Saturation dependency of the Bohr effect: interactions among H-+, CO2, and DPG.

The Bohr effect was measured in normal whole blood and in blood with low DPG concentration as a function of oxygen saturation. pH was changed by varying CO2 concentration (CO2 Bohr effect) or by addition of isotonic NaOH or HC1 at constant PCO2 (fixed acid Bohr effect). At nornal DPG concentration CO2 Bohr effect was -0.52 at 50% blood oxygen saturation, increasing in magnitude at lower saturation and decreasing in magnitude at higher saturation. In DPG depleted blood with base excess (BE) similar to 0 meq/1, there was similar dependence of CO2 Bohr effect on oxygen saturation. At BE similar to -10 meq/1, influence of saturation was comparable, but the magnitude of the Bohr effect was markedly increased at all saturations. Fixed acid Bohr effect at normal DPG concentration was -0.45 at saturations of 50-90% but decreased at lower saturations. In DPG-depleted blood fixed acid Bohr effect averaged about -0.33 with minimal variation with saturation. Influence of DPG on oxygen affinity was greater at intermediate saturations and less at saturations below 20% and above 80%. Effect of CO2, independent of pH, was many fold greater at lower oxygen saturations than at higher saturations. These results support the suggestion that the alpha chain of hemoglobin is the site of the initial oxygenation reaction. Physiologically they indicate that the relative contribution of CO2 and fixed acid, as well as the level of oxygen saturation and DPG concentration, may be important in determining PO2 of capillary blood and resulting oxygen delivery.     

Increased C availability at elevated carbon dioxide concentration improves N assimilation in a legume

Plant growth is typically stimulated at elevated carbon dioxide concentration ([CO2]), but a sustained and maximal stimulation of growth requires acquisition of additional N in proportion to the additional C fixed at elevated [CO2]. We hypothesized that legumes would be able to avoid N limitation at elevated [CO2]. Soybean was grown without N fertilizer from germination to final senescence at elevated [CO2] over two growing seasons under fully open-air conditions, providing a model legume system. Measurements of photosynthesis and foliar carbohydrate content showed that plants growing at elevated [CO2] had a c. 25% increase in the daily integral of photosynthesis and c. 58% increase in foliar carbohydrate content, suggesting that plants at elevated [CO2] had a surplus of photosynthate. Soybeans had a low leaf N content at the beginning of the season, which was a further c. 17% lower at elevated [CO2]. In the middle of the season, ureide, total amino acid and N content increased markedly, and the effect of elevated [CO2] on leaf N content disappeared. Analysis of individual amino acid levels supported the conclusion that plants at elevated [CO2] overcame an early-season N limitation. These soybean plants showed a c. 16% increase in dry mass at final harvest and showed no significant effect of elevated [CO2] on leaf N, protein or total amino acid content in the latter part of the season. One possible explanation for these findings is that N fixation had increased, and that these plants had acclimated to the increased N demand at elevated [CO2].     

Hemoglobin-oxygen affinity in anemia

In blood of 21 anemic patients and 8 normal subjects (N) three oxygen dissociation curves each were measured at different pH values to calculate Bohr coefficients after acidification with CO2 (BCCO2) or fixed acid (BCFA), and other important parameters of oxygen affinity. The patients had either low hemoglobin or red cell production (L: n = 11, 7.3 g/dl Hb) or high erythrocyte production combined with high loss (H: n = 10, 7.8 g/dl Hb). The standard half saturation pressure P50 (pH 7.4, 37 degrees C) was equally elevated in both anemic groups (L: 30.5, H: 30.8, N: 26.7 mmHg), as well as the diphosphoglycerate concentration (DPG) (L: 18.7, H: 18.6, N: 12.7 mumol/g Hb). The red cell pH of the anemics was lower than for the N (approximately 0.045 units) causing part of the difference in P50. Hill's "n" tended to high values in the anemics except at low O2-saturation in the H. For BCCO2 no significant difference among the groups was observed. BCFA, however, increased in the H at low SO2 compared to the N and L. The cause for most of the changes in hemoglobin oxygen affinity in anemics was the high [DPG]. The combination of high P50 and high "n" value as in the L seems to be most advantageous for tissue oxygenation.     

