The transition in hemoglobin proton-binding characteristics within the basal actinopterygian fishes

Carbon dioxide (CO₂) transport in the blood of fishes is aided by the proton-binding properties of hemoglobin (Hb) through either a high-intrinsic buffer value and small oxylabile proton binding (Haldane effect), or a low buffer value and large Haldane effect. Primitive species, such as elasmobranchs and sarcopterygians have been shown to rely on the former, while derived species, such as teleosts rely on the latter. Both strategies are effective in the transport of CO₂ in the blood. However, there is a paucity of information on the nature of the transition between these two strategies that appears to occur within the intermediate group of fishes, the basal actinopterygians. The objective of the present study was to simultaneously assess the intrinsic Hb buffer values and Haldane effects of species within the basal actinopterygian lineage to characterize the transition in Hb-proton-binding strategy seen among the fishes. Expressed in order of most basal to most derived, the species investigated included American paddlefish (Polyodon spathula), white sturgeon (Acipenser transmontanus), spotted gar (Lepisosteus oculatus), alligator gar (Atractosteus spatula), bowfin (Amia calva), and mooneye (Hiodon tergisus). Hemolysates from these species were prepared and Hb titrations (under oxygenated and deoxygenated conditions) were performed in both the presence and absence of saturating levels of organic phosphates (GTP). The findings suggest that the nature of the Hb-proton-binding transition may have been punctuated rather than gradual, with the Hb buffer value decreasing and the Haldane effect increasing significantly in bowfin from fairly steady ancestral levels in the four more basal species. This change is coupled with the initial appearance of the choroid rete, as well as an increase in the magnitude and onset pH of the Root effect in bowfin, suggesting that the change in Hb-proton-binding strategy may be associated with the evolution of enhanced O₂ delivery to the eye and an in vivo operational Root effect.     

Responses of the red blood cells from two high-energy-demand teleosts, yellowfin tuna (Thunnus albacares) and skipjack tuna (Katsuwonus pelamis), to catecholamines

In fishes, catecholamines increase red blood cell intracellular pH through stimulation of a sodium/proton (Na⁺/H⁺) antiporter. This response can counteract potential reductions in blood O₂ carrying capacity (due to Bohr and Root effects) when plasma pH and intracellular pH decrease during hypoxia, hypercapnia, or following exhaustive exercise. Tuna physiology and behavior dictate exceptionally high rates of O₂ delivery to the tissues often under adverse conditions, but especially during recovery from exhaustive exercise when plasma pH may be reduced by as much as 0.4 pH units. We hypothesize that blood O₂ transport during periods of metabolic acidosis could be especially critical in tunas and the response of rbc to catecholamines elevated to an extreme. We therefore investigated the in vitro response of red blood cells from yellowfin tuna (Thunnus albacares) and skipjack tuna (Katsuwonus pelamis) to catecholamines. Tuna red blood cells had a typical response to catecholamines, indicated by a rapid decrease in plasma pH. Amiloride reduced the response, whereas 4,4′diisothiocyanatostilbene-2,2′-disulphonic acid enhanced both the decrease in plasma pH and the increase in intracellular pH. Changes in plasma [Na⁺], [Cl⁻], and [K⁺] were consistent with the hypothesis that tuna red blood cells have a Na⁺/H⁺ antiporter similar to that described for other teleost red blood cells. Red blood cells from both tuna species were more responsive to noradrenaline than adrenaline. At identical catecholamine concentrations, the decrease in plasma pH was greater in skipjack tuna blood, the more active of the two tuna species. Based on changes in plasma pH, the response of red blood cells to catecholamines from both tuna species was less than that of rainbow trout (Oncorhynchus mykiss) red blood cells, but greater than that of cod (Gadus morhua) red blood cells. Noradrenaline had no measurable influence on the O₂ affinity of skipjack tuna blood and only slightly increased the O₂ affinity of yellowfin tuna blood. Our results, therefore, do not support our original hypothesis. The catecholamine response of red blood cells from high-energy-demand teleosts (i.e., tunas) is not enhanced compared to other teleosts. There are data on changes in cardio-respiratory function in tunas caused by acute hypoxia and modest increases in activity, but there are no data on the changes in cardio-respiratory function in tunas accompanying the large increases in metabolic rate seen during recovery from exhaustive exercise. However, we conclude that during those instances where high rates of O₂ delivery to the tissues are needed, tunas' ability to increase cardiac output, ventilation volume, blood O₂ carrying capacity, and effective respiratory (i.e., gill) surface area are probably more important than are the responses of red blood cells to catecholamines. We also use our data to investigate the extent of the Haldane effect and its relationship to blood O₂ and CO₂ transport in yellowfin tuna. Yellowfin tuna blood shows a large Haldane effect; intracellular pH increases 0.20 units during oxygenation. The largest change in intracellular pH occurs between 40–100% O₂ saturation, indicating that yellowfin tuna, like other teleosts, fully exploit the Haldane effect over the normal physiological range of blood O₂ saturation. Accepted: 27 March 1998     

