha 1 beta 1 contact in human hemoglobin (Hb), are altered in bar-headed and Andean geese, respectively, which suggests that loss of this contact increases O2 affinity. Two mutant human Hbs with equivalent mutations at these sites prepared by site-directed mutagenesis show the same increase in O2 affinity compared with Hb A, which indicates that these mutations are responsible for the changes in the protein. The intrinsic affinity difference compared with native Hb A is amplified by organic phosphates. Whereas the recombinant and native Hbs displayed similar sensitivities to pH, chloride, and 2,3-diphosphoglycerate, the oxygenation heat of the alpha-chain mutant decreased in the presence of 2,3-diphosphoglycerate. O2 association constants for the deoxygenated state of the alpha-mutant were about three times those for Hb A. The mutant Hb analogously exhibited higher affinity constants for binding the first three O2 molecules. Calculated heme-heme interaction energies indicated that loss of a single contact, resulting in destabilization of the deoxy (tense) structure, underlies the increased O2 affinity. Adaptations securing Hb-O2 binding at extreme altitude are discussed.

2,3-Diphosphoglycerate↗

PubMed1992

Use of ionic and zwitterionic (Tris/BisTris and HEPES) buffers in studies on hemoglobin function.

The functional characteristics of hemoglobin (Hb) depend on oxygenation-linked proton and anion binding and thus on solvent buffer groups and ionic composition. This study compares the oxygenation properties of human Hb in ionic [tris(hydroxymethyl)aminomethane (Tris) and BisTris] buffers with those in zwitterionic N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer under strictly controlled chloride concentrations at different pH values, two temperatures, and in the absence and presence of the erythrocytic cofactor, 2,3-diphosphoglycerate (DPG). In contrast to earlier studies (carried out at the same or different chloride concentrations) it shows only small buffer effects that are manifested at low chloride concentration and high pH. These observations suggest chloride binding to the Tris buffers, which reduces the interaction with specific chloride binding sites in the Hb. The findings indicate that HEPES allows for more accurate assessment of Hb-oxygen affinity and its anion and temperature sensitivities than ionic buffers and advocates standard use of HEPES in studies on Hb function. Precise oxygen affinities of Hb dissolved in both buffers are defined under standard conditions.

Binding Sites↗

PubMed1991

Adaptation of bird hemoglobins to high altitudes: demonstration of molecular mechanism by protein engineering.

Of two closely related species of geese, one, the greylag goose, lives in the Indian plains all year round, while the other, the bar-headed goose, lives at the Tibetan lakes and migrates across the Himalayas to winter in India. Another species, the Andean goose, lives in the High Andes all year round. Possession of a Hb with high oxygen affinity helps to adapt bar-headed and Andean geese to high altitudes. The Hb amino acid sequences of the bar-headed and the greylag geese differ by four substitutions, of which only one is unique among bird sequences: Pro-119 alpha (H2)----Ala. Perutz proposed that the two-carbon gap left by this substitution at the alpha 1 beta 1 contact raises the oxygen affinity, because it relaxes the tension in the deoxy or T structure [Perutz, M. F. (1983) Mol. Biol. Evol. 1, 1-28]. It was later found that the Hb of the Andean goose has a gap in the same position, due to the complementary substitution Leu-55 beta (D6)----Ser. We have tested Perutz's hypothesis by introducing each of these substitutions into human globin synthesized in Escherichia coli. The reconstituted Hbs combine cooperatively with oxygen. Their oxygen affinities exceed that of normal human Hb by an even larger factor than that found between the high-flying geese and the greylag goose. The mutant Hb Met-55 beta (D6)----Ser was crystallized. Its structure is the same as that of HbA, except in the immediate environment of the gap left by the substitution of the serine for the methionine side chain, which evidently causes the increased oxygen affinity of this Hb.

Adaptation, Physiological↗

PubMed1991

Respiratory impairment in crustaceans and molluscs due to exposure to heavy metals.

1. We have assessed, using current literature, the respiratory consequences of water-borne heavy metal exposure in crustaceans and molluscs. 2. We suggest that in lethal and sub-lethal concentrations the essential metals Cu and Zn act on the respiratory system primarily by disrupting gill function which results in the development of internal hypoxia, although reparation can be accomplished even at "high" sub-lethal concentrations. 3. The more toxic xenobiotes such as Hg (and perhaps to a lesser extent Cd) may interfere with the respiratory system at every level of organisation including cellular respiration itself.

Animals↗

PubMed1991

Primary structure and oxygen-binding properties of the hemoglobin from the lesser hedgehog tenrec (Echinops telfairi, Zalambdodonta). Evidence for phylogenetic isolation.

The primary structures of the alpha- and beta-hemoglobin chains of the lesser hedgehog tenrec (Echinops telfairi, Zalambdodonta) are presented. Chain separation was performed by carboxymethyl-cellulose chromatography. The peptides, obtained by tryptic digestion of the oxidized chains, were prefractionated by gel chromatography and isolated by reversed-phase HPLC. For sequence analysis gas and liquid phase sequencers were employed. The tenrec hemoglobin consists of one alpha- and two beta-chains the latter occurring in a 1:1 ratio and differing in beta 16 Gly/Cys and beta 118 Phe/Leu. Two external cysteine residues at beta 16 and beta 52 cause reversible polymerization to octamers and most likely irreversible formation of higher polymers. A comparison of the whole chains and certain positions of tenrec hemoglobin with those of Insectivora sensu strictu, Scandentia and Proto- and Metatheria corroborates a long and independent evolution of tenrec and its phylogenetic isolation from the Insectivora s.str. (hedgehog, musk shrew and mole). Replacements at positions involved in heme and subunit interface contacts are discussed. Compared to human hemoglobin the tenrec pigment shows a low intrinsic oxygen affinity as well as lower chloride and temperature sensitivities, a reduced Bohr effect and a strong response to 2,3-DPG. The possible adaptive significance of these properties is discussed in relation to the large diurnal body temperature variations seen in tenrecs.

Amino Acid Sequence↗

PubMed1990

Crystallization of the rainbow trout (Salmo gairdneri) haemoglobin IV.

Crystals of rainbow trout (Salmo gairdneri) haemoglobin IV were grown in mini batches from a solution of ammonium sulphate. Large single crystals grew over five days and were up to 2 mm in length. X-ray diffraction experiments indicated a space group of C222(1) with unit cell dimensions of a = 85.3 A, b = 94.6 A and c = 105.7 A. The crystals diffract to better than 2.5 A but exhibit some mosaicity along the c axis.

Animals↗