Influence of allosteric effectors and temperature on oxygen binding properties and the Bohr effect of bovine hemoglobin

The O2 binding properties of bovine Hb were examined. The increase in Cl- and DPG concentration enhanced P50. A reduction in n(max) was observed at high Cl- concentration, while DPG had little effect on n(max). An increase in Cl- concentration enhanced the Bohr effect, the magnitude of which reached a maximum at 0.1 M Cl- and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [Cl-] plot, and also equal to the physiological Cl- concentration (0.1 M) of bovine blood. Furthermore, the influence of Cl- concentration on the Bohr effect is independent of temperature. On the other hand, in the absence of Cl-, bovine Hb is sensitive to DPG; an increase in DPG concentration enhanced the Bohr effect, which reached a maximum at 3 mM DPG and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [DPG] plot. At low DPG concentrations, the DPG effect on the Bohr effect became small with increasing temperature, whereas at high DPG concentrations, the DPG effect was insensitive to temperature changes. At the physiological concentration of DPG (0.5 mM), increases in both Cl- concentration and temperature diminished the DPG effect. At the physiological concentrations of Cl- and DPG, the Bohr effect was -0.36 at 37 degrees C. The deltaH value at the physiological concentrations of Cl- and DPG was approximately -5.8 kcal/mol at pH 7.4. These results indicate that Cl- and temperature are important determinants of the O2 binding properties of bovine Hb.     

Predicting the carboxyhemoglobin levels resulting from carbon monoxide exposures.

Data from a series of human exposures to carbon monoxide (CO) were analyzed to determine the fit to the theoretical Coburn-Forster-Kane (CFK) equation which describes CO absorption and excretion. The equation was found to predict carboxyhemoglobin (HbCO) saturations for both men and women at exercise rates ranging from sedentary to 300 kpm/min when they were exposed to steady CO concentrations of 50, 100, and 200 ppm for 0.33-5.25 h. Methods for determining values of each of the variables in the CFK equation were collected and a rational, efficient procedure for solving the equation by trial and error was outlined. The CFK equation was then used to prepare a graph, relating HbCO saturation to exposure duration and concentration, and also to describe the effect of several variables on the rate of CO uptake and equilibrium HbCO levels, important considerations in the determination of permissible public, occupational, and experimental exposure to CO.     

The effect of 4-isothiocyanatobenzoic acid on the oxygen-linked chloride binding sites in human hemoglobin: Influence on the alkaline Bohr effect

Human oxyhemoglobin reacted with 4-isothiocyanatobenzoic acid shows a decreased oxygen affinity that does not change with increasing chloride concentration indicating that all of the oxygen-linked chloride binding sites are blocked in the modified protein. By contrast, reaction of oxyhemoglobin with 4-isothiocyanatobenzenesulfonamide produces a modified protein with increased oxygen affinity below pH 7.3 that shows the expected decrease in oxygen affinity with increasing chloride concentration. The latter result demonstrates the importance of the negatively charged moiety in producing both the decrease in oxygen affinity and the effect on the oxygen-linked chloride binding sites produced by 4-isothiocyanatobenzoic acid. Reduction in the alkaline Bohr effect by 50% in the protein modified by 4-isothiocyanatobenzoic acid indicates that contribution to the alkaline Bohr effect is evenly divided between chloride dependent and chloride independent groups.     

Properties of carboxymethylated cross-linked hemoglobin A

The selective carboxymethylation of the N-terminal amino groups of hemoglobin A with glyoxylic acid and sodium cyanoborohydride has been studied as a function of the state of ligation of hemoglobin. The N-terminal residues have been established as the primary sites of reaction by peptide mapping of the tryptic digest of each chain and subsequent amino acid analysis of the modified peptides. With oxyhemoglobin, the desired derivatives with a carboxymethyl group at the N-terminal of either or both chains amounted to 55% [Di Donato, A., Fantl, W. J., Acharya, A. S., & Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895]. In the present study it is shown that with deoxyhemoglobin the amount of the desired derivative is increased to 75%. The oxygen equilibrium curve of hemoglobin A carboxymethylated on its four N-terminal residues [0.5 mM as tetramer in 50 mM [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.5, 37 degrees C] had a P50 value of 30 mmHg (Hill coefficient n = 2.8, alkaline Bohr value = 0.4) compared to a P50 of 9 mmHg for unmodified hemoglobin under the same conditions (n = 2.5, alkaline Bohr value = 0.5). In carboxymethylated oxyhemoglobin A, cross-linked with the mild agent glycolaldehyde for 3.5 h, there was 85% of Mr 64,000 species and 15% of Mr 128,000 or higher species. For the former, the extent of cross-linking between two subunits was 19%. For the latter, there was 29% of two cross-linked subunits and 13% of three cross-linked subunits. Termination of cross-linking, which may be desirable in some circumstances, can be successfully achieved with isonicotinic acid hydrazide.(ABSTRACT TRUNCATED AT 250 WORDS)     