Nitrogen binding to deoxyhemoglobin at high pressures and its relation to changes in hemoglobin-oxygen affinity

The stoichiometric and thermodynamic properties of nitrogen (N2) binding to human deoxyhemoglobin (Hb) at N2 saturation pressures up to 400 atm were derived from measured N2 solubilities in protein-free buffers (pH 7.1) and in corresponding buffer + Hb (6.5% w/w) solutions at 20.0, 25.0, and 37.0 degrees C. At each temperature, approximately 3 N2 molecules bind per Hb tetramer at N2 pressures of 100 atm, while about 7 N2 molecules bind per tetramer at 400 atm N2 pressure, where available binding sites are still not fully saturated. Calculated N2-Hb binding isotherms are well described by a simple binding model with 12 independent and equivalent binding sites/Hb tetramer. N2 binding at each of the sites is hydrophobic, exhibiting the typical increase of the dissociation enthalpy with temperature. Enthalpies of dissociation are slightly more negative, while corresponding unitary entropies are somewhat less negative than those for the transfer of N2 from olive oil to water. Calculated partial molar volumes of N2 in Hb are positive but less than the corresponding partial molar volumes of N2 in buffer. Results indicate that N2 binding to Hb is accompanied by only small protein conformational changes which entail slight structural destabilization and loss of free volume in the protein that partially accommodates the volume of the N2 ligand. Results are related to previously reported effects of high pressure and high-pressure N2 on HbO2 affinity, illuminating essential features of the molecular mechanisms for these effects.     

Self-association of haemoglobin Olympia (alpha 2 beta 2 20 (B2) Val----Met). A human haemoglobin bearing a substitution at the surface of the molecule

Oxygenation measurements at equilibrium were carried out for solutions of pure haemoglobin (Hb) Olympia (alpha 2 beta 2 20 (B2) Val----Met) at 200 microM (haem) and revealed a high oxygen affinity (P50 = 4.2 torr at pH 7.20, 25 degrees C) compared to HbA (P50 = 5.6 torr), with the Hill coefficient (eta H) reduced from the normal value of 2.9 to 2.5 for Hb Olympia at neutral pH. 2,3-Diphosphoglycerate and chloride effects were normal, but measurements of the alkaline Bohr effect indicated an excess Bohr effect of about 20% for Hb Olympia. Precise determinations of the oxygen binding curves gave the unexpected finding of a dependence of co-operativity on pH with eta H rising from 2.4 at pH 6.8 to 3.0 at pH 8. Moreover, the Hill coefficient was dependent upon the concentration at alkaline pH and fell to 1.8 in low concentration solutions (approximately 30 microM-haem) of the haemoglobin variant; at this concentration the Bohr effect was normal. This effect of concentration on co-operativity could be accounted for fully by the allosteric model, with introduction of Hb Olympia self-association. In this case the allosteric constant L' for the supramolecular species has the value of the allosteric constant L for the tetramer species, raised to a power equal to the number of molecules in the aggregates and modulated by the ratio of the dissociation constants of the aggregates, DNR/DNT. Model curves with N tetramers per aggregate (where N approximately 2 at pH 7.5 and N approximately 4 at pH 8.0) fully represented the concentration dependence for Hb Olympia of the eta H values and the detailed shape of the experimental curves for eta H as a function of log[y/(1-y)], the first derivative of the Hill plot. These curves are much steeper when supramolecular species are present. Direct measurements of the protein aggregation by centrifugation confirmed the presence of aggregates in the solutions of Hb Olympia. Hb Olympia is therefore one of the few examples of mutant human haemoglobins that self-associate with functional consequences in terms of oxygen binding properties.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

A calorimetric investigation of the interaction of the lac repressor with inducer

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.     

