Biomedical subjects

R E Weber

Publications and source records attributed to R E Weber.

At least 19 recordsLinked to original sources

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↗

PubMed1989

Gas exchange in fish gills with parallel inhomogeneities.

Using a mathematical model, the effects of water and blood flow inequality on gas exchange in the fish gill were investigated. The results show that O2 exchange is more sensitive to flow inequalities than CO2 exchange. Oxygen exchange is most sensitive to blood flow inequality, whereas carbon dioxide exchange is equally sensitive to inequalities in water and blood flow. Furthermore, it is shown that the effects of flow inequalities cannot be abolished by matching the two flows exactly. The results suggest that the greatest potential for regulation of gas exchange lies on the blood side of the exchanger, and that adjustments in flow distributions to maintain homeostasis in oxygen exchange in the face of changing environmental conditions and/or changing organismic demands, most likely will be accompanied by a change in the acid-base status of the animal.

Animals↗

PubMed1989

Deposition of cigarette smoke particles in the rat.

The fractional deposition of cigarette smoke particles in the respiratory tracts of rats was studied. Male and female rats were conditioned in nose-only exposure tubes 25 min/day for 2 days, exposed to cigarette smoke at mass concentrations of 95 or 341 mg/m3, 25 min/day for 3 days, and then exposed to smoke at mass concentrations of 212 and 657 mg/m3, 25 min/day for 5 days. Mainstream cigarette smoke was generated by a modified Walton smoking machine from two 1R3 research cigarettes burned sequentially for each exposure. Deposition studies were conducted by placing the rats in plethysmograph tubes to allow respiratory minute volume measurements during exposure, then exposing them to [14C]dotriacontane-labeled cigarette smoke at mass concentrations of 202 or 624 mg/m3 for 25 min, using the same smoking machine. Size distribution, real-time concentration, and 14C activity of the smoke particles were determined using a multijet Mercer impactor, a real-time aerosol monitor, and filter samples, respectively. Immediately after the exposure, the rats were terminated to determine the distribution of the 14C. Individual lung lobes, trachea and lobar bronchi, head, larynx, kidneys, liver, gastrointestinal (GI) tract, blood, and depleted carcass of each rat were analyzed for 14C content. Results showed that the GI tract contained 16-31% of the total activity, indicating significant clearance from the large airways and nose to the GI tract during the exposure and during the 10-15 min between the cessation of the exposure and the removal of the organs. Total deposition of the inhaled 14C activity was 20.1 +/- 1.6% for both exposure concentrations. The intrapulmonary deposition fractions (lung lobes plus airways below the lobar bronchi) were 12.4 +/- 0.9 and 15.9 +/- 1.4% for concentrations of 202 and 624 mg/m3, respectively, suggesting a slight enhancement in upper airway deposition for animals exposed to the higher smoke concentration.

Alkanes↗

PubMed1989

Continuous cardiac output determination using transtracheal Doppler: initial results in humans.

Transtracheal Doppler, a procedure developed in the authors' laboratory, provides an estimate of cardiac output in endotracheally intubated dogs. The present study reports initial results in humans with a Doppler probe incorporated into an endotracheal tube. The system was first calibrated by empirically determining phi, the angle of the ultrasound beam with respect to the blood velocity vector. For a best least squares fit for phi, aortic diameters can be calibrated over a range of 22-37 mm. The calibration was then tested in a separate group of patients using the same empirically derived angle to calculate cardiac outputs. The transtracheal Doppler cardiac outputs show good correlation with those obtained by thermodilution over a range of cardiac outputs, 2.69-8.62 1/min, R2 = 0.835.

Adolescent↗

PubMed1989

Amino-acid sequences and functional differentiation of hemoglobins A and D from swift (Apus apus, Apodiformes).

The blood of the adult swift contains one major (HbA = alpha 2A beta 2) and two minor components (HbD = alpha 2D beta 2 and HbD'). The components were separated by FPLC with a TSK SP-5 PW-column in phosphate buffers, and were eluted with a linear NaCl gradient. HbD' could be detected only in freshly prepared hemolysates with the sensitive FPLC separation method. The globin chains were separated on a cation exchanger (CM-cellulose), the tryptic peptides by HPLC with a RP-2 LiChrosorb column. Their amino-acid sequences were determined by automatic Edman degradation with the film- or gas-phase method. For the alpha A-, alpha D- and beta-chains, peptide alignment was achieved by homologous comparison with the corresponding chains of the greylag goose (Anser anser). The structural significance of the substitutions was examined with the aid of molecular graphics. The oxygen-binding properties of the stripped hemolysate and of HbA and HbD and their dependence on pH, temperature and inositol polyphosphate are presented and discussed with reference to molecular structures and hypothermy that occurs during torpidity.

Amino Acid Sequence↗

PubMed1989

High-altitude respiration of falconiformes. The primary structures and functional properties of the major and minor hemoglobin components of the adult White-Headed Vulture (Trigonoceps occipitalis, Aegypiinae).