New effectors of human hemoglobin: structure and function

We describe the actions of two new allosteric effectors of hemoglobin, 2-[4-(3,5-dichlorophenylureido)phenoxy]-2-methylpropionic acid (L35) and 2-[4-(3,4,5-trichlorophenylureido)phenoxy]-2-methylpropionic acid (L345). Each of them binds to two pairs of symmetry-related sites in the central cavity of human deoxyhemoglobin. One pair of sites overlaps with that occupied by bezafibrate [Perutz et al. (1986) J. Am. Chem. Soc. 108, 1064-1078]. The other sites are new, and the pair occupied by L35 is different from that occupied by L345. All the sites are at least 20 A from the site where organic phosphates are bound. L345 is by far the most potent allosteric effector of hemoglobin ever described. At a concentration of 0.1 mM, it raises the P50 of a suspension of red cells by 50%; at 0.2 mM it raises the P50 2.5-fold. At acid pH, it reduces Hill's coefficient to near unity and prevents complete oxygen saturation even under 1 atm of pure oxygen. In azidemethemoglobin at pH 6, it induces a transition to higher spin. These properties are reminiscent of those of teleost fish hemoglobins that exhibit a Root effect. The influence of L35 and L345 and that of organic phosphates on the oxygen affinity are additive, but they compete with chloride. L35 acts more weakly than L345, but can be made to induce the same effects as L345 alone by adding inositol hexaphosphate. Both compounds increase the alkaline and acid Bohr effects. They alter the bimolecular kinetics of CO recombination after a flash by increasing the slowly reacting fraction of hemoglobin in the T state at the expense of the fast-reacting fraction in the R state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acid-base and respiratory properties of a buffered bovine erythrocyte perfusion medium

Current research in organ physiology often utilizes in situ or isolated perfused tissues. We have characterized a perfusion medium associated with excellent performance characteristics in perfused mammalian skeletal muscle. The perfusion medium consisting of Krebs-Henseleit buffer, bovine serum albumin, and fresh bovine erythrocytes was studied with respect to its gas-carrying relationships and its response to manipulation of acid-base state. Equilibration of the perfusion medium at base excess of -10, -5, 0, 5, and 10 mmol X L-1 to humidified gas mixtures varying in their CO2 and O2 content was followed by measurements of perfusate hematocrit, hemoglobin concentration, pH, Pco2, Cco2, Po2, and percent oxygen saturation. The oxygen dissociation curve was similar to that of mammalian bloods, having a P50 of 32 Torr (1 Torr = 133.3 Pa), Hill's constant n of 2.87 +/- 0.15, and a Bohr factor of -0.47, showing the typical Bohr shifts with respect to CO2 and pH. The oxygen capacity was calculated to be 190 mL X L-1 blood. The carbon dioxide dissociation curve was also similar to that of mammalian blood. The in vitro nonbicarbonate buffer capacity (delta [HCO3-] X delta pH-1) at zero base excess was -24.6 and -29.9 mmol X L-1 X pH-1 for the perfusate and buffer, respectively. The effects of reduced oxygen saturation on base excess and pH of the medium were quantified. The data were used to construct an acid-base alignment diagram for the medium, which may be used to quantify the flux of nonvolatile acid or base added to the venous effluent during tissue perfusions.     

Ligand binding properties of hemoglobin 3 of the trout, Salmo gairdneri. The occurrence of an acid Bohr effect in the absence of heme-heme interaction.

The four components of hemoglobin from the rainbow trout (Salmo gairdneri) have been isolated. The oxygen affinities of the first two components eluted from the DEAE-cellulose column have much smaller pH dependencies than the last two components. These components have very low O2 affinities at low pH. The effect of pH on the equilibrium and kinetics of ligand binding to the third fraction, the pH-dependent component present in greatest amounts, has been studied. Measurements of ligand binding equilibria demonstrate the presence of both an alkaline and an acid Bohr effect. In the region of the alkaline Bohr effect the value of n in the Hill equation is a function of ligand affinity. For CO binding n decreases as the pH is decreased until at pH 6, the minimum ligand affinity is reached. At this pH there is also a complete loss of cooperative ligand binding. Decreasing the pH further results in an increase of ligand affinity, but this acid Bohr effect is not associated with a reappearance of cooperativity. This suggests that Fraction 3 of S. gairdneri is frozen in the low affinity, deoxygenated conformation at low pH and that the quaternary structure does not change even when fully liganded. However, the properties of the low affinity conformation of this hemoglobin are pH-dependent.     