Physiological consequences of oxygen-dependent chloride binding to hemoglobin.

The PO(2)-dependent binding of chloride to Hb decreases the Cl(-) concentration of the red blood cell (RBC) intracellular fluid in venous blood to approximately 1-3 mmol/l less than that in arterial blood. This change is physiologically important because 1) Cl(-) is a negative heterotropic allosteric effector of Hb that competes for binding sites with 2,3-bisphosphoglycerate and CO(2) and decreases oxyhemoglobin affinity in several species; 2) it may help reconcile several longstanding problems with measured values of the Donnan ratios for Cl(-), HCO, and H(+) across the RBC membrane that are used to calculate total CO(2) carriage, ion flux rates, and membrane potentials; 3) it is a factor in the change in the dissociation constant for the combined nonvolatile weak acids of Hb associated with the Haldane effect; and 4) it diminishes the decrease in strong ion difference in the RBC intracellular fluid that would otherwise occur from the chloride shift and prevent the known increase of HCO concentration in that compartment.     

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.     

The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.     

Towards a molecular explanation of the high performance of the tuna heart

The tuna heart is capable of sustaining cardiac outputs that are high relative to other active teleost species. The question as to whether there are known molecular mechanisms to explain this phenomenon is examined. Unfortunately, the evidence at present is scant but the paper attempts to put existing knowledge into a contextual framework. It is known that the high cardiac outputs in tuna are due to the maximum heart rates that are high relative to other active teleost species. While the normalized stroke volume (ml/kg body weight) in tuna is in the same range as that of trout several important points are worth noting. The stroke volume in the tuna heart is generated in the face of an afterload that is two-fold higher (due to high ventral aortic pressure) than that observed in the trout or other teleosts. Since the stroke volume is predicated on the strength of ventricular contraction, the question then arises as to whether this is a consequence of factors intrinsic or extrinsic to the design of the cardiomyocytes that make up the myocardium. Two important extrinsic factors must be noted: the substantially higher normal operating temperatures and the apparent cardiac hyperplasia in the tuna. While the merit of these factors is discussed, the paper focuses on intrinsic factors. There is very little that has been determined to date that would indicate substantive changes in the design of the myocyte in the tuna heart compared to that of trout. Are these extrinsic factors alone then sufficient to account for the impressive cardiac output capabilities in the tuna?     

Effects of pH and Ca2+ on monomer-dimer and monomer-tetramer equilibria of chromogranin A.

Chromogranin A is a high capacity, low affinity Ca2+ binding protein which undergoes Ca2+- and pH-dependent conformational changes, and has recently been suggested to play a Ca2+-buffering role in the secretory vesicle of adrenal medullary chromaffin cell, the major inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store of chromaffin cell (Yoo, S.H., and Albanesi, J.P. (1990) J. Biol. Chem. 265, 13446-13448). In the present study, it is shown that chromogranin A exists in a monomer-dimer equilibrium at pH 7.5 and in a monomer-tetramer equilibrium at pH 5.5. The pH appears monomer-tetramer equilibrium at pH 5.5. The pH appears to be a necessary and sufficient factor determining the types of oligomers formed. Although Ca2+ did not change the type of oligomerization, it had a very significant effect on the values of the thermodynamic parameters characterizing the associations. The delta G0 values for a monomer-dimer equilibrium were -7 to -8 kcal/mol, while those for a monomer-tetramer equilibrium were -20 to -23 kcal/mol. At pH 5.5, the values of delta H0, delta S0, and delta C0p were large and negative in the absence of Ca2+ and large and positive in the presence of 35 mM Ca2+, implying markedly different reaction mechanisms. Extrapolation of the results to 37 degrees C and 1 mM chromogranin A suggests that chromogranin A is virtually 100% tetramer at pH 5.5 in the presence of 35 mM Ca2+ but is 96% dimer at pH 7.5 in the absence of Ca2+, the two conditions resembling those seen in vivo. These results suggest that chromogranin A is mostly dimer in the endoplasmic reticulum and cis-Golgi area and is essentially all tetramer in the vesicle.     