The primary structures of the hemoglobin components Hb A and Hb D of White-Headed Vulture (Trigonoceps occipitalis) are presented. The globin chains were separated on CM-Cellulose in 8M urea buffer, the components by FPLC in phosphate buffers. The amino-acid sequences were established by automatic Edman degradation of the globin chains and of the tryptic peptides in liquid phase and gas-phase sequenators. The sequences differ from those of European Black Vulture by only one mutation in the alpha A-chains (alpha 137). The alpha D-chains and the beta-chains are identical. This means that for the first time identical minor components in birds have been found. An updated list of identical globin chains is presented. Hb D exhibited a higher oxygen affinity than Hb A. At pH 7.5 and 38 degrees C P50 values of 0.80 and 0.64 kPa (6.0 and 4.8 mm Hg), respectively. Both hemoglobins showed similar Bohr factors displayed a pronounced sensitivity to inositol hexakis(phosphate), which increased P50 values of Hbs A and D to 4.0 and 3.6 kPa (30 and 26 mm Hg), respectively. The molecular and physiological significance of the findings is discussed with special reference to oxygen transport by hemoglobin at high altitude.

Altitude↗

PubMed1989

Allosteric transition in hemoglobin (alpha 2A beta 2I) from the rhynchocephalian reptile relict Sphenodon punctatus.

The major hemoglobin component Hb A of the tuatara, Sphenodon punctatus, a relict of the rhynochocephalian 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 (Hill's cooperativity coefficient at half-saturation, n50, attaining 1.3-1.5 above pH 7.5), 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.

Adenosine Triphosphate↗

PubMed1989

Transtracheal Doppler: a new procedure for continuous cardiac output measurement.

An endotracheal tube with a 5 mm diameter ultrasonic transducer incorporated into its distal end was inserted into the tracheas of six anesthetized dogs. The device provides for ultrasound measurement of aortic diameter and blood velocity in the ascending aorta to obtain cardiac outputs. Because measurements are made proximal to the origins of the aortic arch vessels, total cardiac output is obtained. Procedures for measurement of aortic diameter, aortic blood velocity, and cardiac output are discussed. The resulting data were used to empirically determine the angle of incidence of the ultrasound beam that gave the best least squares fit over a range of thermodilution cardiac outputs. For an angle of incidence of 62.3 degrees, the relationship between cardiac output determined by thermal dilution and by transtracheal Doppler was transtracheal Doppler cardiac output = -.69 + 1.19 thermal dilution cardiac output. R2 = .82. The results from this preliminary assessment of the feasibility of measuring cardiac output in dogs using transtracheal Doppler suggest that additional studies should be pursued. Additional issues that require investigation include confirmation of constancy of human anatomy and demonstration of lack of tracheal trauma from the Doppler equipped endotracheal tube.

Animals↗

PubMed1987

Thermodynamic analysis of precisely measured oxygen equilibria of tench (Tinca tinca) hemoglobin and their dependence on ATP and protons.

Precise oxygen equilibria including extreme, high and low saturation values were determined for hemoglobin (Hb) from the freshwater teleost Tinca tinca at three temperatures, each at two pH levels and in the presence and absence of the erythrocytic cofactor ATP, at twofold molar excess over Hb. Analysis of the data in terms of Adair's successive oxygenation theory shows that in the absence of ATP, each of the four oxygenation steps are exothermic, but that net heat release decreases as pH falls from 8.2 to 7.4. ATP greatly depresses the temperature sensitivity of oxygenation particularly at physiological erythrocytic pH, where endothermic cofactor dissociation finds expression in a reverse temperature sensitivity for binding of the 3rd oxygen molecule to the tetrameric Hb. Enthalpy (delta Hi) and entropy (delta Si) changes of oxygenation vary with oxygenation step, i, as well as with pH and ATP addition, but the variations of delta Hi are similar to those of delta Si reflecting enthalpy-entropy compensation. The data show that the cooperative effects in tench Hb can be dominated either by entropic or enthalpic contributions, depending on the experimental condition and the oxygenation step.

Adenosine Triphosphate↗

PubMed1987

Embryonic pig 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): 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.

2,3-Diphosphoglycerate↗

PubMed1987

Primary structure and functional properties of the hemoglobin from the free-tailed bat Tadarida brasiliensis (Chiroptera). Small effect of carbon dioxide on oxygen affinity.

The hemoglobin of the Free-Tailed Bat Tadarida brasiliensis (Microchiroptera) comprises two components (Hb I and Hb II) in nearly equal amounts. Both hemoglobins have identical beta-chains, whereas the alpha-chains differ in having glycine (Hb I) or aspartic acid (Hb II) in position 115 (GH3). The components could be isolated by DEAE-Sephacel chromatography and separated into the globin chains by chromatography on carboxymethyl-cellulose CM-52. The sequences have been determined by Edman degradation with the film technique or the gas phase method (the alpha I-chains with the latter method only), using the native chains and tryptic peptides, as well as the C-terminal prolyl-peptide obtained by acid hydrolysis of the Asp-Pro bond in the beta-chains. The comparison with human hemoglobin showed 18 substitutions in the alpha-chains and 24 in the beta-chains. In the alpha-chains one amino-acid exchange involves an alpha 1/beta 1-contact. In the beta-chains one heme contact, three alpha 1/beta 1- and one alpha 1/beta 2-contacts are substituted. A comparison with other chiropteran hemoglobin sequences shows similar distances to Micro- and Megachiroptera. The oxygenation characteristics of the composite hemolysate and the two components, measured in relation to pH, Cl-, and 2,3-bis-phosphoglycerate, are described. The effect of carbon dioxide on oxygen affinity is considerably smaller than that observed in human hemoglobin, which might be an adaptation to life under hypercapnic conditions.