The CO and NO Bohr effect of human hemoglobin with and without inositolhexaphosphate.

Using NO and CO as ligands the Bohr effect of human hemoglobin has been measured with and without inositolhexophosphate. It appears that in the absence and presence of inositolhexaphosphate hemoglobin shows a distinct ligand specificity with respect to the Bohr effect. Ligation with NO is accompanied by release of a larger number of Bohr effect. It is shown that this latter result is due to the fact that the number of protons taken up upon binding of inositolhexaphosphate to ligated hemoglobin is larger for HbNO than for HbCO. It is suggested that this additional proton uptake is partially due to a restoration of the saltbridge between His 146beta and Asp 94beta upon addition of IHP.     

Functional behaviour of dilute solutions of Hb-Kempsey: beta 99 (G-1) Asp--Asn.

The authors report that a diluted solution of Hb-Kempsey, beta 99 (G-1) Asp-Asn, can be chromatographically separated from the coexistent Hb-A and functionally examined if progressively depleted in O2 by bubbling pure nitrogen in the solution. Next, at fixed times, the O2 saturations of Hb are compared with the pO2s measured. Hb-Kempsey has a p50 of 1 torr, with an n-value of 1 and a Bohr effect of -0.2. Normal Hb-A of the same patient, examined with identical methods, presents: p50 = 4.5 torr; n = 2.7; Bohr effect = -0.412. Therefore, Hb-Kempsey is strongly hyperaffinic, does not display any heme-heme interaction, and has a half-normal Bohr effect.     

Decomposition of hydroxylamine by hemoglobin

The reaction between hydroxylamine (NH2OH) and human hemoglobin (Hb) at pH 6-8 and the reaction between NH2OH and methemoglobin (Hb+) chiefly at pH 7 were studied under anaerobic conditions at 25 degrees C. In presence of cyanide, which was used to trap Hb+, Hb was oxidized by NH2OH to methemoglobin cyanide with production of about 0.5 mol NH+4/mol of heme oxidized at pH 7. The conversion of Hb to Hb+ was first order in [Hb] (or nearly so) but the pseudo-first-order rate constant was not strictly proportional to [NH2OH]. Thus, the apparent second-order rate constant at pH 7 decreased from about 30 M-1 X s-1 to a limiting value of 11.3 M-1 X s-1 with increasing [NH2OH]. The rate of Hb oxidation was not much affected by cyanide, whereas there was no reaction between NH2OH and carbonmonoxyhemoglobin (HbCO). The pseudo-first-order rate constant for Hb oxidation at 500 microM NH2OH increased from about 0.008 s-1 at pH 6 to 0.02 s-1 at pH 8. The oxidation of Hb by NH2OH terminated prematurely at 75-90% completion at pH 7 and at 30-35% completion at pH 8. Data on the premature termination of reaction fit the titration curve for a group with pK = 7.5-7.7. NH2OH was decomposed by Hb+ to N2, NH+4, and a small amount of N2O in what appears to be a dismutation reaction. Nitrite and hydrazine were not detected, and N2 and NH+4 were produced in nearly equimolar amounts. The dismutation reaction was first order in [Hb+] and [NH2OH] only at low concentrations of reactants and was cleanly inhibited by cyanide. The spectrum of Hb+ remained unchanged during the reaction, except for the gradual formation of some choleglobin-like (green) pigment, whereas in the presence of CO, HbCO was formed. Kinetics are consistent with the view advanced previously by J. S. Colter and J. H. Quastel [1950) Arch. Biochem. 27, 368-389) that the decomposition of NH2OH proceeds by a mechanism involving a Hb/Hb+ cycle (reactions [1] and [2]) in which Hb is oxidized to Hb+ by NH2OH.     