Tetramer-dimer dissociation in homoglobin and the Bohr effect.

The pH dependence of the apparent tetramer to dimer dissociation constant has been determined at 20 degrees for both oxy- and deoxyhemoglobins A and Kansas. These measurements were made by three different procedures: gel chromatography, sedimentation velocity, and kinetic methods in either of three buffer systems: 0.05 M cacodylate, Tris, or glycine with 1 mM EDTA and 0.1 M NaCl between pH 6.5 and 11. The tetramer-dimer dissociation constant of human oxyhemoglobin A decreases from about 3.2 X 10(-6) M at pH 6.0 to about 3.2 X 10(-8) M at pH 8.5. The slope of this line indicates that the dissociation of tetramer to dimer is accompanied by the uptake of about 0.6 protons per mol of tetramer in this region. The corresponding dissociation constant for deoxyhemoglobin in the same pH region increases apparently almost linearly from 1.0 x 10(-12) M at pH 6.5 to about 1.0 x 10(-5) M at pH 11. To dimer is associated with the release of about 1.6 protons per mol of tetramer. Comparison of these data with the known proton release accompanying the oxygenation of tetramers confirms that the pH dependence of oxygen binding by dimers must be very small. The present data predict that the overall proton release or uptake per oxygen bound by dimer should be less than 0.1. The tetramer-dimer dissociation equilibria of oxy- and deoxyhemoglobins above pH 8.5 have identical pH dependences. In this range the dissociation constant of deoxy-Hb is about one-tenth that of oxyhemoglobin. Human oxyhemoglobin Kansas is known to have an enhanced tetramer-dimer dissociation compared with that of hemoglobin A. Below pH 8.5 the tetramer-dimer dissociation constant of Hb Kansas is about 400 times greater than that of HbA in the absence of phosphate buffers. In contrast, the tetramer-dimer dissociation constants of deoxyhemoglobins A and Kansas appear to be identical. These findings are consistent with previous structural observations on these hemoglobins. The data on the tetramer-dimer dissociation of human hemoglobin were used to calculate the total free energy of binding of oxygen to the tetramer and the median oxygen pressure on the basis of fundamental linkage relations and a pH-independent estimate of the total free energy of binding oxygen to dimer. Simulated oxygen binding curves were generated with the equations of Ackers and Halvorson (Ackers, G. K., and Halvorson, H. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4312-4316) by making two assumptions: (a) that the dimers are noncooperative and pH-independent in O2 binding and (b) that the distribution of cooperative energy in the oxygenation of tetramers is independent of pH. We have compared these simulations with experimental data obtained at low protein concentrations (30 to 124 muM heme) to show that the variation in oxygen affinity with pH can be described in terms of the subunit equilibria. We conclude that an accurate analysis of the contributions of individual oxygen binding steps to the Bohr effect cannot be made without considering the contributions of the dimers to oxygen binding...     

Enhanced nitrite reductase activity associated with the haptoglobin complexed hemoglobin dimer: functional and antioxidative implications

The presence of acellular hemoglobin (Hb) within the circulation is generally viewed as a pathological state that can result in toxic consequences. Haptoglobin (Hp), a globular protein found in the plasma, binds with high avidity the αβ dimers derived from the dissociation of Hb tetramer and thus helps clear free Hb. More recently there have been compelling indications that the redox properties of the Hp bound dimer (Hb-Hp) may play a more active role in controlling toxicity by limiting the potential tissue damage caused by propagation of the free-radicals generated within the heme containing globin chains. The present study further examines the potential protective effect of Hp through its impact on the production of nitric oxide (NO) from nitrite through nitrite reductase activity of the Hp bound αβ Hb dimer. The presented results show that the Hb dimer in the Hb-Hp complex has oxygen binding, CO recombination and spectroscopic properties consistent with an Hb species having properties similar to but not exactly the same as the R quaternary state of the Hb tetramer. Consistent with these observations is the finding that the initial nitrite reductase rate for Hb-Hp is approximately ten times that of HbA under the same conditions. These results in conjunction with the earlier redox properties of the Hb-Hp are discussed in terms of limiting the pathophysiological consequences of acellular Hb in the circulation.     