Amino Acid Sequence↗

PubMed1986

Oxygen binding functions of blood and hemoglobin from the Chinese pangolin, Manis pentadactyla: possible implications of burrowing and low body temperature.

We measured O2 equilibria of adult blood and of 'stripped' (cofactor-free) hemolysates from adult and newborn Manis pentadactyla, in order to assess the implications of the burrowing habit and the low deep-core temperature in pangolins, and to discern the mechanisms for maternal-fetal O2 transfer. At pH 7.4 and body temperature (33 degrees C) the blood O2 affinity was significantly higher than in similarly sized non-burrowing, 'normothermic' mammals (P50 = 21 and 33 mm Hg, respectively) indicating an adaptation to hypoxic burrow conditions. This difference is not attributable to a higher intrinsic O2 affinity in the pangolin Hb or to significant differences in its sensitivity to temperature and erythrocytic 2,3 diphosphoglycerate (DPG), but tallies with lower DPG/Hb ratios than generally found in mammals. Stripped adult and newborn hemolysates show similar O2 affinities and pH and DPG sensitivities, but reveal a specific adult Hb that develops after birth, in sharp contrast with the ontogenetic changes in other mammals where specific fetal Hbs are lost after birth.

2,3-Diphosphoglycerate↗

PubMed1986

Oxygen binding in alligator blood related to temperature, diving, and "alkaline tide".

Blood of Alligator mississippiensis has a relatively high O2 affinity, the half-saturation O2 tension (P50) = 24.5 Torr at pH 7.495, the arterial, normocapnic pH at 25 degrees C. Although the overall temperature sensitivity of P50 at 15, 25, and 35 degrees C and constant pH is low, the effect on P50 almost doubles when measured at the in vivo pH of each temperature (delta Happ = -24 and -47 kJ/mol, respectively). The CO2 Bohr effect (theta CO2 = -0.95) is 5.5 times greater than the fixed acid Bohr effect (theta FA), and the Haldane effect is small (approximately 0.03 pH units). The relatively high O2 affinity may ensure efficient utilization of the lung O2 reserve during breath holding and diving, whereas its pronounced in vivo temperature sensitivity may be adaptive to the high temperature quotients of the organismic O2 requirement. The large difference between theta CO2 and theta FA will favor constancy in blood O2 affinity in the face of large activity-induced increases in blood lactate and pronounced feeding-induced alkaloses. These "alkaline tides," which result from an exchange of plasma Cl- for HCO-3 across the gut wall, appear to be only slightly compensated by increased blood CO2 tensions. The results are additionally discussed in terms of allosteric modulation of hemoglobin-O2 affinity in crocodilians.

Alkalosis↗

PubMed1977

Oxygen-binding properties of hemoglobins from estivating and active African lungfish.

The oxygen-binding characteristics and the multiplicity of the stripped hemoglobiin from active lungfish Protopterus amphibius, are the same as in specimens that have been estivating for about 30 months, showing that alteration in the hemoglobin molecules is not involved in the earlier reported increase in oxygen affinity of whole blood during estivation (Johansen et al., '76). At pH 7.0 and 26 degrees C the hemolysates show a high oxygen affinity (P50 = 3.1 Torr), a Bohr factor (delta log P50/delta pH) of - 0.33, and a cooperativity coefficient (n) of 1.7. Between 15 and 26 degrees C, the apparent heat of oxygenation (delta H) is - 8.6 Kcal-mole-1 at pH 7.0, corresponding with data for other fish. A low sensitivity of oxygen affinity to urea appears to be adaptive to the high urea concentrations in estivating lungfish. The salt sensitivity is, however, similar to human hemoglobin. The hemoglobin consists of two major (electrophoretically anodal) components, which differ slightly in oxygen affinity but are both sensitive to pH and nucleoside triphosphates (NTP). Guanosine triphosphate (GTP), the major erythrocytic organic phosphate, however, depresses the oxygen affinity of the composite and separated hemoglobins more effectively than ATP suggesting that GTP is the primary modulator of oxygen affinity. Comparative measurements reveal only one major hemoglobin component in P. annectens which has a markedly lower oxygen affinity and phosphate sensitivity than P. amphibius hemoglobins and thus seems less pliable to phosphate-mediated variation in oxygen affinity. The data are discussed in relation to the hemoglobin systems of other fish.

Adaptation, Physiological↗