Functional characterization of the single hemoglobin of the migratory bird Ciconia ciconia

Hemolysate from white stork displayed a single hemoglobin component, thus resulting into two bands and two globin peaks in dissociating PAGE and reversed phase-HPLC, respectively. Stripped hemoglobin showed an oxygen affinity higher than that of human HbA, a small Bohr effect, and a cooperative oxygen binding. A small decrease of oxygen affinity, of the same extent in all the pH range examined, was observed by addition of chloride, thus indicating an unusual chloride-independent Bohr effect (DeltalogP50/Deltalog pH=-0.24). Saturating amounts of inositol hexakisphosphate, largely decreased hemoglobin-oxygen affinity (DeltalogP(50)=1.17 at pH 7.0), and increased the extent of its Bohr effect (DeltalogP50/DeltalogpH=-0.45). The phosphate binding curve allowed to measure a very high overall binding constant (K=1.18 x 10(5) M(-1)). The effect of temperature on the oxygen affinity was measured, and the enthalpy change of oxygenation resulted almost independent on pH. Structural-functional relationships are discussed by considering some amino acid residues situated at alpha1/beta1 and alpha1/beta2 interfaces, such as alpha38 and alpha89 positions. The presence of only one hemoglobin component, a rare event among birds, and its functional properties have been related to the physiological oxygen requirements of this soaring migrant bird and to its technique of flight during migration.     

Carbon dioxide concentration at night affects translocation from soybean leaves

Studies have indicated that the concentration of carbon dioxide [CO2] during the dark period may influence plant dry matter accumulation. It is often suggested that these effects on growth result from effects of [CO2] on rates of respiration, but responses of respiration to [CO2] remain controversial, and connections between changes in respiration rate and altered growth rate have not always been clear. The present experiments tested whether translocation, a major consumer of energy from respiration in exporting leaves, was sensitive to [CO2]. Nineteen-day-old soybean plants grown initially at a constant [CO2] of 350 micromol mol(-1) were exposed to three consecutive nights with a [CO2] of 220-1400 micromol mol(-1), with a daytime [CO2] of 350 micromol mol(-1). Change in dry mass of the individual second, third and fourth trifoliate leaves over the 3-d period was determined, along with rates of respiration and photosynthesis of second leaves, measured by net CO2 exchange. Translocation was determined from mass balance for second leaves. Additional experiments were conducted where the [CO2] around individual leaves was controlled separately from that of the rest of the plant. Results indicated that low [CO2] at night increased both respiration and translocation and elevated [CO2] decreased both processes, to similar relative extents. The effect of [CO2] during the dark on the change in leaf mass over 3 d was largest in second leaves, where the change in mass was about 50% greater at 1400 micromol mol(-1) CO2 than at 220 micromol mol(-1) CO2. The response of translocation to [CO2] was localized in individual leaves. Results indicated that effects of [CO2] on net carbon dioxide exchange rate in the dark either caused or reflected a change in a physiologically important process which is known to depend on energy supplied by respiration. Thus, it is unlikely that the observed effects of [CO2] on respiration were artefacts of the measurement process in this case.     

Effect of CO2 concentration on phosphatidylcholine and phosphatidylglycerol metabolism in surfactant and residual lung fractions.

An investigation of the effect of change of total CO(2) concentration from 7 to 43 mM at pH 7.35 in the medium perfusing isolated rat lungs on [U-(14)C]glucose incorporation into lung phospholipids has been carried out. The incorporation of [U-(14)C]glucose into phosphatidylcholine and phosphatidylglycerol of the surfactant fraction and of the remaining lung tissue (residual fraction) was observed. Increased CO(2) concentration increased [U-(14)C]glucose incorporation into phosphatidylcholine of the surfactant fraction and residual fraction by 43 and 50%, respectively, during a 2 hr perfusion. Likewise, incorporation of [U-(14)C]glucose into phosphatidylglycerol was increased 22 and 34% into the surfactant and residual fractions, respectively. The percentage of [U-(14)C]glucose incorporated into the fatty acid moieties of phosphatidylcholine of both fractions increased as a result of increased CO(2) concentration. The increase in the incorporation of [U-(14)C]glucose into the fatty acid moieties of phosphatidylcholine was confirmed by an average increase of 56 and 77% in the specific activity of palmitic acid isolated from phosphatidylcholine of the surfactant and residual fraction, respectively, as a result of increased CO(2) concentration. The results suggest that alteration in extracellular CO(2) concentration affects the de novo synthesis from glucose of phosphatidylcholine and phosphatidylglycerol of the surfactant-lipoprotein fraction of lung.     