The nicotinamide adenine dinucleotides as allosteric effectors of human hemoglobin

The oxygen binding properties of human hemoglobin are appreciably altered by the nicotinamide dinucleotides NADH, NADP+, and NADPH. These cofactors are important in the control of many metabolic pathways and in providing reductive potential for a number of enzymatic reactions, including in vivo reduction of methemoglobin. Specific binding of these cofactors to hemoglobin and their potential for acting as allosteric modifiers of hemoglobin function have not been previously recognized. Detailed oxygen binding studies utilizing a thin-layer method suggest that the nicotinamide dinucleotides bind with high affinity to the deoxyhemoglobin tetramer at the beta chain anion-binding site and stabilize the low affinity "T-state" conformation. Stripped Hb A in 0.05 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 6.5, at 20 degrees C is half-saturated at a pO2 of 1.6 mm Hg. In the presence of 0.5 mM NADH, NADP+, or NADPH, the P50 is raised to 3.8, 7.1, and 12.5 mm Hg, respectively. The Bohr factor for stripped Hb A in 0.05 M HEPES buffer is sensitive to these effectors and is raised from 0.25 to about 0.65 by the addition of NADPH. The data suggest that routine use of these effectors in studies of human hemoglobin variants or the allosteric mechanism of Hb A be considered carefully. The relatively low intraerythrocytic levels of the nicotinamide dinucleotides in relation to hemoglobin dictate that these cofactors cannot significantly affect in vivo oxygen delivery. However, the converse is theoretically possible. The binding of the cofactors to hemoglobin and the preferential binding of their reduced forms may affect cofactor-dependent metabolic processes in red blood cells.     

Ammonia excretion by the skin of zebrafish (Danio rerio) larvae

The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique was applied to measure H(+) and NH(4)(+) fluxes in specific cells on the skin of zebrafish larvae. NH(4)(+) extrusion was relatively high in H(+) pump-rich cells, which were identified as the H(+)-secreting ionocyte in zebrafish. Minor NH(4)(+) extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH(4)(+) extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H(+) and NH(4)(+) gradients at the larval surface. Moreover, coupled decreases in NH(4)(+) and H(+) extrusion were found in larvae treated with an H(+)-pump inhibitor (bafilomycin A1) or H(+)-pump gene (atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligonucleotides also decreased NH(4)(+) excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and it provides direct evidence for the involvement of the H(+) pump and an Rh glycoprotein (Rhcg1) in ammonia excretion.     

Hemoglobins of the tadpole of the bullfrog, Rana catesbeiana. Temperature dependence of oxygen binding and pH dependence of subunit dissociation.

The temperature dependence of the oxygen equilibrium of tadpole hemoglobin has been determined between 0 degrees and 32 degrees for the unfractionated but phosphate-free lysate and between 12 degrees and 32 degrees for each of the four isolated components between pH 6 and 10 in 0.05 M cacodylate, Tris, or glycine buffers containing 0.1 M NaCl and 1 mM EDTA. Under these conditions the Bohr effect (defined as deltalog p50/deltapH) of the unfractionated lysate is positive at low temperatures between pH 6 and 8.5 and is negative above pH 8.5 to 8.8 at any temperature. As the temperature rises the Bohr effect below pH 8.5 changes greatly. In the interval pH 7.0 to 7.5, the magnitude of the Bohr effect decreases from + 0.28 at 0 degrees to zero at about 24 degrees and becomes negative, as in mammalian hemoglobins, above this temperature. Measurements with the isolated components show that the temperature dependence of oxygen binding for Components I and II and for Components III and IV is very similar. For both sets of components the apparent overall enthalpy of oxygenation at pH 7.5 is about -16.4 kcal/mol and -12.6 kcal/mol at pH 9.5. The measured enthalpies include contributions from the active Bohr groups, the buffer ions themselves, the hemoglobin groups contributing buffering, and any pH-dependent, oxygenation-dependent binding of ions such as chloride by the hemoglobin. The apportioning of the total enthalpy among these various processes remains to be determined. Between pH 8 and 10.5 tadpole oxyhemoglobin undergoes a pH-dependent dissociation from tetramer to dimer. The pH dependence of the apparent tetramer-dimer dissociation constant indicates that at pH 9.5 the dissociation of each tetramer is accompanied by the release of approximately 2 protons. In this pH range the oxygen equilibrium measurements indicate that about 0.5 proton is released for each oxygen molecule bound. The results are consistent with the conclusion that one acid group per alphabeta dimer changes its pK from about 10 to 8 or below upon dissociation of the tetramer.     