Photosynthesis, productivity, and yield of maize are not affected by open-air elevation of CO2 concentration in the absence of drought

While increasing temperatures and altered soil moisture arising from climate change in the next 50 years are projected to decrease yield of food crops, elevated CO2 concentration ([CO2]) is predicted to enhance yield and offset these detrimental factors. However, C4 photosynthesis is usually saturated at current [CO2] and theoretically should not be stimulated under elevated [CO2]. Nevertheless, some controlled environment studies have reported direct stimulation of C4 photosynthesis and productivity, as well as physiological acclimation, under elevated [CO2]. To test if these effects occur in the open air and within the Corn Belt, maize (Zea mays) was grown in ambient [CO2] (376 micromol mol(-1)) and elevated [CO2] (550 micromol mol(-1)) using Free-Air Concentration Enrichment technology. The 2004 season had ideal growing conditions in which the crop did not experience water stress. In the absence of water stress, growth at elevated [CO2] did not stimulate photosynthesis, biomass, or yield. Nor was there any CO2 effect on the activity of key photosynthetic enzymes, or metabolic markers of carbon and nitrogen status. Stomatal conductance was lower (-34%) and soil moisture was higher (up to 31%), consistent with reduced crop water use. The results provide unique field evidence that photosynthesis and production of maize may be unaffected by rising [CO2] in the absence of drought. This suggests that rising [CO2] may not provide the full dividend to North American maize production anticipated in projections of future global food supply.     

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)     

Increased protein carbonylation in leaves of Arabidopsis and soybean in response to elevated [CO2]

While exposure of C3 plants to elevated [CO2] would be expected to reduce production of reactive oxygen species (ROS) in leaves because of reduced photorespiratory metabolism, results obtained in the present study suggest that exposure of plants to elevated [CO2] can result in increased oxidative stress. First, in Arabidopsis and soybean, leaf protein carbonylation, a marker of oxidative stress, was often increased when plants were exposed to elevated [CO2]. In soybean, increased carbonyl content was often associated with loss of leaf chlorophyll and reduced enhancement of leaf photosynthetic rate (Pn) by elevated [CO2]. Second, two-dimensional (2-DE) difference gel electrophoresis (DIGE) analysis of proteins extracted from leaves of soybean plants grown at elevated [CO2] or [O3] revealed that both treatments altered the abundance of a similar subset of proteins, consistent with the idea that both conditions may involve an oxidative stress. The 2-DE analysis of leaf proteins was facilitated by a novel and simple procedure to remove ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from soluble soybean leaf extracts. Collectively, these findings add a new dimension to our understanding of global change biology and raise the possibility that oxidative signals can be an unexpected component of plant response to elevated [CO2].     

ATP sulfurylase from Penicillium chrysogenum. Molecular basis of the sigmoidal velocity curves induced by sulfhydryl group modification

ATP sulfurylase from Penicillium chrysogenum is a noncooperative homooligomer containing three free sulfhydryl groups per subunit. Under nondenaturing conditions, one SH group per subunit was modified by 5,5'-dithiobis-(2-nitrobenzoate), or N-ethylmaleimide. Modification had only a small effect on kcat, but markedly increased the [S]0.5 values for the substrates, MgATP and SO4(2-). MgATP and adenosine-5'-phosphosulfate protected against modification. The SH-modified enzyme displayed sigmoidal velocity curves for both substrates with Hill coefficients (nH) of 2. Fluorosulfonate (FSO3-) and other dead-end inhibitors competitive with SO4(2-) activated the SH-modified enzyme at low SO4(2-) concentration. In order to determine whether the sigmoidicity resulted from true cooperative binding (as opposed to a kinetically based mechanism), the shapes of the binding curves were established from the degree of protection provided by a ligand against phenylglyoxal-dependent irreversible inactivation under noncatalytic conditions. Under standard conditions (0.05 M Na-N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid buffer, pH 8, 30 degrees C, and 3mM phenylglyoxal) the native enzyme was inactivated with a k of 2.67 +/- 0.25 X 10-3 s-1, whereas k for the SH-modified enzyme was 5.44 +/- 0.27 X 10-3 s-1. The increased sensitivity of the modified enzyme resulted from increased reactivity of ligand-protectable groups. Both the native and the SH-modified enzyme displayed hyperbolic plots of delta k (i.e. protection) versus [MgATP], or [FSO3-], or [S2O3(2-]) in the absence of coligand (nH = 0.98 +/- 0.06). The plots of delta k versus [ligand] for the native enzyme were also hyperbolic in the presence of a fixed concentration of coligand. However, in the presence of a fixed [FSO3-] or [S2O3(2-]), the delta k versus [MgATP] plot for the SH-modified enzyme was sigmoidal, as was the plot of delta k versus [FSO3-] or [S2O3(2-]) in the presence of a fixed [MgATP]. The nH values were 1.92 +/- 0.09. The results indicate that substrates (or analogs) bind hyperbolically to unoccupied SH-modified subunits, but in a subunit-cooperative fashion to form a ternary complex.