Heats of carbon monoxide binding by hemoglobin M Iwate.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.     

Asymmetric (deoxy dimer/azido-met dimer) hemoglobin hybrids dissociate within seconds.

Double mixing stopped-flow experiments have been performed to study the stability of asymmetric hemoglobin (Hb) hybrids, consisting of a deoxy and a liganded dimer. The doubly liganded [deoxy/cyano-met] hybrid (species 21) was reported to have an enhanced stability, with tetramer to dimer dissociation requiring over 100 seconds, based on a method that required an incubation of over two days. However, kinetic experiments revealed rapid ligand binding to species 21, as for triply liganded tetramers, which dissociate within a few seconds. For the present study, [deoxy dimer/azido-met dimer] hybrids are formed within 200 ms by stopped-flow mixing of dithionite with a solution containing oxyHb and azido-metHb. The dithionite scavenges oxygen, thus transforming oxyHb to deoxyHb, and the [oxy dimer/azido-met dimer] hybrid to the asymmetric [deoxy/azido-met] hybrid (species 21). After a variable aging time of the asymmetric hybrids, their allosteric state is probed by CO binding in a second mixing. As previously observed the freshly produced asymmetric hybrids bind CO rapidly as for R-state Hb. As the hybrids are aged from 0.1 to 10 seconds, the fraction of slow CO binding increases, consistent with a dissociation of the asymmetric hybrid to form the more stable deoxy Hb tetramer which reacts slowly with CO. Control experiments showed a predominantly slow phase for deoxy Hb, and fast rebinding for the symmetric hybrids.The kinetic data can be simulated with a tetramer to dimer dissociation rate for species 21 of 1.5/second at 100 mM NaCl (pH 7.2) and 1.9/second at 180 mM NaCl (pH 7.4). These values are similar to those reported for liganded Hb, as opposed to deoxy (T-state) tetramers which dissociate over four orders of magnitude more slowly. As expected from simulations of dimer exchange, the observed transition rate depends on the initial fractions of oxy- and metHb; this effect is not consistent with a slow R to T transition. These results, showing a lifetime of about one second for species 21, do not support the symmetry rule which is based on an enhanced stability of the asymmetric hybrid.     

Effect of 2,3-diphosphoglycerate on the Bohr effect of human adult hemoglobin

The effect of 2,3-diphosphoglycerate (DPG) on the Bohr effect of human hemoglobin has been studied by means of hydrogen ion titration techniques. The results indicate a) that both the acid and the alkaline Bohr effect are equally affected, b) that the DPG binding to deoxyhemoglobin (Hb) is much stronger than to carboxyhemoglobin (HbCO) and c) that Hb binds effectively one DPG molecule. The effect on the Bohr effect can roughly be described by assuming that upon binding two groups per tetramer change their pK from 6.8 to 7.8 and two others from 6.8 to 5.8. These groups very probably are the imidazole groups of the two histidines H21 (143)β and the two phosphate groups of DPG (second dissociation). From the experiments a value for the dissociation constant K of the Hb-DPG complex of about 10⁻⁵ M⁻¹ could be estimated at pH 6.2 and pH 7.5.     

Effect of carbonic acid on glutamate dehydrogenase interaction with NADH

The method of fluorescent titration was used to study the effect of carbonic acid on the process of NADH binding with the purified preparation of glutamate dehydrogenase as well as on fluorescent properties of the above mentioned coenzyme. It is shown that in bicarbonate buffer at the alkaline values of pH there is a decrease in the maximal capacity of the binding sites of the enzyme with low affinity to co-enzyme. The capacity of high-affinity binding sites in this case is unchanged. It is found that pCO2, HCO3- and H+ exert an effect on the dissociation constants of the NADH-glutamate dehydrogenase complex as well as on fluorescent properties of bound NADH. It is supposed that the effects observed are a result of the interaction of dissolved carbon dioxide with free amino groups of protein molecule.